The CRHR1→PN→PVI Pathway in Medial Prefrontal Cortex Mediates Early-life Stress-induced Cognitive Deficits in Adolescent Mice

The CRHR1→PN→PVI Pathway in Medial Prefrontal Cortex Mediates Early-life Stress-induced Cognitive Deficits in Adolescent Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The CRHR1→PN→PVI Pathway in Medial Prefrontal Cortex Mediates Early-life Stress-induced Cognitive Deficits in Adolescent Mice Jitao Li, Yu-Nu Ma, Chao-Juan Yang, Chen-Chen Zhang, Ya-Xin Sun, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3572074/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Nov, 2024 Read the published version in Molecular Psychiatry → Version 1 posted 13 You are reading this latest preprint version Abstract Cognitive impairment, one core symptom of psychiatric disorders, is frequently observed in adolescents exposed to early-life stress (ES). However, the underlying neural mechanisms are unclear and the therapeutic efficacy is limited. Targeting at parvalbumin-expressing interneurons (PVIs) in the medial prefrontal cortex (mPFC), we report that mPFC PVI activity was reduced by ES and causally mediated ES-induced cognitive deficits in adolescent mice through chemogenetic or optogenetic experiments. We then demonstrate that ES reduced the excitatory inputs onto PVIs and pyramidal neuron (PN) activity and that ES negative effects were reversed by the knockout of corticotropin-releasing hormone receptor 1 (CRHR1, mainly expressed in PNs) in mouse mPFC, supporting the prefrontal CRHR1→PN→PVI pathway in mediating ES-induced cognitive deficits. Finally, antalarmin (a CRHR1 antagonist) treatment and environmental enrichment successfully restored PVI activity and cognitive deficits induced by ES. These findings highlight the critical role of PVIs in mediating and preventing ES-induced cognitive deficits in adolescent mice. Biological sciences/Psychology Biological sciences/Neuroscience Biological sciences/Molecular biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Adolescence often witnesses the first episodes of several psychiatric disorders, which has been associated with various genetic and environmental risk factors [ 1 , 2 ]. One such risk factor is early-life stress (ES). For instance, meta-analyses show that exposure to adverse life events increases the diagnosis of depression in childhood or adolescence by 2.5 times [ 3 ]. Cognitive impairment is one of the core symptoms of several psychiatric disorders and is frequently observed in adolescents exposed to ES [ 4 ]. However, the neural mechanisms underlying cognitive impairment remain unclear and the efficacy of current first-line therapeutic drugs for cognitive deficits is limited [ 5 , 6 ]. Importantly, several recent studies have provided behavioral evidence that ES could significantly impair cognition in adolescent animals [ 7 – 9 ], paving the way for identifying potential neural correlates for early and effective intervention. The prefrontal cortex (PFC) plays crucial roles in cognitive behaviors, is a late-developing structure and is highly vulnerable to ES [ 10 , 11 ]. Within the PFC, parvalbumin (PV)-expressing interneurons (PVIs) are the largest class of inhibitory neurons (accounting for about 40% of interneurons) [ 12 ]. They form perisomatic projections onto excitatory pyramidal neurons (PNs), controlling neural network synchrony that are crucial for learning and memory [ 13 – 15 ]. Genetic manipulation experiments to inactivate [ 16 , 17 ] or activate [ 18 , 19 ] PVIs in the mPFC have supported the causal link between PVI activity and prefrontal-dependent cognitive abilities in adult animals. PVIs undergo a protracted period of maturation during adolescence and contribute significantly to the stability of cortical excitatory-inhibitory microcircuits [ 20 ] and to the development of PFC-dependent cognitive abilities [ 21 , 22 ]. For instance, sustained inhibition of prefrontal PVIs activity during adolescence was found to disrupt cognitive flexibility in adult mice [ 18 ]. Only a few studies have examined the effects of ES on PVIs in adolescence, which have largely focused on the number (density) of PVIs [ 23 – 26 ]. It remains unknown whether and how ES would affect the activity of adolescent PFC PVIs and how this alteration links with ES-induced cognitive deficits. Moreover, several mechanisms may contribute to the maturation of PVIs during adolescence, one of which is the increased glutamatergic inputs onto PVIs [ 27 ]. Although ES has been reported to impair the structure [ 28 ] and function [ 29 ] of PNs in the adult mPFC, few studies have examined how ES would affect PNs activity and their inputs onto PVIs in the adolescent mPFC. For the treatment of cognitive impairment, the current pharmacological (e.g., first-line antidepressants or antipsychotics) and non-pharmacological (e.g., repeated transcranial magnetic stimulation (rTMS), or deep brain stimulation (DBS)) strategies have not yielded satisfactory results [ 5 , 30 , 31 ]. Recent clinical and basic studies have highlighted the association between PFC-dependent cognitive dysfunction and the corticotropin-releasing hormone (CRH) and its receptor 1 (CRHR1) [ 32 ]. Genetic polymorphisms of the CRHR1 gene have been found to be associated with cognitive functioning in both healthy adults and patients with psychiatric disorders [ 33 , 34 ]. In animal models, downregulating the CRH-CRHR1 system using pharmacological (e.g., CRH antagonist D-Phe-CRF5; CRHR1 antagonist, antalarmin) or genetic (e.g., knockdown of prefrontal CRHR1) methods could reverse cognitive deficits [ 28 , 35 , 36 ]. However, in adolescent animals, it remains unknown whether CRHR1-based intervention is effective for ES-induced cognitive deficits and how the CRH-CRHR1 system interacts with PFC PVIs. On the other hand, as a non-pharmacological intervention environmental enrichment (EE) has been shown to have beneficial effects on stress-related negative outcomes [ 37 ], and importantly on ES-induced cognitive impairments in adolescent animals [ 38 , 39 ]. However, the underlying mechanisms are still unclear. In this study, we systematically test the involvement of mPFC PVI activity in ES-induced cognitive deficits in adolescent mice. We adopted the well-established ES paradigm, the limited nesting and bedding material (LBN), which we have previously reported to induce deficits in PFC-dependent cognitive functions and pyramidal neuronal structural plasticity in adult male mice [ 28 ]. We first evaluated the effects of ES on cognition and mPFC PVI activity in adolescent mice. We then manipulated mPFC PVI activity through optogenetic and chemogenetic methods to examine the causal link between reduced mPFC PVI activity and ES-induced cognitive deficits. To understand the possible causes of PVI activity reduction following ES, considering the interaction between PVIs and PNs, the involvement of the CRH-CRHR1 system in ES adverse effects revealed in our previous studies [ 28 , 40 – 42 ], and the localization of CRHR1 in PNs[ 43 ], we proposed the following hypothesis: early-life stress may first upregulate CRH-CRHR1 signaling in prefrontal PNs, which reduces PN activity, inhibits excitatory input to PVIs, and in turn downregulates PVI activity and ultimately leads to PFC-dependent cognitive impairment. To test this hypothesis, we then carried out chemogenetic and pharmacological experiments to demonstrate the involvement of PNs and the CRH-CRHR1 system in ES-induced cognitive deficits and PVI reduction. Finally, we tested the therapeutic effects of a non-pharmacological treatment– EE–for cognitive deficits in adolescents. Results Early-Life Stress Specifically Impaired Cognition in Adolescent Male Mice To investigate the effects of ES on cognition in adolescent mice, we first established the LBN model during PND 2–9 and then compared stressed and control mice on a series of stress-related physiological and behavioral measures (Fig. 1 A-B). Immediately after stress (PND9), stressed mice had significantly lower body weight gain (Fig. S1 A left) and the effect persisted into adolescence (Fig. S1 A right). Mice exposed to ES also showed significant adrenal atrophy (Fig. S1 B) and a tendency for thymus atrophy (Fig. S1 C) in adolescence. Cognitive performance was assessed in four tasks. In the temporary order memory task (TOM, Fig. 1 C), unlike the control mice, stressed mice failed to distinguish the “remote” object from the “recent” object and had significantly lower discrimination index than control mice. In the Y-maze spontaneous alternation test (Fig. 1 D), stressed mice showed lower spontaneous alternation rates and higher error rates of same arm return, indicative of spatial working memory deficits. In the novel object recognition test, stressed mice did not distinguish the “novel” object from the “familiar” object as control mice did (Fig. 1 E), despite of no group differences in the discrimination index. Stressed mice also exhibited spatial object recognition deficits, as in this task they failed to discriminate the “displaced” object from the “stationary” object, and showed significantly lower discrimination index than control mice (Fig. 1 F). The total probe time and distance traveled during the test phase of the recognition tasks or the total arm entries in the Y-maze were not affected by ES (Fig. S2A-D). Besides cognitive behaviors, we also evaluated anxiety-like, social approach, and depression-like behaviors. In three tasks of anxiety-like behaviors, no significant differences were observed between stressed and control mice (Fig. 1 G-I and S2 E-G). Social approach was not affected by ES (Fig. 1 J and S2 H). ES did not significantly affect depression-like behaviors in the tail suspension test (Fig. 1 K and S2 I), sucrose preference test (Fig. 1 M and S2 K), or the latency to immobility in the forced swimming test (Fig. S2J), except that increased immobility was observed in the stressed mice in the forced swimming test (Fig. 1 L). Together, these behavioral and stress-related physiological results indicate that the adverse effects of ES emerge as early as in adolescent male mice, with the cognitive behaviors being particularly vulnerable. Early-Life Stress Reduced PVI (not SST-INs) Interneuron Activity in mPFC in Adolescent Male Mice To examine the involvement of mPFC PVI in the adverse effects of ES during adolescence, we quantified PVI density and activity in the following three analyses. First, immunohistochemistry revealed that ES significantly decreased the density of PVI in the mPFC, irrespective of subregions examined (Fig. 2 A and S3 A). The density of somatostatin-expressing interneurons (SST-INs) was not affected (Fig. 2 B and S3 B). Next, using immediate early gene c-fos immunostaining (which reflects neural activation[ 44 ]) and colocalization analysis, we found that stressed mice showed reduced density of activated PVIs in mPFC during the TOM test (Fig. 2 C-D and S3 C). Again, no stress effects were observed for SST-INs (Fig. 2 E and S3 D). Finally, to validate the effects of ES on mPFC PVI activity, we recorded the evoked and spontaneous action potential in mPFC PVIs in PV-cre::Ai14 mice through whole-cell voltage clamp (Fig. 2 F). PVIs of stressed mice showed a significantly lower frequency of evoked action potentials in response to current injection compared with controls (Fig. 2 G) and the interaction reflected that ES-induced suppression was mainly observed at currents greater than 200 pA. The spontaneous action potentials of PVIs were not altered by ES (Fig. 2 H). Together, these results indicate that ES specifically reduced PVI density and activity (not SST-INs) in mPFC in adolescent male mice. Prefrontal PVI Activity Mediates Early-Life Stress-Induced Cognitive Deficits in Adolescent Mice Having shown that ES elicited both cognitive deficits and mPFC PVI activity reduction in adolescent mice, in this section we examined whether the reduced mPFC PVI activity causally mediates ES-induced cognitive deficits in adolescent mice using chemogenetic or optogenetic techniques. To start with, we carried out the following experiments to mimic ES-induced reduction PVI density and activity in mPFC and to evaluate the corresponding behavioral consequences. First, a loss-of-function experiment was performed to selectively ablate mPFC PVIs using PV-Cre mice by injecting adeno-associated virus (AAV) expressing Cre-dependent Casp3 (a cell apoptosis effector molecule) into the mPFC at PND22 (Fig. S4A-B). This manipulation resulted in cognitive deficits, including lower discrimination indices in the TOM test (Fig. S4C) and in the novel object recognition test (Fig. S4E), and higher error rates of SAR in the Y-maze spontaneous alternation test (Fig. S4D). The spatial object recognition task was not affected by mPFC PVIs ablation (Fig. S4F). Anxiety-like behaviors in three tasks were also largely unaffected (Fig. S4G-I), except for reduced time spent in the light box in the light-dark box test (Fig. S4H). Second, we inhibited mPFC PVI activity with a chemogenetic DREADDs manipulation, i.e., by bilateral injection of the AAV vector carrying the Cre-dependent hM4Di (Gi) into the mPFC in PV-Cre mice (Fig. 3 A-B). Immunofluorescence staining combined with colocalization verified that the CNO group showed significantly lower percentage of virus-infected PVIs that co-express c-fos, compared with the Veh group (Fig. 3 C), indicative of PVI activity inhibition. This manipulation again resulted in cognitive deficits in TOM and Y-maze spontaneous alternation tests. In the TOM task (Fig. 3 D), mice in the CNO group showed reduced discrimination index than the vehicle group and they were unable to discriminate the “remote” object and the “recent” object. In the Y-maze spontaneous alternation test (Fig. 3 E), CNO-treated mice showed higher error rates of SAR; no group differences were observed in SA or AAR. The novel object recognition and spatial object recognition tasks were not significantly affected by CNO treatment (Fig. S5A-B). CNO treatment did not affect anxiety-like behaviors in the open field test (Fig. S5C), but reduced the time spent in the light box in the light-dark box test (Fig. S5D) and the time spent in the open arms in the elevated plus maze (Fig. S5E), indicative of increased anxiety levels following PVI inhibition in mPFC, which are consistent with previous findings [ 16 ]. Optogenetic manipulation was then carried out to validate the above-mentioned chemogenetic results (Fig. 3 F-I and S6 A-B). As the previous two experiments support the consistent involvement of mPFC PVIs in the TOM and Y-maze spontaneous alternation tests, these two behavioral tests were carried out in the following experiments. Optogenetic inhibition of mPFC PVIs disrupted temporal order memory and spatial working memory (Fig. 3 H-I). In the TOM task (Fig. 3 H), eNpHR3.0-infected mice failed to discriminate the “remote” object and the “recent” object and exhibited lower discrimination index than control mice. In the Y-maze spontaneous alternation test (Fig. 3 I), eNpHR3.0-infected mice displayed more errors of alternate arm return than EGFP-infected mice; no group differences were found for SA or SAR. Finally, based on the three experiments above showing that PVI inhibition reproduced ES-induced impairments of PFC-dependent cognitive functions, we continued to investigate whether upregulating mPFC PVI activity could reverse the cognition-impairing effects of ES by bilateral injection of the AAV vector carrying the Cre-dependent hM3Dq (Gq) into the mPFC in PV-Cre mice (Fig. 3 J-K). To test the efficiency and specificity of the DREADDs system, c-fos immunoreactivity was detected in mCherry-infected neurons (Fig. S7A) and PVIs (Fig. 3 L) after a single CNO dose. The percentage of mCherry-infected neurons co-labeled with c-fos was significantly elevated by DREADDs (Fig. S7A). For the immunofluorescence staining for c-fos and PV co-labeling, two-way ANOVA revealed a significant stress × drug interaction (Fig. 3 L). Further group comparisons revealed that c-fos expression in PVIs were decreased by ES, which was reversed by DREADDs. In terms of behavioral consequences, for the TOM task, two-way ANOVA revealed a significant stress × drug interaction (Fig. 3 M). Selective activation of mPFC PVIs restored ES-induced impairment. No stress or CNO effects were observed in total probe time or total distances traveled in the acquisition phase (Fig. S7B). For the Y-maze spontaneous alternation task (Fig. 3 N and Fig. S7C), two-way ANOVA showed significant stress × CNO interactions for SA and SAR. The negative stress effects induced by ES were attenuated by activation of mPFC PVIs. These results indicate that mPFC PVI upregulation is sufficient to alleviate ES-induced cognitive deficits in temporal order memory and spatial working memory. By selectively downregulating and upregulating mPFC PVI activity, the four experiments above provide causal evidence that mPFC PVI activity mediates ES-induced cognitive deficits in adolescent mice. Prefrontal Pyramidal Neurons Are Involved in Early-Life Stress-Induced Cognitive Deficits through PVI How does ES reduce mPFC PVI activity in adolescent male mice? The functional maturation of the PVIs during adolescence involves several mechanisms [ 21 , 27 ], including increased glutamatergic inputs, increased density of perineuronal net (PNN) proteins, etc. PNNs as one component of the extracellular matrix preferentially surround PVIs and modulate their excitability [ 45 ]. We quantified the expression of PNNs in stressed and control mice at PND35 and did not observe significant group differences (Fig. S8A). As pyramidal neurons (PNs) form robust functional synapses onto PVIs, it is possible that ES may first reduce the PN activity, which in turn limits the excitatory inputs to PVIs. So, we examined the effects of ES on the excitatory inputs onto PVIs and the PN activity. The excitatory inputs onto PVIs were quantified by the expression levels of vesicular glutamate transporter-1 (VGluT1, responsible for loading glutamate into synaptic vesicles for future release [ 46 ] and involved in the regulation of excitatory neurotransmission [ 47 ] at different distances from the soma of PVIs (Fig. 4 A-C). Two-way ANOVA revealed a main effect of stress for VGluT1 fluorescence intensity as well as a significant stress × distance interaction, which was driven by decreased VGluT1 expression in stressed group at the distances larger than 8.784 um. We also quantified the soma radius of PVIs, which was not significantly affected by ES and approximately 8.662 ± 1.211 um (Fig. S8B), which is consistent with VGluT1 result above to suggest that ES-induced excitatory input reduction occurred surrounding the soma of PVIs. We then measured the mPFC PN activity during TOM test using immunofluorescence staining combined with co-localization of c-fos and CamkIIa or neurogranin (two excitatory neurons markers [ 48 , 49 ]). Compared with control mice, stressed mice showed reduced density of neurons showing the co-labeling of c-fos and CamkIIa (Fig. 4 D) or neurogranin (Fig. 4 E), indicative of ES-induced inhibition of PN activity. To examine the causal involvement of PNs in ES adverse effects in adolescent mice, we carried out the following chemogenetic experiments. First, we tested whether inhibiting PN activity in adolescent mice could reproduce the cognitive deficits of ES (Fig. S9A-F). Specifically, we injected the AAV vector expressing Cre-dependent hM4Di (Gi) bilaterally into the mPFC of CamkIIa-Cre mice. Immunofluorescence staining for the co-labeling of mCherry + and c-fos + (Fig. S9B) showed that, compared with the Veh group, the CNO group showed decreased percentage of neurons co-expressing mCherry + and c-fos + . In terms of cognitive performances, in the TOM task, CNO-treated mice failed to discriminate the “remote” object and the “recent” object, and exhibited lower discrimination index (Fig. S9C). In the Y-maze test, CNO-treated mice showed lower SA and higher error rates of alternate arm return compared with Veh-treated mice (Fig. S9E). No significant group differences were observed in the test phase of TOM task (Fig. S9D) or the total arm entries in the Y-maze test (Fig. S9F). That is, selective inhibition of mPFC PNs reproduces ES-induced deficits of temporal order memory and spatial working memory. Second, we investigated whether activating PNs (Fig. 4 F) could reverse ES-induced cognitive impairment. Immunofluorescence staining showed that the virus-infected (mCherry + ) neurons co-labeled with excitatory neurons marker CamkIIa (Fig. 4 G) and that the percentage of mCherry-infected neurons co-labeled with c-fos was significantly elevated by DREADDs (Fig. 4 H-I), indicative of selective activation of PNs in vivo . For the cognitive tests, we observed a significant stress × CNO interaction (Fig. 4 J) in the TOM task in that PN activation restored the discrimination index decreased in ES-treated mice. No stress or CNO effects were observed in the test phase (Fig. S10A). For the Y-maze spontaneous alternation task (Fig. 4 K), two-way ANOVA showed significant stress × drug interactions on SA and SAR. The negative stress effects on these measures were attenuated by activation of prefrontal PNs. No differences were observed in total arm entries (Fig. S10B). That is, selective activation of mPFC PNs reverses ES-induced deficits of temporal order memory and spatial working memory. Based on the above-mentioned results that ES reduced the functional activity of both PNs and PVIs and the excitatory inputs onto PVIs and that both types of neurons are causally involved in ES-induced cognitive impairment in adolescent mice, we then hypothesized that the cognition-improving effects of PN activation may be mediated by PVI. To test this hypothesis, we carried out an experiment to activate PNs and inhibit PVIs in mPFC and examined whether PVI inhibition could block the reversal effect of PN activation on ES-induced cognitive deficits. The double chemogenetic manipulation was achieved by bilateral injection of a mixture of two viruses (AAV-CamkIIa-Gq; Cre-dependent AAV-DIO-Gi) into the mPFC in adolescent PV-Cre mice (Fig. 4 L). Immunofluorescence staining for mCherry, PV, and c-fos was performed to examine the c-fos-positive neurons that are mCherry + (non-PV + ) and PV + for validation. For mCherry + (non-PV + ) & c-fos + neurons (Fig. 4 M), we observed both the main effect of CNO and the stress × CNO interaction. Post hoc tests with Bonferroni correction showed that activation of PNs was observed in both control and stressed mice. For PV + & c-fos + neurons (Fig. 4 N), we also observed both the main effect of CNO and the stress × CNO interaction. That is, PVI activity was inhibited in control mice after CNO administration and was reduced in ES-exposed vehicle mice; CNO did not further downregulate the PVI activity in stressed mice. For cognitive effects, in the TOM task (Fig. 4 O), two-way ANOVA revealed a tendency for main effect of CNO and stress × CNO interaction. That is, selective inhibition of mPFC PVIs blocked the reversal effect of activation of PNs on the TOM impairment induced by ES. Besides, the double chemogenetic manipulation significantly reduced the discrimination index in control mice, which resembled the previous results of PVI inhibition. No stress or CNO effects were observed in the total probe time and distance traveled (Fig. S10C). For the Y-maze spontaneous alternation task (Fig. 4 P), two-way ANOVA showed a significant main effect of stress on SA, indicating the absence of the reversal effects of PN activation after PVI inhibition. Main effects of CNO were also observed for AAR and SAR in that CNO significantly increased AAR and decreased SAR. No differences were observed in total arm entries (Fig. S10D). Together, these results indicate that inhibition of the PVIs in mPFC could block the reversal effect of the PN activation on ES-induced deficits in temporal order memory and spatial working memory, supporting the indispensable role of PVIs in the cognition-improving effects of PNs. CRHR1 Downregulation Reverses Early-Life Stress-Induced Cognitive Deficits in Adolescent Mice by Restoring PVI Activity in mPFC Our previous studies have highlighted the critical role of the CRH-CRHR1 system in ES-induced behavioral and neural abnormalities [ 28 , 40 – 42 , 50 ], including corticolimbic neurons [ 28 ], in postnatal and adult mice. Here we examined whether the CRH-CRHR1 system is involved in ES-induced negative effects in adolescent mice. As CRHR1 has been found to be mainly expressed in PNs in cortex [ 51 ], we first validated the localization of Crhr1 mRNA in the mPFC using single molecule fluorescence in situ hybridization (RNAscope ISH). Colocalization of Crhr1 mRNA with Slc17a7 (the mRNA of VGluT1, an excitatory neuron marker) and Slc32a1 (the mRNA of GABA vesicular transporter, an inhibitory neuron marker) in the mPFC (Fig. 5 A) demonstrated that Crhr1 + neurons mainly co-localized with Slc17a7 + neurons (2313 out of 2800 Crhr1 + neurons, 82.61% and out of 2489 Slc17a7 + neurons, 92.93%). Only a small fraction of Crhr1 + neurons co-localized with Slc32a1 + neurons (43 out of 2800 Crhr1 + neurons, 1.54% and out of 520 Slc32a1 + neurons, 8.27%). This observation confirmed that Crhr1 mRNA is primarily expressed in pyramidal, not inhibitory, neurons, in the mPFC. We then investigated whether blocking the mPFC CRH-CRHR1 system could reverse ES-induced deficits on PVIs and temporal order memory. We constructed an AAV vector carrying sgRNA targeting Crhr1 to achieve CRISPR-Cas9-mediated deletion of CRHR1 (Fig. 5 B). Five gRNAs were screened using in vitro cellular assays and the sgRNA3 sequence showing the most effective transfection was chosen for packaging (Fig. S11A). HA tag staining confirmed that the virus was mainly infected in the mPFC (Fig. S11B). For the TOM task, significant reversal effects were observed: CRHR1 deletion restored temporary order memory impaired by ES (Fig. 5 C). No significant effects of ES or CRHR1 deletion were observed in the test phase (Fig. S11C). For the PVI activity (Fig. 5 D-E), two-way ANOVA revealed a significant stress × virus interaction in the mPFC: CRHR1 deletion in the mPFC reversed ES-induced reduction of PVI activity. Furthermore, discrimination index in the TOM task significantly correlated with PVI activity in the mPFC across all the animals (Fig. 5 F). Together, these results support our hypothesis that early-life stress may first upregulate CRH-CRHR1 signaling in prefrontal PNs, which reduces PN activity, inhibits excitatory inputs to PVIs, and in turn downregulates PVI activity and leads to cognitive impairments. Motivated by the recently established association between the CRH-CRHR1 system and cognition in the literature [ 32 ], we then carried out two pharmacological intervention experiments using the CRHR1 antagonist, antalarmin. In the first experiment, intraperitoneal injection of antalarmin was performed daily during stress procedure (PND2-8, Fig. 5 G). The TOM task and the activity of PVIs were then assessed in adolescent mice. For TOM, two-way ANOVA revealed a significant stress × drug interaction, as antalarmin restored the ES-induced reduction of discrimination index (Fig. 5 H). No significant group differences were observed in the test stage (Fig. S11D). For PVI activity in the mPFC, immunofluorescence staining for c-fos and PV showed significant main effects of stress and antalarmin, without stress × drug interaction (Fig. 5 I-J). That is, the percentage of PVIs co-labeled with c-fos was significantly reduced by ES and upregulated by antalarmin. Similar ES and antalarmin effects were observed for the density of neurogranin + neurons co-labeled with c-fos (Fig. S11E-F). Furthermore, discrimination index in the TOM task significantly correlated with PVI (Fig. 5 K) and PN (Fig. S11G) activity in the mPFC. In the second experiment, to examine the acute pharmacological effects of antalarmin, the drug was given 30 minutes prior to the TOM test in adolescent stressed and control mice (Fig. 5 L). Similar stress × drug interactions were observed for TOM and mPFC PVIs. Acute antalarmin injection blocked ES-induced reduction of discrimination index (Fig. 5 M and S11 H) and of mPFC PVIs (Fig. 5 N-O). Significant correlation between the discrimination index and PVI activity in the mPFC was also observed (Fig. 5 P). To further test the hypothesis that the cognition-improving effects of antalarmin may be mediated by PVI activity in the mPFC, we carried out an experiment to examine whether PVI inhibition could block the reversal effects of acute antalarmin treatment on ES-induced cognitive deficits (Fig. 5 Q-R). For PVI activity in the mPFC in stressed mice receiving antalarmin treatment, immunofluorescence staining for c-fos and PV showed that the percentage of PVIs co-labeled with c-fos (Fig. 5 O) was significantly reduced by DREADDs (Fig. 5 S). In the TOM task, compared with the stressed mice that received antalarmin treatment and exhibited intact temporal order memory, the CNO mice could not discriminate the “remote” object from the “recent” object and had significantly lower percentage time exploring the “remote” object (Fig. 5 T), indicating that the reversal effect of antalarmin on ES-induced TOM impairment (Fig. 5 M) was blocked by mPFC PVI inhibition. Together, these pharmacological experiments indicate that CRHR1 blockade could successfully reverse ES-induced temporal order memory deficits by restoring mPFC PVI activity, supporting the therapeutic potentials of antalarmin on ES-related cognitive impairments. Environmental Enrichment Alleviates Early-life Stress-induced Cognitive Deficits through Activation of Prefrontal PNs and PVIs in Adolescent Mice Besides pharmacological intervention, here we also tested whether EE, a commonly used non-pharmacological intervention for animals exposed to ES, could reverse ES-induced negative effects in adolescent mice. Stressed and control mice were exposed to three-week enriched or standard housing environments after weaning (PND21-42) and were then tested in the TOM task (Fig. 6 A-B). As shown in Fig. 6 C, despite lack of significant main effects and interactions in the two-way ANOVA, only the stressed mice kept in a standard environment failed to distinguish the “remote” object from the “recent” object, while mice in the other three groups exhibited intact recognition memory, which indicates that EE partially reversed ES-induced temporal order memory deficits. We then investigated the neural correlates underlying EE cognition-improving effects by measuring the activity of PNs and PVIs in mPFC. Regarding the density of neurogranin + cells that are c-fos + (indicative of the PN activity), we found a significant stress × environment interaction in the mPFC (Fig. 6 D-E). Specifically, ES significantly reduced neurogranin + neuron activity, which was reversed by environment enrichment. Similar with the results of PNs, for the density of PVIs that are c-fos + (indicative of the PVI activity), we also observed a significant stress × environment interaction (6G-H). ES significantly reduced PVI activity and EE significantly upregulated the activity of PVIs in stressed mice. Importantly, the discrimination indices in the temporal order memory test were significantly correlated with both the density of activated PNs (Fig. 6 F) and PVIs (Fig. 6 I) in the mPFC. These data support the beneficial effects of EE in alleviating ES-induced temporal order memory deficits and reduced activity of PNs and PVIs. Discussion In this study, we examined whether and how mPFC PVIs causally mediate ES-induced cognitive deficits in adolescent mice. We first demonstrated that exposure to resource scarcity environment in early-life led to selective reduction of PVI activity in the mPFC in adolescent male mice, along with cognitive deficits in temporal order memory and spatial working memory. Ablating or inhibiting PVIs in the mPFC in adolescent mice phenocopied the deficits observed in ES-exposed mice, and activating PVIs rescued ES-induced cognitive deficits, supporting the causal relationship between PVI activity and cognitive deficits in mice exposed to ES. We further demonstrated that ES also reduced excitatory inputs onto PVIs and the PN activity in the mPFC and that PN activity causally contributed to cognitive deficits, which required the activation of PVIs. In addition, genetic knockout of CRHR1 (mainly expressed in PNs) in the mPFC in adolescent mice improved ES-induced cognitive impairment and PVI activity reduction. These results collectively support the prefrontal CRHR1→PN→PVI pathway in mediating the adverse effects ES on cognition in adolescent mice. Finally, our intervention experiments revealed the beneficial effects of pharmacological (antalarmin) and non-pharmacological (EE) treatment on ES-induced cognitive deficits and mPFC PVI and PN activity, providing insight into early treatment and prevention of cognitive impairments in stress-related psychiatric diseases. While the effects of ES on cognition have been extensively studied in adult animals in our own and other research groups [ 4 , 52 ], it is worth highlighting that several recent studies have started to examine this issue in adolescent rodents. Studies with maternal separation or deprivation models reported that ES impaired spatial learning and memory in morris water maze test[ 38 ], novel object recognition memory [ 53 ], and temporary order memory [ 54 ] in adolescent rodents. To our knowledge, only one study has used the LBN paradigm and found that exposure to LBN during PND4-11 led to spatial object recognition memory loss in adolescent male mice [ 7 ]. Different from these studies adopting one cognitive task for a given model, we tested the effects of the LBN paradigm in adolescent mice on a larger battery of cognitive tasks, including temporary order memory, spatial working memory, novel object recognition, and spatial object recognition. We found that ES-induced cognitive impairments we observed in adult mice [ 28 , 50 , 55 ] are already present in adolescence. Intriguingly, unlike previous studies showing both cognitive and emotional deficits following ES [ 8 , 29 , 38 ], here we observed cognitive deficits only, without significant alterations of anxiety-like, depression-like or social behaviors, which are consistent with a previous study [ 56 ]. Some LBN studies did report emotional alterations in adolescent animals, such as reduced sucrose preference [ 57 ], increased immobility time in FST test [ 58 , 59 ], and increased anxiety-like behaviors [ 60 , 61 ]. The inconsistency among these studies may be related to species (mouse or rat), stress mode, and stress exposure time. Together, our observation of ES-induced cognitive deficits in adolescent mice provides further evidence that the LBN paradigm is a valid model for the early onset of cognitive impairments, one of the core symptoms of psychiatric disorders, and for studying the underlying neural mechanisms. Considering the pivotal role of PVIs in mPFC-dependent cognitive behaviors and their prolonged maturation during adolescence, we hypothesized that PVIs may causally mediate ES-induced cognitive deficits we observed. We first examined the effects of ES on mPFC PVI activity. As mentioned in Introduction, current studies have largely focused on the effects of ES on the number (density) of PVIs or the expression of PV mRNA or PV protein, some of which reported downregulation in adolescent animals [ 23 – 26 ]. To our knowledge, direct evidence is still lacking regarding how ES affects the functional activity of prefrontal PVIs. One recent study found that ES (maternal separation and early weaning) downregulated evoked action potentials of mPFC GABAergic neurons in adult mice [ 62 ], yet it remains unknown which type of interneurons was affected by ES and whether such effects emerge in adolescence. In this study, by the immediate early gene c-fos colocalization technique we demonstrated that ES significantly decreased the number of activated PVIs, not SST-INs, during TOM test in adolescent mice, which was validated by electrophysiological findings of the lower frequency of evoked action potentials in PVIs of stressed mice. These results indicate for the first time that ES specifically reduces PV-expressing (not SST-expressing) interneuron activity in mPFC in adolescent male mice. Next, regarding the link between reduced mPFC PVI activity and cognitive deficits in adolescent mice exposed to ES, previous studies in adult animals (not adopting the ES models) have provided supporting evidence. For example, inactivation of prefrontal PVIs impaired prefrontal-dependent cognitive abilities, including spatial working memory, reversal learning[ 63 ], rule-shift learning[ 16 , 17 , 64 ], whereas enhancing the activity of PVIs in the mPFC could alleviate these cognitive deficits in various animal models [ 19 , 65 , 66 ]. Two recent studies in adolescent animals also suggest the crucial involvement of mPFC PVIs in cognitive behaviors. One study found that sustained inhibition of prefrontal PVIs activity during adolescence disrupted cognitive flexibility in adult mice [ 18 ]. The other study reported that selective activation of prefrontal PVIs during adolescence rescued deficits in novel object recognition induced by chronic MK801 treatment [ 67 ]. Here, by manipulating mPFC PVI functional activity through chemogenetic and optogenetic methods, we found that ablating or inhibiting PVIs in the mPFC in adolescent mice mimicked the deficits observed in ES-exposed mice, and activating PVIs rescued ES-induced cognitive deficits. These results provide causal evidence that mPFC PVI activity mediates the cognitive deficits induced by ES. Finally, to understand the mechanisms by which ES modulates functional activity changes in PVIs during adolescence, we targeted at the CRH-CRHR1 system and PNs. As mentioned in Introduction, the association between the CRH-CRHR1 system and PFC-dependent cognitive dysfunction has been highlighted in recent clinical and animal studies [ 32 , 36 ]. For PNs, numerous studies have reported that in adult animals, ES reduces their functional activity (measured as the action potential frequency, amplitude and frequency of excitatory postsynaptic currents [ 62 , 68 ]) and impairs their structural plasticity (e.g., dendritic retraction and spine loss [ 28 , 69 ]). Considering that CRHR1 is mainly expressed in excitatory neurons rather than inhibitory neurons in mPFC[ 51 ], we speculate that ES may first act on CRHR1 on PNs, reducing PN neuronal activity and their excitatory inputs to PVIs, leading to decreased activity of PVIs and ultimately leading to cognitive impairment in adolescent mice. This speculation was supported by the following experimental results: 1) we observed decreased excitatory inputs onto PVIs (measured by reduced VGluT1 expression levels) and decreased activation of mPFC PNs during the TOM task. 2) Inhibition of mPFC PNs mimicked ES-induced TOM damage and activation of mPFC PNs reversed ES-induced cognitive impairments; this reversal could be blocked by inhibiting mPFC PVI activity, indicating that PVIs play indispensable roles in the pyramidal neuron modulation. 3) ES-induced cognitive impairment and PVI activity reduction could be restored by genetic knockout of CRHR1 in the mPFC in adolescent mice. Together, these results indicate that the prefrontal CRHR1→PN→PVI pathway may underlie the cognitive deficits in adolescent mice exposed to ES. For cognitive impairment, the efficacy of current first-line therapeutic drugs and non-pharmacological treatments such as psychotherapy and physical therapy (e.g., rTMS, DBS) are limited [ 5 ], and the effective measures for early intervention are still lacking in clinical practice[ 31 ]. Here targeting at ES-induced cognitive deficits and mPFC PVI activity reduction, we examined the efficacy of pharmacological (i.e., antalarmin, a CRHR1 antagonist) and non-pharmacological (i.e., environmental enrichment) interventions. There were several clinical trials on drugs targeting the CRH-CRHR1 system; they have largely focused on improving symptoms of depression and anxiety, not cognition, and have ended in failure [ 70 , 71 ]. Our series of studies have consistently shown the cognition-improving effects of antalarmin in animal models of early-life stress in adulthood, such as PFC-dependent temporary order memory and spatial working memory [ 28 , 41 ], and hippocampus-dependent cognitive changes [ 50 , 55 ]. In this study, we found that either co-administration or acute intraperitoneal injection of the CRHR1 antagonist antalarmin could reverse ES-induced cognitive impairment and mPFC PVI activity downregulation in adolescent mice. These results highlight the therapeutic potential of CRHR1 antagonism on cognitive dysfunctions (compared with emotional symptoms) of stress-related psychiatric disorders and also support its potential in early intervention. Compared with medications, non-pharmacological interventions for psychiatric disorders have advantages in terms of fewer adverse effects and better acceptability. EE is a commonly used non-pharmacological intervention for animals exposed to ES and has beneficial effects on ES-induced behavioral and neural changes [ 37 , 72 ]. For cognitive impairment, two studies using maternal separation have reported the beneficial effects of EE during adolescence (i.e., reversal learning[ 38 ], spatial working memory [ 39 ]). Our study found that cognitive impairment caused by LBN can also be reversed by EE. More importantly, EE significantly upregulated mPFC PVI activity, similar to antalarmin intervention, which may underlie its cognitive improvement effect. Previous studies have shown that inhibition of PVIs could block the improvement of EE on cognitive deficits [ 67 , 73 ], supporting the hypothesis that PVIs mediate the cognitive improvement of EE. Taken together, these intervention experiments indicate that mPFC PVIs could serve as a potential target for early intervention in both pharmacological and non-pharmacological treatments of cognitive impairment. In summary, our study uncovers the crucial role of prefrontal PVIs, along with PNs and CRHR1, in mediating cognitive deficits induced by early-life stress in adolescent male mice. Our findings also suggest that interventions to enhance mPFC PVI activity could ameliorate the cognitive impairment of stress-related psychiatric disorders, which should be highlighted in developing early treatment and prevention strategies. Materials And Methods Animals Adult C57BL/6 (10–12 weeks old) male and female mice were purchased from Vital River Laboratories (Beijing, China). The mice were transferred to individually ventilated cages (IVC) and 3–4 mice were housed normally. The Pvalb tm1(cre)Arbr /J (PV-Cre) mice express Cre recombinase in parvalbumin-expressing interneurons (PVI) [ 74 ]. The Gt (ROSA)26Sor tm14(CAG−tdTomato)Hze /J (Ai14) mice express robust tdTomato fluorescence following Cre-mediated recombination [ 75 ]. The Tg (Camk2a-cre) T29−1Stl /J (CamKIIa-Cre) mice express Cre recombinase in pyramidal neurons (PNs) in the forebrain [ 76 ]. All transgenic mice were purchased from the Jackson Laboratory (CA, USA) and maintained fully back-crossed onto C57BL/6 mice, and adult male heterozygous mice were used. For breeding, male and female mice were mated 1:2 for 2 weeks and separated. Pregnant females were monitored daily for pup delivery, and the day of parturition was defined as postnatal day 0 (PND0). Only male offspring were used in the following experiments. All mice were housed under a 12-h light/dark cycle (lights on at 8:00 a.m.) and constant temperature (23 ± 1℃) conditions with plenty of food and water. All experiments were approved by the Peking University Committee on Animal Care, and they were performed in compliance with the NIH’s Guide for the Use and Care of Laboratory Animals. Early-life stress paradigm The limited nesting and bedding paradigm was used as an early-life stressor and was conducted as previously described [ 50 ]. The procedure was as follows: At PND2, the number of pups per cage was adjusted to ensure 6–8 pups with a male-to-female ratio of 1:1. Control dams received 500 ml of sawdust bedding and 4.8 g of nesting material (2 squares of Nestlets, Ancare, New York, USA), while in the "stress" cages, dams were provided with a fine-gauge aluminum mesh platform (McNichols, Tampa, FL, USA) with 200 mL of corncob bedding on the bottom to collect droppings. A limited amount of nesting material [1/2 square (1.2 g) nestlets] was put on top of the mesh. After one week of stress treatment (PND2-9), all mice were returned to standard environment. Male offspring were weaned on PND21 and housed in groups of 3–4 per cage for further study. Behavioral Assays A battery of behavioral tests, including cognitive behaviors, anxiety-like behaviors, depressive-like behaviors and social approach, were performed in adolescent mice (PND35-42) between 09:00 and 15:00 as described previously [ 28 , 77 ]. Mice were handled for at least 3 days prior to behavioral testing. Behavioral data in the open field, elevated plus maze, and light-dark box tasks were automatically analyzed by ANY-maze 7.0 (Stoelting, Wood Dale, IL, USA). The remaining behavioral tests were scored by an experimenter blind to experimental conditions. Temporary Order Memory Test The temporary order memory (TOM) test was to assess animals’ ability to differentiate between two familiar objects presented at different time intervals, which relies on the medial prefrontal cortex (mPFC) [ 78 ]. The test was carried out in the open field arena illuminated at 10 lux, including 3 trials with 1-hour intertrial interval (ITI). The first two trials were the acquisition trials where two identical triangular prisms or triangular pyramids were placed in the arena and the animals were allowed to freely explore the arena for 10 min. In the test phase, a triangular prism (the “remote” object) and a triangular pyramid (the “recent” object) were placed in the arena. During the 10 min test phase, the discrimination index was calculated as: 100% × time probing the “remote” object /time probing both objects. Novel Object Recognition Test The novel object recognition (NOR) task was designed to assess recognition memory based on the familiarity with the object itself, which depends on several brain regions, including the perirhinal cortex, the hippocampus, and mPFC [ 79 ]. The NOR was also performed in the open field arena, illuminated at 10 lux, and consisted of 2 trials separated by an ITI of 1 hour. During the acquisition phase, two identical cubes were presented. In the test phase, one of the cubes (the “familiar” object) was replaced by a hexagonal column (the “novel” object). During the 10 min test phase, the discrimination index was calculated as: 100% × time probing the “novel” object /time probing both objects. Spatial Object Recognition Test The spatial object recognition (SOR) task was designed to assess the animals’ ability to detect the location changes of familiar objects, which depends on the hippocampus [ 80 ]. The test was conducted in the open field arena with a 10-lux illumination and consisted of two acquisition trials and one test trial with 1-hour ITI. During the first two acquisition trials, two identical objects (cylinders) were placed approximately 15 cm apart. One hour later, one object was moved diagonally (the “displaced” object), and the other object was left in its original position (the “stationary” object). During the 10-min test phase, the discrimination index was calculated as: 100% × time probing the “displaced” object /time probing both objects. Y-maze Spontaneous Alternation Test Y-maze spontaneous alternation task was designed to assess spatial working memory [ 81 ]. The Y-maze apparatus consisted of gray polyvinyl chloride with three symmetrical arms (30 × 10 × 15 cm 3 , 10 lux) with spatial cues surrounding the maze. Mice were placed in the end of one arm and were allowed to explore freely for 8 min. The percentage of spontaneous alternations (SA: A→B→C), alternative arm returns (AAR: A→B→A) and same arm returns (SAR: A→A) were recorded manually. Open Field Test Open field test was performed in a gray polyvinyl chloride chamber (50 cm × 50 cm × 50 cm) with smooth interior walls and evenly illuminated at 60 lux. During the test, mice were placed in one corner, facing the wall and permitted to explore the environment freely for 10 min. Time spent in the center area of the open field (20 cm in diameter), the latency and the number of entries to the center area were measured to reflect animals’ anxiety levels. The total distance traveled was also quantified. Elevated plus maze test Elevated plus maze test was carried out in an elevated plus maze, consisting of a central platform (5 × 5 cm 2 ) with two opposing open arms (30 × 5 × 0.5 cm 3 , 40 lux) and two opposing closed arms (30 × 5 × 15 cm 3 , 10 lux) extending from it in a plus shape. The maze was elevated 50 cm above the floor. Mice were individually placed in the center with their heads facing a closed arm and allowed to explore for 5 min. Time spent in open arms, the latency and the number of entries to open arms were recorded. Light-dark box test Light-dark box test was performed in a plastic box containing a dark chamber (15 × 20 × 25 cm 3 , 10 lux) and a brightly illuminated chamber (30 × 20 × 25 cm 3 , 700 lux), connected by a 4-cm long tunnel. Mice were placed in the dark chamber, facing the other chamber. The time spent in the light chamber during 5 min, the latency and the number of entries to the light chamber were measured. Tail Suspension Test The test was carried out in a plastic enclosure (15 cm × 17 cm × 50 cm). Mice were suspended by the distal end of the tail for 6 min using a suspension hook. Mice were considered immobile when they were passively suspended and completely immobile. The total time spent immobile and the latency of immobilization were manually scored for each animal. Forced Swimming Test Mice were individually placed for 6 min in a transparent cylinder (25 cm high, 10 cm diameter) filled with water to a depth of 18 cm and maintained at 25 ± 1℃. After the test, the mouse was wiped dry with a towel and returned to its home cage. Mice were considered immobile if they did not make any active movements. The total immobility time was recorded and analyzed. Sucrose Preference Test Four days prior to the test, the mice were individually housed in home cage with two bottles of tap water to avoid place preference bias. On the fourth day, two bottles of 1% sucrose solution were given. The test began at 20:00, during which the mice were given one bottle of 1% sucrose solution and one bottle of water, and the weight of each bottle was recorded. The positions of the two bottles were changed every 12 hours, and the bottle weights were recorded at 24, 48, and 72 hours later. Sucrose preference index (%) = 100 × (sucrose solution consumption) / (sucrose solution consumption + water consumption). Social Approach Test Social approach test was performed in a gray polyvinyl chloride chamber (50 cm × 50 cm × 50 cm) illuminated at 10 lux. Mice were acclimated to the chamber with an empty wire mesh cage in the center for 10 min. One hour later, a strange mouse of the same sex and age (defined as “tool” mice) was placed in the empty wire mesh cage and then testing mouse was placed in one corner, facing the wall and allowed to explore freely for 10 min. The time spent interacting with the “tool” mouse was measured manually. Immunohistochemistry and Image Analysis Mice were anesthetized with 2,2,2-Tribromoethanol (250 mg/kg, i.p., T48402, Sigma-Aldrich) and transcardially perfused with 0.9% saline followed by 4% buffered paraformaldehyde. Then, mouse brains were dissected, post-fixed for 10–12 h at 4 ℃ in 4% buffered paraformaldehyde, and dehydrated in a 30% sucrose solution for 72 h at 4 ℃. Using a cryostat (Leica, Wetzlar, Germany), serial coronal sections through the mPFC (1.98 mm-1.54 mm from bregma) were obtained at 30-um by a 180-um interval. For immunofluorescence, sections were washed into 0.1 M PBS three times (10 min each time), and permeabilized with 0.3% Triton X-100 in 0.1 M PB three times for 10 min each time, followed by 1% normal donkey serum blocking solution at room temperature for 1 h. The following primary antibodies were used: goat anti-somatostatin (1:100, sc-7819, SANTA Cruz), goat anti-parvalbumin (1:2000, PVG-213, SWANT), mouse anti-CamKIIa (1:1000, 22609, Abcam), mouse anti-parvalbumin (1:2000, PV-235, SWANT), mouse anti-VGluT1 (1:1000, 135511, Synaptic Systems), rabbit anti-parvalbumin (1:2000, PV-25, SWANT), rabbit anti-c-fos (1:1000, 2250S, Cell Signaling), rabbit anti-neurogranin (1:1000, 217672, Abcam), pig anti-c-fos (1:1000, 226005, Synaptic Systems). After rinsing in 0.1 M PB containing 0.3% Triton X-100 three times (10 min each time), sections were incubated with secondary antibody (Alexa Fluor 488-, 594- and or 647-conjugated donkey) diluted 1:500 in 1% normal donkey serum blocking solution for 2 h at room temperature. After rinsing in 0.1 M PB three times (10 min each time), sections were mounted on slides and covered with Vectashield containing 40,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). For immunohistochemistry, sections were washed in 0.1 M PBS three times (5 min each time) and treated with 3% hydrogen peroxide (10 min), followed by 1% normal goat serum (1 h). They were labeled with rabbit anti-parvalbumin (1:20000, PV-25, SWANT) or goat anti somatostatin (1:200, sc-7819, SANTA Cruz) antibody at 4°C (overnight). The next day, sections were rinsed and incubated with a biotinylated goat anti-rabbit or rabbit anti-goat secondary antibody (Zhongshan Golden Bridge Biotechnology, Beijing, China) at room temperature (2 h). After rinsing, sections were stained with the 3,3’-Diaminobenzidine Horseradish Peroxidase Color Development Kit (Zhongshan Golden Bridge, ZLI-9019, Beijing, China), transferred onto slides and coverslipped with mounting medium (Zhongshan Golden Bridge, ZLI-9516, Beijing, China). For image analysis, brain sections were assigned random numbers so that investigators were blind to the experimental conditions. To count the number of PVIs and somatostatin-expressing interneurons (SST-INs) in the mPFC, images were obtained from 3 sections per animal at 10× objective using an Olympus VS120-S6-W automated slide scanner (Olympus, Tokyo, Japan) and three subregions of mPFC (Cg, PrL, IL) were defined as ROI (Region of Interest) for analysis using NIH ImageJ software. For colocalization analysis, brain sections were captured at 20× objective from 3 sections per animal using an Olympus FV1000 laser-scanning confocal microscope (Olympus, Tokyo, Japan). To examine the optical density of VGluT1 at different distances from the soma of PVIs, sections were acquired at 20× objective from 3 sections per animal using an Olympus FV3000 laser-scanning confocal microscope (Olympus, Tokyo, Japan); for each group, 30–40 PVIs were chosen and sholl analysis was performed using NIH ImageJ software at different distances (step by 1.0 um) from the soma of PVIs. To validate the virus- or optical fiber-target regions, images were photographed at 10× objective using an Olympus VS200-S6-W automated slide scanner (Olympus, Tokyo, Japan). Images were adjusted for optimal brightness and contrast using FV10-ASW 4.2 software or OlyVIA 3.4.1 (Olympus, Tokyo, Japan). Brain-slice Preparation and Electrophysiological recordings Adolescent male PV::Ai14 mice (PND35-42) were anesthetized with isoflurane rapidly and decapitated. The brain slices (200 µm) containing mPFC were obtained using standard techniques and incubated in artificial cerebrospinal fluid (ACSF)at room temperature (20–24℃) for 1 hour. The slices were then transferred to a recording chamber at room temperature (20–24℃) and continuously perfused with oxygenated-standard and ACSF heated at 37℃ containing (in mM) 10 glucose, 125 NaCl, 5 KCl, 2 NaH 2 PO 4 , 2.6 CaCl 2 , 1.3 MgCl 2 , 26 NaHCO 3 (pH: 7.3–7.4, osmolarity: 300–310 mOsm/kg), at a rate of 2 mL/min. Target mPFC PVIs were identified by tdTomato fluorescence using an Olympus BX-51 microscope equipped with DIC optics, a water-immersion objective (×60 NA 1.1). To measure the evoked firing of PVIs, incremental current steps (0.5 s duration, 50 pA step size) were injected through the recording pipette. Recordings were obtained with an EPC-10 amplifier and Patchmaster software (HEKA Elektronik, Lambrecht /Pfalz, Germany). Signals were analyzed with Clampfit 10.3 (Molecular Devices, Union City, CA, USA). Stereotaxic Surgery and Viral Microinjection Adolescent mice (PND22-23) were anesthetized with isoflurane (induction 2.5%, maintenance 1-1.5%) with perioperative meloxicam analgesia (3 mg/kg, i.p.) and placed in a stereotaxic frame (RWD Life Science Co., LTD, Shenzhen, China). Mice received viral microinjections into the mPFC (250 nL per side, 30 nL/min) through a glass micropipette. The injection coordinates (relative to bregma) were anterior + 1.9 mm, lateral ± 0.3 mm, and ventral-1.8 mm. The micropipette was left in the site for another 5 min. Mice were allowed to recover until the beginning of behavioral tests (PND35). For manipulation of PVIs in mPFC, an adeno-associated virus (AAV) of DIO-hM3Dq-mCherry (1.14 × 10 13 genome copies/mL, Vigene Biotechnology, China), DIO-hM4Di-mCherry (3.00 × 10 13 genome copies/mL, Vigene Biotechnology, China), DIO-eNpHR3.0-EYFP (1.81 × 10 14 genome copies/mL, Vigene Biotechnology, China), flex-taCasp3-TEVp (3.04 × 10 12 genome copies/mL, ObioTechnology, China), or ACSF was delivered bilaterally to the mPFC of male PV-Cre mice. For manipulation of pyramidal neurons in mPFC, an AAV virus of DIO-hM4Di-mCherry was bilaterally injected into the mPFC of male CamkIIa-Cre mice or AAV virus of CamkIIa-hM3Dq-mCherry (7.07 × 10 13 genome copies/mL, Vigene Biotechnology, China) was bilaterally injected into the mPFC of male C57BL/6 mice. For simultaneous manipulation of mPFC PNs and PVIs, a mixture of CamkIIa-hM3Dq-mCherry and DIO-hM4Di-mCherry or a mixture of CamkIIa-hM4Di-mCherry and DIO-hM3Dq-mCherry were bilaterally injected into the mPFC of male PV-Cre mice. To achieve CRISPR-mediated deletion of Crhr1 , an AAV vector carrying sgRNA targeting Crhr1 (AAV2/9-CMV-SaCas9-U6-crhr1.gRNA, 7.22 × 10 13 genome copies/mL, Vigene Biotechnology, China) and scrambled sgRNA (AAV2/9-CMV-SaCas9-U6-scrambled. gRNA, 4.13 × 10 13 genome copies/mL, Vigene Biotechnology, China) was bilaterally injected into the mPFC of male C57BL/6 mice. Chemogenetic Manipulation of Neurons Clozapine-N-oxide (CNO, HY-17366, MEC) was dissolved in 100% DMSO to a storage concentration of 20 mg/mL and stored at -20℃. For chemogenetic inactivation of PNs or PVIs, storage solution of CNO was diluted into a working solution (0.3 mg/mL) and then the working solution of CNO was delivered intraperitoneally (i.p. 3 mg/kg body weight) to hM4Di transfected mice 30 min prior to each behavioral test. For chemogenetic activation of PNs or PVIs storage solution of CNO (20 mg/mL) was diluted into a working solution (0.1 mg/mL) and then the working solution of CNO was administered intraperitoneally (i.p. 1 mg/kg) to hM3Dq transfected mice 30 min prior to each behavioral test. To simultaneously manipulate PNs and PVIs, the working solution of CNO (0.3 mg/mL) was administered intraperitoneally (i.p. 3 mg/kg) 30 min prior to each behavioral test. For vehicle groups, 3 mg/kg body weight of 1.5% DMSO or 1 mg/kg body weight of 0.5% DMSO were injected intraperitoneally in the inactivation and activation experiments, respectively. Optogenetic Inactivation of PV-expressing Interneurons For optogenetic inactivation of prefrontal PVIs, an AAV carrying a DIO-eNpHR3.0-EYFP or DIO-EGFP was injected bilaterally into the mPFC and optic-fiber cannula (200-um-diameter; 0.37 NA) was implanted above the mPFC with − 1.55-mm DV coordinate. Light was provided by a 594 nm laser diode (ThinkerTech, Nanjing, China). The light intensity at the fiber tip was measured with a light sensor (Thorlabs, Newton, NJ, USA). A 4–5 mW laser pulse (ON-OFF-ON-OFF, 2 min/section) was delivered by a Master-8 pulse stimulator (AMPI, Jerusalem, Israel) through the optical fiber embedded in the mPFC. RNAscope in Situ Hybridization The mPFC mRNA expression was visualized using RNAscope (Advanced Cell Diagnostics). Following the manufacturer's instructions, mouse brains were rapidly dissected, dehydrated, and frozen. After cryoprotection, serial coronal sections (bregma 1.98–1.54 mm) of the mPFC at 15 µm thickness and 180 µm interval were obtained using a cryostat (Leica), which were dried at -20 ℃ for 1 h and then stored at -80 ℃ for up to one week. Then, slices were processed following the RNAscope protocol using a fluorescent multiplex reagent kit (ACD: 323100) and probes for Crhr1 (Mm-Crhr1-C1; ACD, catalog #418011), Slc32a1 (Mm-Slc32a1-C2; ACD, catalog #319198-C2), and Slc17a7 (Mm-Slc17a7-C3; ACD, catalog #416631). For co-localization analysis, images (1024 × 1024 pixel 2 ) were captured at 20 × objective using an Olympus FV3000 laser-scanning confocal microscope (Olympus, Tokyo, Japan). Images were then separated into multiple color channels and cell nuclei were identified in the DAPI channel. Signals in the red, green, and magenta channels were thresholded, identified, and filtered by the locations of nuclei. If a signal was found in a nucleus, the cell was defined as ‘‘positive’’ for the respective RNA species. Nuclei positive for Slc32a1 or Slc17a7 were finally filtered to determine whether they co-expressed Crhr1 . CRHR1 Antagonist Antalarmin Treatment To investigate the intervention effects of CRHR1 blockade on the ELS negative effects, we intraperitoneally administered the CRHR1 antagonist antalarmin (20 µg/g of body weight; Sigma-Aldrich, USA) or vehicle (15% β-cyclodextrin in sterile normal saline, Solarbio, Beijing, China) in two treatment strategies. First, the concurrent blockade of CRHR1 receptors during early-life stress exposure was achieved by daily intraperitoneal injections of antalarmin or vehicle at 09:00–12:00 during PND2-8. The entire injection procedure did not exceed 10 min per cage to avoid maternal separation stress. Second, the acute pharmacological effects of antalarmin were tested in adolescent mice. That is, antalarmin or vehicle was injected 30 min before behavioral tests. Environmental Enrichment Stressed and control mice were exposed to environmental enrichment (EE) with 3–4 mice per cage from PND 22–42. EE cage is an acrylic cage with a volume of 36 cm × 25 cm × 60 cm, divided into 3 layers (Fig. 6 B). Each layer is connected by a tunnel, with facilities such as a running wheel, a swing, a pipe and a house, and toys of different shapes and colors. To ensure the novelty of the objects for the mice, the type, number, and location of the toys were changed every three days. Quantification and Statistical Analysis GraphPad Prism 9.0 (GraphPad Software, Inc., USA) was used for the statistical analyses and graphing. Signals obtained from electrophysiological recordings were analyzed with Clampfit 10.3 (Molecular Devices, Union City, CA, USA). Kolmogorov-Smirnov test was used to checked for normality before t test, ANOVA and descriptive statistics. Comparisons between two groups were analyzed by unpaired t test (with same variance) or unpaired t test with Welch's correction (with unequal variance). Paired t test was used to compare the percentage of time probing two objects in the NOR, SOR and TOM test. For multiple group comparisons, data were analyzed by two-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test when yielded a significant interaction. Repeated measures ANOVA was used for detecting the effect of ES on the frequency of evoked action potential and VGluT1 expression on PVIs. Data are shown as individual values or expressed as the mean ± SEM, and significance levels are indicated as p < 0.05, p < 0.01, p < 0.001 and not significant (n.s.). All sample size, statistical methods and results are specified in Table S1 and Table S2. Declarations Author contributions TS, JL, CZ designed research; YM, CY, Y-XS, C-CZ, XL, and HW performed research; YM, CY, TW, Y-AS and X-XL analyzed data; JT, TS, Y-AS and YM wrote the manuscript. Acknowledgments This work was supported by the Beijing National Science Foundation (grant No., 7222236), the National Natural Science Foundation of China (grant No., 82271569, 82171529, 82071528, 82001145, and 82001418), the Capital Medical Development Research Fund (2022-1-4111). The funders have no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Graphical abstract and cartoons in Figures 1(A, C) were created with BioRender.com Conflict of Interest The authors report no conflict of interests. References Solmi M, Radua J, Olivola M, Croce E, Soardo L, de Pablo GS et al. Age at onset of mental disorders worldwide: large-scale meta-analysis of 192 epidemiological studies. Mol Psychiatr 2022; 27(1): 281–295. Tomáš Paus MK, Jay N. Giedd. Why do many psychiatric disorders emerge during adolescence. Nature reviews neuroscience 2008; 9: 947–957. LeMoult J, Humphreys KL, Tracy A, Hoffmeister JA, Ip E, Gotlib IH. 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University Six Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tian-mei","middleName":"","lastName":"Si","suffix":""}],"badges":[],"createdAt":"2023-11-07 06:00:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3572074/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3572074/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41380-024-02845-6","type":"published","date":"2024-11-22T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49535180,"identity":"edd1b39c-aae2-4151-b58a-c1037b71b923","added_by":"auto","created_at":"2024-01-12 15:25:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":667491,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEarly-life Stress Specifically Impaired Cognition in Adolescent Male Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eThe experimental timeline of ES, behavioral tests, and brain tissue acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eA schematic illustration of control (left) and ES (right) housing conditions. While control mice live in cages with sufficient bedding/nesting conditions, ES dam and pups live in cages with limited nesting and bedding materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C-F) \u003c/strong\u003eCognitive behavioral tests and results. The left panels in (C, E, F) are the representative maps showing the time spent in each location for CT and ES mice; warm colors represent more time. (C) Temporary order memory test. Right, the percentage of time probing the “remote” and “recent” object (Paired \u003cem\u003et\u003c/em\u003e test between two objects: CT: \u003cem\u003et\u003c/em\u003e\u003csub\u003e11\u003c/sub\u003e = 4.475, \u003cem\u003ep\u003c/em\u003e = 0.001; ES: \u003cem\u003et\u003c/em\u003e\u003csub\u003e11\u003c/sub\u003e = 0.006, \u003cem\u003ep \u003c/em\u003e= 0.996; unpaired \u003cem\u003et\u003c/em\u003e test: \u003cem\u003et\u003c/em\u003e\u003csub\u003e22\u003c/sub\u003e = 2.911, \u003cem\u003ep\u003c/em\u003e = 0.008). (D) Y-maze spontaneous alternations. Left, representative motion path in the test; middle, spontaneous alternation ratio (\u003cem\u003et\u003c/em\u003e\u003csub\u003e22\u003c/sub\u003e = 2.676, \u003cem\u003ep\u003c/em\u003e = 0.014, unpaired \u003cem\u003et \u003c/em\u003etest). Right, the erroneous alternations: alternative arm return (AAR: \u003cem\u003et\u003c/em\u003e\u003csub\u003e22\u003c/sub\u003e = 1.549, \u003cem\u003ep\u003c/em\u003e = 0.136, unpaired \u003cem\u003et\u003c/em\u003e test) and same arm return (SAR: \u003cem\u003et\u003c/em\u003e\u003csub\u003e22\u003c/sub\u003e = 2.381, \u003cem\u003ep\u003c/em\u003e = 0.026, unpaired \u003cem\u003et\u003c/em\u003e test). (E) Novel object recognition test. Right, the percentage of time probing the “novel” object and “familiar” object (Paired \u003cem\u003et\u003c/em\u003e test between two objects: CT: \u003cem\u003et\u003c/em\u003e\u003csub\u003e11\u003c/sub\u003e = 4.839, \u003cem\u003ep\u003c/em\u003e = 0.001; ES: \u003cem\u003et\u003c/em\u003e\u003csub\u003e11\u003c/sub\u003e = 2.003, \u003cem\u003ep\u003c/em\u003e = 0.0704; unpaired \u003cem\u003et\u003c/em\u003e test: \u003cem\u003et\u003c/em\u003e\u003csub\u003e22\u003c/sub\u003e = 0.806, \u003cem\u003ep\u003c/em\u003e = 0.008). (F) Spatial object recognition test. Right, the percentage of time probing the “displaced” and “stationary” object (Paired \u003cem\u003et\u003c/em\u003e test: CT: \u003cem\u003et\u003c/em\u003e\u003csub\u003e11\u003c/sub\u003e = 3.426, \u003cem\u003ep\u003c/em\u003e = 0.006; ES: \u003cem\u003et\u003c/em\u003e\u003csub\u003e11\u003c/sub\u003e = 0.358, \u003cem\u003ep\u003c/em\u003e = 0.727; unpaired \u003cem\u003et\u003c/em\u003e test: \u003cem\u003et\u003c/em\u003e\u003csub\u003e22\u003c/sub\u003e = 2.819, \u003cem\u003ep\u003c/em\u003e = 0.010).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G-I) \u003c/strong\u003eAnxiety-like behavioral tests and results. (G) Open field test. Left, representative motion path for CT and\u003cem\u003e \u003c/em\u003eES mice. Middle, total distance traveled in 10 min (\u003cem\u003et\u003c/em\u003e\u003csub\u003e22\u003c/sub\u003e = 1.073, \u003cem\u003ep\u003c/em\u003e = 0.295, unpaired \u003cem\u003et \u003c/em\u003etest). Right, time spent in center zone (\u003cem\u003et\u003c/em\u003e\u003csub\u003e12.73\u003c/sub\u003e = 2.095, \u003cem\u003ep\u003c/em\u003e = 0.057, unpaired \u003cem\u003et\u003c/em\u003e test with Welch's correction). (H) Light-dark box test. Left, representative motion path in the light chamber for CT and\u003cem\u003e \u003c/em\u003eES mice. Middle, time in the light chamber (\u003cem\u003et\u003c/em\u003e\u003csub\u003e17\u003c/sub\u003e = 1.051, \u003cem\u003ep\u003c/em\u003e = 0.308, unpaired \u003cem\u003et\u003c/em\u003e test). Right, latency to the light chamber (\u003cem\u003et\u003c/em\u003e\u003csub\u003e17\u003c/sub\u003e = 0.482, \u003cem\u003ep\u003c/em\u003e = 0.636, unpaired \u003cem\u003et\u003c/em\u003e test).\u003cstrong\u003e \u003c/strong\u003e(I)\u003cstrong\u003e \u003c/strong\u003eElevated plus maze test. Left, representative map showing the time spent in each location for CT and\u003cem\u003e \u003c/em\u003eES mice; warm colors represent more time. Middle, time in open arms (\u003cem\u003et\u003c/em\u003e\u003csub\u003e10.17\u003c/sub\u003e = 1.184, \u003cem\u003ep\u003c/em\u003e = 0.263, unpaired \u003cem\u003et\u003c/em\u003e test with Welch's correction). Right, latency to open arms (\u003cem\u003et\u003c/em\u003e\u003csub\u003e10.52\u003c/sub\u003e = 1.053, \u003cem\u003ep\u003c/em\u003e = 0.316, unpaired \u003cem\u003et\u003c/em\u003e test with Welch's correction).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J) \u003c/strong\u003eSocial approach test. Left, representative map showing the time spent in each location for CT and\u003cem\u003e \u003c/em\u003eES mice; warm colors represent more time. Right, time spent interacting with the stranger mice (\u003cem\u003et\u003c/em\u003e\u003csub\u003e22\u003c/sub\u003e = 1.320, \u003cem\u003ep\u003c/em\u003e = 0.201, unpaired \u003cem\u003et\u003c/em\u003e test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(K-M) \u003c/strong\u003eDepressive-like behavioral tests and results. (K) Tail suspension test. Left, diagram of the test. Right, immobility time in 6 min (\u003cem\u003et\u003c/em\u003e\u003csub\u003e22\u003c/sub\u003e = 2.095, \u003cem\u003ep\u003c/em\u003e = 1.299, unpaired \u003cem\u003et\u003c/em\u003e test). (L)\u003cstrong\u003e \u003c/strong\u003eForced swimming test. Left, diagram of the test; middle. Right, immobility time in 6 min (\u003cem\u003et\u003c/em\u003e\u003csub\u003e20\u003c/sub\u003e = 2.260, \u003cem\u003ep\u003c/em\u003e = 0.035, unpaired \u003cem\u003et\u003c/em\u003e test).\u003cstrong\u003e \u003c/strong\u003e(M) Sucrose preference test. The percentage of sucrose preference (stress effect, \u003cem\u003eF \u003c/em\u003e\u003csub\u003e(1, 63)\u003c/sub\u003e\u003cem\u003e = \u003c/em\u003e0.001, \u003cem\u003ep\u003c/em\u003e = 0.976; time effect, \u003cem\u003eF\u003c/em\u003e \u003csub\u003e(2, 63)\u003c/sub\u003e = 0.493, \u003cem\u003ep\u003c/em\u003e = 0.613; stress × time interaction, \u003cem\u003eF\u003c/em\u003e \u003csub\u003e(2, 63)\u003c/sub\u003e = 0.148, \u003cem\u003ep\u003c/em\u003e = 0.863).\u003c/p\u003e\n\u003cp\u003eData are represented as mean ± SEM. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, comparisons between CT and ES group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, paired \u003cem\u003et\u003c/em\u003e test. AAR, alternative arm return; CT, control; ES, early-life stress; PND, postnatal day; SAR, same arm return. See also Figures S1-S2\u003c/p\u003e","description":"","filename":"Fig161.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3572074/v1/f8a2a207794277452573435a.jpg"},{"id":49535026,"identity":"cf51d54c-443c-40cd-9f1b-3caae9eadf68","added_by":"auto","created_at":"2024-01-12 15:17:44","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":429422,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEarly-life Stress Reduced PVIs (not SST-INs) Activity in the mPFC in Adolescent Male Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-B)\u003c/strong\u003e Left,\u003cstrong\u003e \u003c/strong\u003erepresentative immunostaining images showing the PVI (arrowheads) (A) and SST-INs (arrowheads) (B) in the mPFC; Scale bar, 500 μm or 30 μm. Right, the density of PVIs (A), not SST-Ins (B), in the three subregions of mPFC was decreased in ES mice (PVIs: stress effect, \u003cem\u003eF \u003c/em\u003e\u003csub\u003e(1, 24)\u003c/sub\u003e\u003cem\u003e = \u003c/em\u003e10.34, \u003cem\u003ep\u003c/em\u003e = 0.004, two-way ANOVA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C-E)\u003c/strong\u003e Quantification of the density of PVI or SST-INs that were c-fos-positive during the TOM test in CT and ES mice. (C) The experimental timeline of ES exposure, behavioral tests and brain tissue acquisition in C57BL/6 mice. (D) Top, representative images show the co-expression of c-fos and PV in the mPFC of CT and ES mice. Asterisks indicate neurons that co-express c-fos and PV; arrowheads indicate PV-expressing cells without detectable c-fos expression. Scale bar, 100 µm or 20 µm. Bottom: the density of neurons showing PV and c-fos colocalization in the three subfields of mPFC in two groups (Stress effect, \u003cem\u003eF \u003c/em\u003e\u003csub\u003e(1, 24)\u003c/sub\u003e\u003cem\u003e = \u003c/em\u003e17.78, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003cstrong\u003e \u003c/strong\u003e(E) Top, representative images show the co-expression of c-fos and SST in the mPFC of CT and ES mice. Asterisks indicate neurons that co-express c-fos and SST; arrowheads indicate SST-expressing cells without detectable c-fos expression. Scale bar, 100 µm or 20 µm. Bottom: the density of neurons showing SST and c-fos colocalization in the three subfields of mPFC in two groups (Stress effect, \u003cem\u003eF \u003c/em\u003e\u003csub\u003e(1, 24)\u003c/sub\u003e\u003cem\u003e = \u003c/em\u003e1.969, \u003cem\u003ep\u003c/em\u003e = 0.170).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F-H)\u003c/strong\u003e ES effects on intrinsic excitability of PVIs in the mPFC. (F) The experimental timeline of electrophysiological recordings in the mPFC in adolescent male PV::Ai14 mice. (G)\u003cstrong\u003e \u003c/strong\u003eEvoked action potential. Top panel, sample traces in response to a 500-pA current step of CT and ES mice. Bottom panel, ES reduced frequency of evoked action potential in response to current injection (≥ 200 μA, all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.044, unpaired \u003cem\u003et \u003c/em\u003etest). (H)\u003cstrong\u003e \u003c/strong\u003eSpontaneous action potential. Top panel, sample traces of spontaneous potential of CT and ES mice. Bottom panel, ES did not alter the frequency and amplitude of spontaneous action potential of PVIs.\u003c/p\u003e\n\u003cp\u003eData are represented as mean ± SEM. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, comparisons between CT and ES group; \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e\u0026amp;\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, stress effect of two-way ANOVA. Cg, cingulate cortex; CT, control; ES, early-life stress; IL, infralimbic cortex; mPFC, medial prefrontal cortex; PrL, prelimbic cortex; PVI, parvalbumin-expressing interneuron; SST-IN, somatostatin-expressing interneuron. See also Figures S3\u003c/p\u003e","description":"","filename":"Fig162.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3572074/v1/76004274dfe90cccdc06d86b.jpg"},{"id":49535024,"identity":"a0302d8d-7d13-41f9-b964-c241368a933e","added_by":"auto","created_at":"2024-01-12 15:17:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":756230,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrefrontal PVI Activity Mediates Early-Life Stress-Induced Cognitive Deficits in Adolescent Male Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-E) \u003c/strong\u003eChemogenetic inhibition of mPFC PVI activity impairs cognition. (A) The experimental timeline of the behavioral tests, CNO injection, and brain tissue acquisition after hM4D(Gi) viral injection in adolescent PV-Cre mice. (B) Left panel, representative image showing region-specific expression of mCherry in the mPFC; right panel, representative image showing that the majority of PVIs express hM4Di in the mPFC. Asterisks indicate neurons that co-express mCherry and PV; arrowheads indicate PV-expressing cells without detectable mCherry expression. Scale bar, 500 μm or 20 μm. (C) Representative images show the expression of c-fos and mCherry in the mPFC of Veh and CNO mice. Asterisks indicate neurons that co-express mCherry and c-fos, while arrowheads indicate mCherry-expressing cells without detectable c-fos expression. Scale bar, 100 µm or 20 µm. Immunostaining analyses confirmed that Gi decreased in the expression of c-fos in PVIs in the mPFC (\u003cem\u003et\u003c/em\u003e\u003csub\u003e10\u003c/sub\u003e = 2.748, \u003cem\u003ep\u003c/em\u003e = 0.021, unpaired \u003cem\u003et\u003c/em\u003e test). Chemogenetic inhibition of mPFC PVI activity impaired (D) temporal order memory (Paired \u003cem\u003et\u003c/em\u003e test: Veh: \u003cem\u003et\u003c/em\u003e\u003csub\u003e9\u003c/sub\u003e = 2.617, \u003cem\u003ep\u003c/em\u003e = 0.028; CNO: \u003cem\u003et\u003c/em\u003e\u003csub\u003e9\u003c/sub\u003e = 1.495, \u003cem\u003ep \u003c/em\u003e= 0.169; unpaired \u003cem\u003et\u003c/em\u003e test: \u003cem\u003et\u003c/em\u003e\u003csub\u003e18\u003c/sub\u003e = 3.010, \u003cem\u003ep\u003c/em\u003e = 0.008) and (E) spatial working memory in the Y-maze spontaneous alternation test (SAR: \u003cem\u003et\u003c/em\u003e\u003csub\u003e15\u003c/sub\u003e = 2.135, \u003cem\u003ep\u003c/em\u003e = 0.050, unpaired \u003cem\u003et\u003c/em\u003e test) in adolescent mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F-I) \u003c/strong\u003eOptogenetic inhibition of mPFC PVI activity impairs cognition. (F) The experimental timeline of the behavioral tests, light stimulation, and brain tissue acquisition after eNpHR3.0 viral injection in adolescent PV-Cre mice. (G) Representative image shows the location of bilateral viral infection and optic-fiber implantation in mPFC. Scale bar, 500 μm.\u003cstrong\u003e \u003c/strong\u003eOptogenetic inhibition of mPFC PVI activity impaired (H) temporal order memory (Paired \u003cem\u003et\u003c/em\u003e test: EGFP: \u003cem\u003et\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e = 4.301, \u003cem\u003ep\u003c/em\u003e = 0.013; eNpHR3.0: \u003cem\u003et\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e = 1.542, \u003cem\u003ep \u003c/em\u003e= 0.198; unpaired \u003cem\u003et\u003c/em\u003e test: \u003cem\u003et\u003c/em\u003e\u003csub\u003e8\u003c/sub\u003e = 3.414, \u003cem\u003ep\u003c/em\u003e = 0.009) and (I) increased AAR in the Y-maze spontaneous alternation test (\u003cem\u003et\u003c/em\u003e\u003csub\u003e8\u003c/sub\u003e = 2.709, \u003cem\u003ep\u003c/em\u003e = 0.027, unpaired \u003cem\u003et\u003c/em\u003e test) in adolescent mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J-N)\u003c/strong\u003e Chemogenetic activation of mPFC PVI activity reverses ES-induced cognitive deficits. (J) The experimental timeline of the behavioral tests, CNO injection, and brain tissue acquisition after hM3D(Gq) viral injection in adolescent PV-Cre mice. (K) Left panel, representative image showing region-specific expression of mCherry in mPFC; right panel, representative image showing that the majority of PVIs express hM3Dq in the mPFC. Asterisks indicate neurons that co-express mCherry and PV; arrowheads indicate PV-expressing cells without detectable mCherry expression. Scale bar, 500 μm or 20 μm. (L) Representative images showed the expression of c-fos and PV in the mPFC of the four groups of mice. Immunostaining analyses show that the number of activated PVI neurons in the mPFC in the temporal order memory test was decreased by ES (\u003cem\u003ep\u003c/em\u003e = 0.040, Bonferroni’s test) and increased by CNO injection (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, Bonferroni’s test). Asterisks indicate neurons that co-express PV and c-fos; arrowheads indicate PV-expressing cells without detectable c-fos expression. Scale bar, 100 µm or 20 µm. (M-N) Activation of PVIs in the mPFC reverses the ES-induced deficits of temporal order memory (M, CT-Veh \u003cem\u003evs\u003c/em\u003e. ES-Veh: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ES-Veh \u003cem\u003evs\u003c/em\u003e. ES-CNO: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, Bonferroni’s test) and spatial working memory (N, SA: CT-Veh \u003cem\u003evs\u003c/em\u003e. ES-Veh: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, Bonferroni’s test; ES-Veh \u003cem\u003evs\u003c/em\u003e. ES-CNO: \u003cem\u003ep\u003c/em\u003e = 0.001, unpaired \u003cem\u003et\u003c/em\u003e test; SAR: CT-Veh \u003cem\u003evs\u003c/em\u003e. ES-Veh: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ES-Veh \u003cem\u003evs\u003c/em\u003e. ES-CNO: \u003cem\u003ep\u003c/em\u003e = 0.002, Bonferroni’s test).\u003c/p\u003e\n\u003cp\u003eData are represented as mean ± SEM. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, unpaired \u003cem\u003et\u003c/em\u003e test or Bonferroni’s \u003cem\u003epost hoc\u003c/em\u003e test; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, paired \u003cem\u003et\u003c/em\u003e test. AAR, alternative arm return; CNO, clozapine-N-oxide; CT, control; ES, early-life stress; mPFC, medial prefrontal cortex; PND, postnatal day; PVI, parvalbumin-expressing interneuron; SA, spontaneous alternation; SAR, same arm return; Veh, vehicle. See also Figures S3–S7.\u003c/p\u003e","description":"","filename":"Fig163.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3572074/v1/b01807a7e7bc6bb4eb72e832.jpg"},{"id":49535027,"identity":"5295270a-aebb-429d-b5c6-f3d914c5a2eb","added_by":"auto","created_at":"2024-01-12 15:17:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":997750,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrefrontal PN Activity Mediates Early-Life Stress-Induced Cognitive Deficits by Enhancing PVI Activity in Adolescent Male Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-C) \u003c/strong\u003eES reduced the VGluT1 expression surrounding the soma of PVIs in mPFC in adolescent mice. (A) Representative images showing VGluT1 and PV fluorescence staining of PVIs in the mPFC in CT and ES mice. The dotted circle represents the soma of PVIs. Scale bar, 5 µm. (B) Sholl analysis. Concentric circles (1 µm apart) are drawn centered on the soma of PVIs. Scale bar, 5 µm. (C) Quantification of VGluT1 expression levels at different distances from the soma of PVIs (Interaction effect, \u003cem\u003eF\u003c/em\u003e \u003csub\u003e21, 147\u003c/sub\u003e = 8.021, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; Stress effect, \u003cem\u003eF\u003c/em\u003e \u003csub\u003e1, 7\u003c/sub\u003e = 7.628, \u003cem\u003ep\u003c/em\u003e = 0.028; two-way ANOVA; Distances \u0026gt; 8.784 um, CT \u003cem\u003evs\u003c/em\u003e. ES: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.028, Bonferroni’s test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D-E)\u003c/strong\u003e The effects of ES on PN activity in the mPFC. Left panels in (D-E) are the representative images showing the co-expression of c-fos and c-fos and CamkIIa (D) or Neurogranin (E) in the mPFC of CT and ES mice. Asterisks indicate co-expressing neurons; arrowheads indicate CamkIIa-expressing (D) or Neurogranin-expressing (E) cells without detectable c-fos expression. Scale bar, 100 μm or 10 μm.\u003cstrong\u003e \u003c/strong\u003eRight panels show that\u003cstrong\u003e \u003c/strong\u003eES reduced the density of c-fos- and CamkIIa-co-expressing neurons (D, \u003cem\u003et\u003c/em\u003e\u003csub\u003e8\u003c/sub\u003e = 1.475, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, unpaired \u003cem\u003et\u003c/em\u003e test) and c-fos- and neurogranin-co-expressing neurons (E, \u003cem\u003et\u003c/em\u003e\u003csub\u003e9\u003c/sub\u003e = 2.352, \u003cem\u003ep\u003c/em\u003e = 0.043, unpaired \u003cem\u003et\u003c/em\u003e test), during the TOM test, in the mPFC in adolescent mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F-K) \u003c/strong\u003eChemogenetic activation of mPFC PN activity reverses ES-induced cognitive deficits. (F)\u003cstrong\u003e \u003c/strong\u003eThe experimental timeline of the behavioral tests, CNO injection, and brain tissue acquisition after CamkIIa-hM3D(Gq) viral injection in adolescent C57BL/6 mice.\u003cstrong\u003e \u003c/strong\u003e(G) Representative image shows the co-expression of mCherry and CamkIIa in the mPFC. Scale bar, 10 µm. (H) Representative images show the expression of c-fos and mCherry in the mPFC of the four groups of mice. Asterisks indicate neurons that co-express mCherry and c-fos; arrowheads indicate mCherry-expressing cells without detectable c-fos expression. Scale bar, 100 µm or 20 µm. (I) Immunostaining analyses show that the percentage of mCherry-infected neurons co-labeled with c-fos was significantly elevated in the mPFC by CNO injection (CNO effect: \u003cem\u003eF\u003c/em\u003e \u003csub\u003e1, 20\u003c/sub\u003e = 23.03, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; two-way ANOVA). (J-K) Activation of PNs in the mPFC reversed ES-induced deficits of temporal order memory (J, CT-Veh \u003cem\u003evs\u003c/em\u003e. ES-Veh: \u003cem\u003ep\u003c/em\u003e = 0.003, ES-Veh \u003cem\u003evs\u003c/em\u003e. ES-CNO: \u003cem\u003ep\u003c/em\u003e = 0.038, Bonferroni’s test) and spatial working memory (K, SA: CT-Veh \u003cem\u003evs\u003c/em\u003e. ES-Veh: \u003cem\u003ep\u003c/em\u003e = 0.002; ES-Veh \u003cem\u003evs\u003c/em\u003e. ES-CNO: \u003cem\u003ep\u003c/em\u003e = 0.008, Bonferroni’s test; SAR: CT-Veh \u003cem\u003evs\u003c/em\u003e. ES-Veh: \u003cem\u003ep\u003c/em\u003e = 0.003, ES-Veh \u003cem\u003evs\u003c/em\u003e. ES-CNO: \u003cem\u003ep\u003c/em\u003e = 0.007, Bonferroni’s test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(L) \u003c/strong\u003eThe experimental timeline of the behavioral tests, CNO injection, and brain tissue acquisition after CamkIIa-Gq and DIO-Gi viral infection in adolescent PV-Cre mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(M)\u003c/strong\u003e Representative images show the expression of mCherry and c-fos in the mPFC of the four groups of mice. CNO increased the activated PNs (co-expressing mCherry and c-fos) in both control (CT-Veh \u003cem\u003evs\u003c/em\u003e. CT-CNO: \u003cem\u003ep\u003c/em\u003e = 0.032, Bonferroni’s test) and stressed (ES-Veh \u003cem\u003evs.\u003c/em\u003e ES-CNO, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, Bonferroni’s test) mice. Asterisks indicate neurons that co-express mCherry and c-fos; arrowheads indicate mCherry-expressing cells without detectable c-fos expression. Scale bar, 20 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(N)\u003c/strong\u003e Representative images show the expression of PV and c-fos in the mPFC of the four groups of mice. PVI activity was inhibited in control mice after CNO administration and was reduced in ES-exposed vehicle mice (CT-Veh \u003cem\u003evs.\u003c/em\u003e CT-CNO, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; CT-Veh \u003cem\u003evs.\u003c/em\u003e ES-Veh, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, Bonferroni’s test). Asterisks indicate neurons that co-express PV and c-fos; arrowheads indicate PV-expressing cells without detectable c-fos expression. Scale bar, 20 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(O)\u003c/strong\u003e In temporal order memory test, inhibition of mPFC PVIs blocked the reversal effects of activation of PNs (ES-Veh \u003cem\u003evs.\u003c/em\u003e ES-CNO: \u003cem\u003et\u003c/em\u003e\u003csub\u003e10\u003c/sub\u003e = 0.048, \u003cem\u003ep\u003c/em\u003e = 0.963, unpaired \u003cem\u003et\u003c/em\u003e test) on temporal order memory impairment induced by ES (CT-Veh \u003cem\u003evs.\u003c/em\u003e ES-Veh:\u003cem\u003e t\u003c/em\u003e\u003csub\u003e13\u003c/sub\u003e = 4.556, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, unpaired \u003cem\u003et\u003c/em\u003e test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(P)\u003c/strong\u003e In the Y-maze spontaneous alternation test, a significant main effect of stress was found in SA (\u003cem\u003eF\u003c/em\u003e \u003csub\u003e1, 27\u003c/sub\u003e = 4.859, \u003cem\u003ep\u003c/em\u003e = 0.036), indicating the absence of the reversal effects of PN activation after PVI inhibition. In addition, CNO significantly increased AAR (\u003cem\u003eF\u003c/em\u003e \u003csub\u003e1, 27\u003c/sub\u003e = 11.72, \u003cem\u003ep\u003c/em\u003e = 0.002) and decreased SAR (\u003cem\u003eF\u003c/em\u003e \u003csub\u003e1, 27\u003c/sub\u003e = 8.421, \u003cem\u003ep\u003c/em\u003e = 0.007).\u003c/p\u003e\n\u003cp\u003eData are represented as mean ± SEM. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, unpaired \u003cem\u003et\u003c/em\u003e test or Bonferroni’s \u003cem\u003epost hoc\u003c/em\u003e test; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, paired \u003cem\u003et\u003c/em\u003e test. \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, stress effect of two-way ANOVA. \u003csup\u003e$$\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e$$$\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, CNO effects in two-way ANOVA. AAR, alternative arm return; CNO, clozapine-N-oxide; CT, control; ES, early-life stress; mPFC, medial prefrontal cortex; PND, postnatal day; PVI, parvalbumin-expressing interneuron; SA, spontaneous alternation; SAR, same arm return; Veh, vehicle; VGluT1, Vesicular glutamate transporter-1. See also Figures S8-10.\u003c/p\u003e","description":"","filename":"Fig164.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3572074/v1/9fb0e8e78507e83b5a58c837.jpg"},{"id":49535029,"identity":"e53a00dd-7ed2-42ef-bd0f-90ebc7cb0b87","added_by":"auto","created_at":"2024-01-12 15:17:44","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1007411,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCRHR1 Downregulation Reverses Early-Life Stress-Induced Cognitive Deficits in Adolescent Male Mice by Restoring PVI Activity in Medial Prefrontal Cortex\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003e\u003cem\u003eCrhr1\u003c/em\u003e expression in excitatory and inhibitory neurons in adolescent mice mPFC. Left, representative (top, scale bar, 50 µm) and magnified (bottom, scale bar, 10 µm) images showing the mRNA expression of \u003cem\u003eCrhr1\u003c/em\u003e,\u003cem\u003e Slc32a1\u003c/em\u003e, and \u003cem\u003eSlc17a7\u003c/em\u003e. Right, numbers of neurons that co-express \u003cem\u003eCrhr1\u003c/em\u003e and \u003cem\u003eSlc32a1\u003c/em\u003e, \u003cem\u003eCrhr1\u003c/em\u003e and \u003cem\u003eSlc17a7\u003c/em\u003e, \u003cem\u003eSlc32a1\u003c/em\u003e and \u003cem\u003eSlc17a7\u003c/em\u003e, and \u003cem\u003eCrhr1\u003c/em\u003e,\u003cem\u003e Slc32a1\u003c/em\u003e, and \u003cem\u003eSlc17a7\u003c/em\u003e. Numbers in parentheses indicate the total number of neurons expressing the corresponding mRNA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B, G, and L) \u003c/strong\u003eThe experimental timeline of the behavioral tests and brain tissue acquisition after CRHR1 knockout in mPFC (B), antalarmin administration during ES exposure (F), or acute antalarmin treatment (J) in C57BL/6 mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C, H, and M)\u003c/strong\u003e ES-induced deficits of temporal order memory were reversed by mPFC CRHR1 deletion (C, CT-CV \u003cem\u003evs\u003c/em\u003e. ES-CV: \u003cem\u003ep\u003c/em\u003e = 0.002, ES-CV \u003cem\u003evs\u003c/em\u003e. ES-KD: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, Bonferroni’s test), chronic antalarmin administration during PND2-8 (H, CT-Veh \u003cem\u003evs\u003c/em\u003e. ES-Veh: \u003cem\u003ep\u003c/em\u003e = 0.003, ES-Veh \u003cem\u003evs\u003c/em\u003e. ES-Anta: \u003cem\u003ep\u003c/em\u003e = 0.042, Bonferroni’s test), and acute antalarmin treatment (M, CT-Veh \u003cem\u003evs\u003c/em\u003e. ES-Veh: \u003cem\u003ep\u003c/em\u003e = 0.002, ES-Veh \u003cem\u003evs\u003c/em\u003e. ES-Anta: \u003cem\u003ep\u003c/em\u003e = 0.002, Bonferroni’s test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D, I, and N)\u003c/strong\u003e Representative images show the expression of c-fos and PV in the mPFC of four groups. Asterisks indicate neurons that co-express c-fos and PV; arrowheads indicate PV-expressing cells without detectable c-fos expression. Scale bar, 100 µm or 20 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E, J, and O)\u003c/strong\u003e The effects of CRHR1 blockade on ES-induced PVI activity reduction. (E, O) ES negative effects were reversed by mPFC CRHR1 deletion (E, CT-CV \u003cem\u003evs\u003c/em\u003e. ES-CV: \u003cem\u003ep\u003c/em\u003e = 0.047, ES-CV \u003cem\u003evs\u003c/em\u003e. ES-KD: \u003cem\u003ep\u003c/em\u003e = 0.041; Bonferroni’s test) or acute antalarmin treatment (O, CT-Veh \u003cem\u003evs.\u003c/em\u003e ES-Veh: \u003cem\u003ep \u003c/em\u003e= 0.001; ES-Veh \u003cem\u003evs.\u003c/em\u003e ES-Anta: \u003cem\u003ep \u003c/em\u003e= 0.007; Bonferroni’s test). (J) Chronic antalarmin administration during PND2-8 upregulated the density of PVIs co-labeled with c-fos reduced by ES (\u003cem\u003eF\u003c/em\u003e \u003csub\u003e1, 16\u003c/sub\u003e = 16.97, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, two-way ANOVA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F, K and P) \u003c/strong\u003eCorrelations between the mPFC PVI activity and discrimination index in the temporal order memory test across all animals in each experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(Q) \u003c/strong\u003eThe experimental timeline of the behavioral tests, CNO and antalarmin injection, and brain tissue acquisition after DIO-Gi viral infection in adolescent PV-Cre mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(R)\u003c/strong\u003e Left panel, representative image shows region-specific expression of mCherry in the mPFC; right panel, representative image shows the majority of PVIs express hM4Di in the mPFC. Asterisks indicate neurons that co-express mCherry and PV; arrowheads indicate PV-expressing cells without detectable mCherry expression. Scale bar, 500 μm or 10 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(S)\u003c/strong\u003e In temporal order memory test, inhibition of mPFC PVIs blocked the reversal effects of acute injection of antalarminon temporal order memory impairment induced by ES (ES-Anta-Veh \u003cem\u003evs.\u003c/em\u003e ES-Anta-CNO: \u003cem\u003et\u003c/em\u003e\u003csub\u003e18\u003c/sub\u003e = 2.464, \u003cem\u003ep\u003c/em\u003e = 0.024, unpaired \u003cem\u003et\u003c/em\u003e test; ES-Anta-Veh:\u003cem\u003e t\u003c/em\u003e\u003csub\u003e9\u003c/sub\u003e = 3.122, \u003cem\u003ep\u003c/em\u003e = 0.012, ES-Anta-CNO:\u003cem\u003e t\u003c/em\u003e\u003csub\u003e9\u003c/sub\u003e = 0.242, \u003cem\u003ep\u003c/em\u003e = 0.815, paired \u003cem\u003et\u003c/em\u003e test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(T)\u003c/strong\u003e Representative images show the expression of PV and c-fos in the mPFC of the two groups of mice. PVI activity was inhibited in stressed mice with acute injection of antalarmin after CNO administration (\u003cem\u003et\u003c/em\u003e\u003csub\u003e9\u003c/sub\u003e = 4.898, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, unpaired \u003cem\u003et\u003c/em\u003e test). Asterisks indicate neurons that co-express PV and c-fos; arrowheads indicate PV-expressing cells without detectable c-fos expression. Scale bar, 100 µm or 20 µm.\u003c/p\u003e\n\u003cp\u003eData are represented as mean ± SEM. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, Bonferroni’s \u003cem\u003epost hoc\u003c/em\u003e test; \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, stress effect of two-way ANOVA;\u003csup\u003e $$$\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, drug effect of two-way ANOVA. Anta: antalarmin; Cg, cingulate cortex; CNO, clozapine-N-oxide; CT, control; CV: control virus; ES, early-life stress; KO: knockout; mPFC, medial prefrontal cortex; PND, postnatal day; PrL, prelimbic cortex; PVI, parvalbumin-expressing interneuron; Veh, vehicle. See also Figures S11.\u003c/p\u003e","description":"","filename":"Fig165.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3572074/v1/478ea996ad15de155fb28b93.jpg"},{"id":49535028,"identity":"6aa4712f-4e4c-43e7-939a-5d22355dbd2e","added_by":"auto","created_at":"2024-01-12 15:17:44","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":592598,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnvironmental Enrichment Alleviates Early-life Stress-induced Cognitive Deficits through Activation of Prefrontal PNs and PVIs in Adolescent Male Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eThe experimental timeline of EE, behavioral tests and brain tissue acquisition after ES.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eA schematic illustration (left) and a photograph (right) of the enriched housing environment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e EE partially restored ES-induced temporal order memory deficits. Only the stressed mice in the standard housing environments failed to distinguish the “remote” object from the “recent” object (ES-SE: \u003cem\u003et\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e = 0.396, \u003cem\u003ep\u003c/em\u003e = 0.706, paired \u003cem\u003et\u003c/em\u003e test), while mice in the other three groups exhibited intact temporal order memory (CT-SE: \u003cem\u003et\u003c/em\u003e\u003csub\u003e7\u003c/sub\u003e = 4.818, \u003cem\u003ep \u003c/em\u003e= 0.002; CT-EE: \u003cem\u003et\u003c/em\u003e\u003csub\u003e8\u003c/sub\u003e = 3.316, \u003cem\u003ep \u003c/em\u003e= 0.011; ES-EE: \u003cem\u003et\u003c/em\u003e\u003csub\u003e5\u003c/sub\u003e = 3.609, \u003cem\u003ep \u003c/em\u003e= 0.015; paired \u003cem\u003et\u003c/em\u003e test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e Representative images show the expression of c-fos and neurogranin in the mPFC of four groups. Asterisks indicate neurons that co-express c-fos and neurogranin; arrowheads indicate neurogranin-expressing cells without detectable c-fos expression. Scale bar, 100 µm or 20 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E) \u003c/strong\u003eES significantly reduced neurogranin\u003csup\u003e+\u003c/sup\u003e neuron activity (\u003cem\u003ep\u003c/em\u003e = 0.013, Bonferroni’s test), which was reversed by environment enrichment (\u003cem\u003et\u003c/em\u003e\u003csub\u003e8\u003c/sub\u003e = 2.774, \u003cem\u003ep\u003c/em\u003e = 0.024 unpaired \u003cem\u003et\u003c/em\u003e test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F) \u003c/strong\u003eThe\u003cstrong\u003e \u003c/strong\u003ecorrelation between the mPFC PN activity and discrimination index in the temporal order memory test across all animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Representative images show the expression of c-fos and PV in the mPFC of four groups. Asterisks indicate neurons that co-express c-fos and PV, while arrowheads indicate PV-expressing cells without detectable c-fos expression. Scale bar, 100 µm or 20 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H) \u003c/strong\u003eES significantly reduced PVI activity (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, Bonferroni’s test), which was reversed by environment enrichment (\u003cem\u003ep\u003c/em\u003e = 0.017, Bonferroni’s test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I) \u003c/strong\u003eThe\u003cstrong\u003e \u003c/strong\u003ecorrelation between the mPFC PVI activity and discrimination index in the temporal order memory test across all animals.\u003c/p\u003e\n\u003cp\u003eData are represented as mean ± SEM. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, Bonferroni’s \u003cem\u003epost hoc\u003c/em\u003e test; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, paired \u003cem\u003et\u003c/em\u003e test. CT, control; ES, early-life stress; EE, environmental enrichment; mPFC, medial prefrontal cortex; PND, postnatal day; PVI, parvalbumin-expressing interneuron; SE, standard environment. See also Figures S12.\u003c/p\u003e","description":"","filename":"Fig166.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3572074/v1/4ffecbf8bd188facce12b439.jpg"},{"id":69684827,"identity":"41d5f613-93da-4360-96b9-5795ef9be8de","added_by":"auto","created_at":"2024-11-23 08:07:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5778923,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3572074/v1/31c724a5-3da8-409a-8475-ef86b4009c95.pdf"},{"id":49535030,"identity":"142b8d31-216c-4070-8a38-36e670de838f","added_by":"auto","created_at":"2024-01-12 15:17:44","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":5004478,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-3572074/v1/a56c0d94a7232081c91911a6.docx"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"The CRHR1→PN→PVI Pathway in Medial Prefrontal Cortex Mediates Early-life Stress-induced Cognitive Deficits in Adolescent Mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAdolescence often witnesses the first episodes of several psychiatric disorders, which has been associated with various genetic and environmental risk factors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. One such risk factor is early-life stress (ES). For instance, meta-analyses show that exposure to adverse life events increases the diagnosis of depression in childhood or adolescence by 2.5 times [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Cognitive impairment is one of the core symptoms of several psychiatric disorders and is frequently observed in adolescents exposed to ES [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, the neural mechanisms underlying cognitive impairment remain unclear and the efficacy of current first-line therapeutic drugs for cognitive deficits is limited [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Importantly, several recent studies have provided behavioral evidence that ES could significantly impair cognition in adolescent animals [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], paving the way for identifying potential neural correlates for early and effective intervention.\u003c/p\u003e \u003cp\u003eThe prefrontal cortex (PFC) plays crucial roles in cognitive behaviors, is a late-developing structure and is highly vulnerable to ES [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Within the PFC, parvalbumin (PV)-expressing interneurons (PVIs) are the largest class of inhibitory neurons (accounting for about 40% of interneurons) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. They form perisomatic projections onto excitatory pyramidal neurons (PNs), controlling neural network synchrony that are crucial for learning and memory [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Genetic manipulation experiments to inactivate [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] or activate [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] PVIs in the mPFC have supported the causal link between PVI activity and prefrontal-dependent cognitive abilities in adult animals. PVIs undergo a protracted period of maturation during adolescence and contribute significantly to the stability of cortical excitatory-inhibitory microcircuits [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and to the development of PFC-dependent cognitive abilities [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. For instance, sustained inhibition of prefrontal PVIs activity during adolescence was found to disrupt cognitive flexibility in adult mice [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Only a few studies have examined the effects of ES on PVIs in adolescence, which have largely focused on the number (density) of PVIs [\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. It remains unknown whether and how ES would affect the activity of adolescent PFC PVIs and how this alteration links with ES-induced cognitive deficits. Moreover, several mechanisms may contribute to the maturation of PVIs during adolescence, one of which is the increased glutamatergic inputs onto PVIs [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Although ES has been reported to impair the structure [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and function [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] of PNs in the adult mPFC, few studies have examined how ES would affect PNs activity and their inputs onto PVIs in the adolescent mPFC.\u003c/p\u003e \u003cp\u003eFor the treatment of cognitive impairment, the current pharmacological (e.g., first-line antidepressants or antipsychotics) and non-pharmacological (e.g., repeated transcranial magnetic stimulation (rTMS), or deep brain stimulation (DBS)) strategies have not yielded satisfactory results [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Recent clinical and basic studies have highlighted the association between PFC-dependent cognitive dysfunction and the corticotropin-releasing hormone (CRH) and its receptor 1 (CRHR1) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Genetic polymorphisms of the CRHR1 gene have been found to be associated with cognitive functioning in both healthy adults and patients with psychiatric disorders [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In animal models, downregulating the CRH-CRHR1 system using pharmacological (e.g., CRH antagonist D-Phe-CRF5; CRHR1 antagonist, antalarmin) or genetic (e.g., knockdown of prefrontal CRHR1) methods could reverse cognitive deficits [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, in adolescent animals, it remains unknown whether CRHR1-based intervention is effective for ES-induced cognitive deficits and how the CRH-CRHR1 system interacts with PFC PVIs. On the other hand, as a non-pharmacological intervention environmental enrichment (EE) has been shown to have beneficial effects on stress-related negative outcomes [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], and importantly on ES-induced cognitive impairments in adolescent animals [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, the underlying mechanisms are still unclear.\u003c/p\u003e \u003cp\u003eIn this study, we systematically test the involvement of mPFC PVI activity in ES-induced cognitive deficits in adolescent mice. We adopted the well-established ES paradigm, the limited nesting and bedding material (LBN), which we have previously reported to induce deficits in PFC-dependent cognitive functions and pyramidal neuronal structural plasticity in adult male mice [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. We first evaluated the effects of ES on cognition and mPFC PVI activity in adolescent mice. We then manipulated mPFC PVI activity through optogenetic and chemogenetic methods to examine the causal link between reduced mPFC PVI activity and ES-induced cognitive deficits. To understand the possible causes of PVI activity reduction following ES, considering the interaction between PVIs and PNs, the involvement of the CRH-CRHR1 system in ES adverse effects revealed in our previous studies [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and the localization of CRHR1 in PNs[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], we proposed the following hypothesis: early-life stress may first upregulate CRH-CRHR1 signaling in prefrontal PNs, which reduces PN activity, inhibits excitatory input to PVIs, and in turn downregulates PVI activity and ultimately leads to PFC-dependent cognitive impairment. To test this hypothesis, we then carried out chemogenetic and pharmacological experiments to demonstrate the involvement of PNs and the CRH-CRHR1 system in ES-induced cognitive deficits and PVI reduction. Finally, we tested the therapeutic effects of a non-pharmacological treatment\u0026ndash; EE\u0026ndash;for cognitive deficits in adolescents.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEarly-Life Stress Specifically Impaired Cognition in Adolescent Male Mice\u003c/h2\u003e \u003cp\u003eTo investigate the effects of ES on cognition in adolescent mice, we first established the LBN model during PND 2\u0026ndash;9 and then compared stressed and control mice on a series of stress-related physiological and behavioral measures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). Immediately after stress (PND9), stressed mice had significantly lower body weight gain (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA left) and the effect persisted into adolescence (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA right). Mice exposed to ES also showed significant adrenal atrophy (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB) and a tendency for thymus atrophy (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC) in adolescence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCognitive performance was assessed in four tasks. In the temporary order memory task (TOM, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), unlike the control mice, stressed mice failed to distinguish the \u0026ldquo;remote\u0026rdquo; object from the \u0026ldquo;recent\u0026rdquo; object and had significantly lower discrimination index than control mice. In the Y-maze spontaneous alternation test (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), stressed mice showed lower spontaneous alternation rates and higher error rates of same arm return, indicative of spatial working memory deficits. In the novel object recognition test, stressed mice did not distinguish the \u0026ldquo;novel\u0026rdquo; object from the \u0026ldquo;familiar\u0026rdquo; object as control mice did (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), despite of no group differences in the discrimination index. Stressed mice also exhibited spatial object recognition deficits, as in this task they failed to discriminate the \u0026ldquo;displaced\u0026rdquo; object from the \u0026ldquo;stationary\u0026rdquo; object, and showed significantly lower discrimination index than control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). The total probe time and distance traveled during the test phase of the recognition tasks or the total arm entries in the Y-maze were not affected by ES (Fig. S2A-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBesides cognitive behaviors, we also evaluated anxiety-like, social approach, and depression-like behaviors. In three tasks of anxiety-like behaviors, no significant differences were observed between stressed and control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-I and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS2\u003c/span\u003eE-G). Social approach was not affected by ES (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS2\u003c/span\u003eH). ES did not significantly affect depression-like behaviors in the tail suspension test (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS2\u003c/span\u003eI), sucrose preference test (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS2\u003c/span\u003eK), or the latency to immobility in the forced swimming test (Fig. S2J), except that increased immobility was observed in the stressed mice in the forced swimming test (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL).\u003c/p\u003e \u003cp\u003eTogether, these behavioral and stress-related physiological results indicate that the adverse effects of ES emerge as early as in adolescent male mice, with the cognitive behaviors being particularly vulnerable.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEarly-Life Stress Reduced PVI (not SST-INs) Interneuron Activity in mPFC in Adolescent Male Mice\u003c/h3\u003e\n\u003cp\u003eTo examine the involvement of mPFC PVI in the adverse effects of ES during adolescence, we quantified PVI density and activity in the following three analyses. First, immunohistochemistry revealed that ES significantly decreased the density of PVI in the mPFC, irrespective of subregions examined (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA). The density of somatostatin-expressing interneurons (SST-INs) was not affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB). Next, using immediate early gene c-fos immunostaining (which reflects neural activation[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]) and colocalization analysis, we found that stressed mice showed reduced density of activated PVIs in mPFC during the TOM test (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC). Again, no stress effects were observed for SST-INs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003eS3\u003c/span\u003eD). Finally, to validate the effects of ES on mPFC PVI activity, we recorded the evoked and spontaneous action potential in mPFC PVIs in PV-cre::Ai14 mice through whole-cell voltage clamp (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). PVIs of stressed mice showed a significantly lower frequency of evoked action potentials in response to current injection compared with controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eG) and the interaction reflected that ES-induced suppression was mainly observed at currents greater than 200 pA. The spontaneous action potentials of PVIs were not altered by ES (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Together, these results indicate that ES specifically reduced PVI density and activity (not SST-INs) in mPFC in adolescent male mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePrefrontal PVI Activity Mediates Early-Life Stress-Induced Cognitive Deficits in Adolescent Mice\u003c/h2\u003e \u003cp\u003eHaving shown that ES elicited both cognitive deficits and mPFC PVI activity reduction in adolescent mice, in this section we examined whether the reduced mPFC PVI activity causally mediates ES-induced cognitive deficits in adolescent mice using chemogenetic or optogenetic techniques. To start with, we carried out the following experiments to mimic ES-induced reduction PVI density and activity in mPFC and to evaluate the corresponding behavioral consequences.\u003c/p\u003e \u003cp\u003eFirst, a loss-of-function experiment was performed to selectively ablate mPFC PVIs using PV-Cre mice by injecting adeno-associated virus (AAV) expressing Cre-dependent Casp3 (a cell apoptosis effector molecule) into the mPFC at PND22 (Fig. S4A-B). This manipulation resulted in cognitive deficits, including lower discrimination indices in the TOM test (Fig. S4C) and in the novel object recognition test (Fig. S4E), and higher error rates of SAR in the Y-maze spontaneous alternation test (Fig. S4D). The spatial object recognition task was not affected by mPFC PVIs ablation (Fig. S4F). Anxiety-like behaviors in three tasks were also largely unaffected (Fig. S4G-I), except for reduced time spent in the light box in the light-dark box test (Fig. S4H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSecond, we inhibited mPFC PVI activity with a chemogenetic DREADDs manipulation, i.e., by bilateral injection of the AAV vector carrying the Cre-dependent hM4Di (Gi) into the mPFC in PV-Cre mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). Immunofluorescence staining combined with colocalization verified that the CNO group showed significantly lower percentage of virus-infected PVIs that co-express c-fos, compared with the Veh group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), indicative of PVI activity inhibition. This manipulation again resulted in cognitive deficits in TOM and Y-maze spontaneous alternation tests. In the TOM task (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), mice in the CNO group showed reduced discrimination index than the vehicle group and they were unable to discriminate the \u0026ldquo;remote\u0026rdquo; object and the \u0026ldquo;recent\u0026rdquo; object. In the Y-maze spontaneous alternation test (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), CNO-treated mice showed higher error rates of SAR; no group differences were observed in SA or AAR. The novel object recognition and spatial object recognition tasks were not significantly affected by CNO treatment (Fig. S5A-B). CNO treatment did not affect anxiety-like behaviors in the open field test (Fig. S5C), but reduced the time spent in the light box in the light-dark box test (Fig. S5D) and the time spent in the open arms in the elevated plus maze (Fig. S5E), indicative of increased anxiety levels following PVI inhibition in mPFC, which are consistent with previous findings [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOptogenetic manipulation was then carried out to validate the above-mentioned chemogenetic results (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-I and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003eS6\u003c/span\u003eA-B). As the previous two experiments support the consistent involvement of mPFC PVIs in the TOM and Y-maze spontaneous alternation tests, these two behavioral tests were carried out in the following experiments. Optogenetic inhibition of mPFC PVIs disrupted temporal order memory and spatial working memory (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eH-I). In the TOM task (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eH), eNpHR3.0-infected mice failed to discriminate the \u0026ldquo;remote\u0026rdquo; object and the \u0026ldquo;recent\u0026rdquo; object and exhibited lower discrimination index than control mice. In the Y-maze spontaneous alternation test (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eI), eNpHR3.0-infected mice displayed more errors of alternate arm return than EGFP-infected mice; no group differences were found for SA or SAR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, based on the three experiments above showing that PVI inhibition reproduced ES-induced impairments of PFC-dependent cognitive functions, we continued to investigate whether upregulating mPFC PVI activity could reverse the cognition-impairing effects of ES by bilateral injection of the AAV vector carrying the Cre-dependent hM3Dq (Gq) into the mPFC in PV-Cre mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ-K). To test the efficiency and specificity of the DREADDs system, c-fos immunoreactivity was detected in mCherry-infected neurons (Fig. S7A) and PVIs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eL) after a single CNO dose. The percentage of mCherry-infected neurons co-labeled with c-fos was significantly elevated by DREADDs (Fig. S7A). For the immunofluorescence staining for c-fos and PV co-labeling, two-way ANOVA revealed a significant stress \u0026times; drug interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eL). Further group comparisons revealed that c-fos expression in PVIs were decreased by ES, which was reversed by DREADDs. In terms of behavioral consequences, for the TOM task, two-way ANOVA revealed a significant stress \u0026times; drug interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eM). Selective activation of mPFC PVIs restored ES-induced impairment. No stress or CNO effects were observed in total probe time or total distances traveled in the acquisition phase (Fig. S7B). For the Y-maze spontaneous alternation task (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eN and Fig. S7C), two-way ANOVA showed significant stress \u0026times; CNO interactions for SA and SAR. The negative stress effects induced by ES were attenuated by activation of mPFC PVIs. These results indicate that mPFC PVI upregulation is sufficient to alleviate ES-induced cognitive deficits in temporal order memory and spatial working memory.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBy selectively downregulating and upregulating mPFC PVI activity, the four experiments above provide causal evidence that mPFC PVI activity mediates ES-induced cognitive deficits in adolescent mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePrefrontal Pyramidal Neurons Are Involved in Early-Life Stress-Induced Cognitive Deficits through PVI\u003c/h2\u003e \u003cp\u003eHow does ES reduce mPFC PVI activity in adolescent male mice? The functional maturation of the PVIs during adolescence involves several mechanisms [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], including increased glutamatergic inputs, increased density of perineuronal net (PNN) proteins, etc. PNNs as one component of the extracellular matrix preferentially surround PVIs and modulate their excitability [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. We quantified the expression of PNNs in stressed and control mice at PND35 and did not observe significant group differences (Fig. S8A). As pyramidal neurons (PNs) form robust functional synapses onto PVIs, it is possible that ES may first reduce the PN activity, which in turn limits the excitatory inputs to PVIs. So, we examined the effects of ES on the excitatory inputs onto PVIs and the PN activity. The excitatory inputs onto PVIs were quantified by the expression levels of vesicular glutamate transporter-1 (VGluT1, responsible for loading glutamate into synaptic vesicles for future release [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and involved in the regulation of excitatory neurotransmission [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] at different distances from the soma of PVIs (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). Two-way ANOVA revealed a main effect of stress for VGluT1 fluorescence intensity as well as a significant stress \u0026times; distance interaction, which was driven by decreased VGluT1 expression in stressed group at the distances larger than 8.784 um. We also quantified the soma radius of PVIs, which was not significantly affected by ES and approximately 8.662\u0026thinsp;\u0026plusmn;\u0026thinsp;1.211 um (Fig. S8B), which is consistent with VGluT1 result above to suggest that ES-induced excitatory input reduction occurred surrounding the soma of PVIs. We then measured the mPFC PN activity during TOM test using immunofluorescence staining combined with co-localization of c-fos and CamkIIa or neurogranin (two excitatory neurons markers [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]). Compared with control mice, stressed mice showed reduced density of neurons showing the co-labeling of c-fos and CamkIIa (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) or neurogranin (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), indicative of ES-induced inhibition of PN activity. To examine the causal involvement of PNs in ES adverse effects in adolescent mice, we carried out the following chemogenetic experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFirst, we tested whether inhibiting PN activity in adolescent mice could reproduce the cognitive deficits of ES (Fig. S9A-F). Specifically, we injected the AAV vector expressing Cre-dependent hM4Di (Gi) bilaterally into the mPFC of CamkIIa-Cre mice. Immunofluorescence staining for the co-labeling of mCherry\u003csup\u003e+\u003c/sup\u003e and c-fos\u003csup\u003e+\u003c/sup\u003e (Fig. S9B) showed that, compared with the Veh group, the CNO group showed decreased percentage of neurons co-expressing mCherry\u003csup\u003e+\u003c/sup\u003e and c-fos\u003csup\u003e+\u003c/sup\u003e. In terms of cognitive performances, in the TOM task, CNO-treated mice failed to discriminate the \u0026ldquo;remote\u0026rdquo; object and the \u0026ldquo;recent\u0026rdquo; object, and exhibited lower discrimination index (Fig. S9C). In the Y-maze test, CNO-treated mice showed lower SA and higher error rates of alternate arm return compared with Veh-treated mice (Fig. S9E). No significant group differences were observed in the test phase of TOM task (Fig. S9D) or the total arm entries in the Y-maze test (Fig. S9F). That is, selective inhibition of mPFC PNs reproduces ES-induced deficits of temporal order memory and spatial working memory.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSecond, we investigated whether activating PNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) could reverse ES-induced cognitive impairment. Immunofluorescence staining showed that the virus-infected (mCherry\u003csup\u003e+\u003c/sup\u003e) neurons co-labeled with excitatory neurons marker CamkIIa (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eG) and that the percentage of mCherry-infected neurons co-labeled with c-fos was significantly elevated by DREADDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-I), indicative of selective activation of PNs \u003cem\u003ein vivo\u003c/em\u003e. For the cognitive tests, we observed a significant stress \u0026times; CNO interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ) in the TOM task in that PN activation restored the discrimination index decreased in ES-treated mice. No stress or CNO effects were observed in the test phase (Fig. S10A). For the Y-maze spontaneous alternation task (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eK), two-way ANOVA showed significant stress \u0026times; drug interactions on SA and SAR. The negative stress effects on these measures were attenuated by activation of prefrontal PNs. No differences were observed in total arm entries (Fig. S10B). That is, selective activation of mPFC PNs reverses ES-induced deficits of temporal order memory and spatial working memory.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the above-mentioned results that ES reduced the functional activity of both PNs and PVIs and the excitatory inputs onto PVIs and that both types of neurons are causally involved in ES-induced cognitive impairment in adolescent mice, we then hypothesized that the cognition-improving effects of PN activation may be mediated by PVI. To test this hypothesis, we carried out an experiment to activate PNs and inhibit PVIs in mPFC and examined whether PVI inhibition could block the reversal effect of PN activation on ES-induced cognitive deficits. The double chemogenetic manipulation was achieved by bilateral injection of a mixture of two viruses (AAV-CamkIIa-Gq; Cre-dependent AAV-DIO-Gi) into the mPFC in adolescent PV-Cre mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eL). Immunofluorescence staining for mCherry, PV, and c-fos was performed to examine the c-fos-positive neurons that are mCherry\u003csup\u003e+\u003c/sup\u003e (non-PV\u003csup\u003e+\u003c/sup\u003e) and PV\u003csup\u003e+\u003c/sup\u003e for validation. For mCherry\u003csup\u003e+\u003c/sup\u003e (non-PV\u003csup\u003e+\u003c/sup\u003e) \u0026amp; c-fos\u003csup\u003e+\u003c/sup\u003e neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eM), we observed both the main effect of CNO and the stress \u0026times; CNO interaction. Post hoc tests with Bonferroni correction showed that activation of PNs was observed in both control and stressed mice. For PV\u003csup\u003e+\u003c/sup\u003e \u0026amp; c-fos\u003csup\u003e+\u003c/sup\u003e neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eN), we also observed both the main effect of CNO and the stress \u0026times; CNO interaction. That is, PVI activity was inhibited in control mice after CNO administration and was reduced in ES-exposed vehicle mice; CNO did not further downregulate the PVI activity in stressed mice. For cognitive effects, in the TOM task (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eO), two-way ANOVA revealed a tendency for main effect of CNO and stress \u0026times; CNO interaction. That is, selective inhibition of mPFC PVIs blocked the reversal effect of activation of PNs on the TOM impairment induced by ES. Besides, the double chemogenetic manipulation significantly reduced the discrimination index in control mice, which resembled the previous results of PVI inhibition. No stress or CNO effects were observed in the total probe time and distance traveled (Fig. S10C). For the Y-maze spontaneous alternation task (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eP), two-way ANOVA showed a significant main effect of stress on SA, indicating the absence of the reversal effects of PN activation after PVI inhibition. Main effects of CNO were also observed for AAR and SAR in that CNO significantly increased AAR and decreased SAR. No differences were observed in total arm entries (Fig. S10D). Together, these results indicate that inhibition of the PVIs in mPFC could block the reversal effect of the PN activation on ES-induced deficits in temporal order memory and spatial working memory, supporting the indispensable role of PVIs in the cognition-improving effects of PNs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCRHR1 Downregulation Reverses Early-Life Stress-Induced Cognitive Deficits in Adolescent Mice by Restoring PVI Activity in mPFC\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur previous studies have highlighted the critical role of the CRH-CRHR1 system in ES-induced behavioral and neural abnormalities [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], including corticolimbic neurons [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], in postnatal and adult mice. Here we examined whether the CRH-CRHR1 system is involved in ES-induced negative effects in adolescent mice. As CRHR1 has been found to be mainly expressed in PNs in cortex [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], we first validated the localization of \u003cem\u003eCrhr1\u003c/em\u003e mRNA in the mPFC using single molecule fluorescence in situ hybridization (RNAscope ISH). Colocalization of \u003cem\u003eCrhr1\u003c/em\u003e mRNA with \u003cem\u003eSlc17a7\u003c/em\u003e (the mRNA of VGluT1, an excitatory neuron marker) and \u003cem\u003eSlc32a1\u003c/em\u003e (the mRNA of GABA vesicular transporter, an inhibitory neuron marker) in the mPFC (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) demonstrated that \u003cem\u003eCrhr1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e neurons mainly co-localized with \u003cem\u003eSlc17a7\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e neurons (2313 out of 2800 \u003cem\u003eCrhr1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e neurons, 82.61% and out of 2489 \u003cem\u003eSlc17a7\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e neurons, 92.93%). Only a small fraction of \u003cem\u003eCrhr1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e neurons co-localized with \u003cem\u003eSlc32a1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e neurons (43 out of 2800 \u003cem\u003eCrhr1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e neurons, 1.54% and out of 520 \u003cem\u003eSlc32a1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e neurons, 8.27%). This observation confirmed that \u003cem\u003eCrhr1\u003c/em\u003e mRNA is primarily expressed in pyramidal, not inhibitory, neurons, in the mPFC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then investigated whether blocking the mPFC CRH-CRHR1 system could reverse ES-induced deficits on PVIs and temporal order memory. We constructed an AAV vector carrying sgRNA targeting \u003cem\u003eCrhr1\u003c/em\u003e to achieve CRISPR-Cas9-mediated deletion of CRHR1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Five gRNAs were screened using in vitro cellular assays and the sgRNA3 sequence showing the most effective transfection was chosen for packaging (Fig. S11A). HA tag staining confirmed that the virus was mainly infected in the mPFC (Fig. S11B). For the TOM task, significant reversal effects were observed: CRHR1 deletion restored temporary order memory impaired by ES (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). No significant effects of ES or CRHR1 deletion were observed in the test phase (Fig. S11C). For the PVI activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E), two-way ANOVA revealed a significant stress \u0026times; virus interaction in the mPFC: CRHR1 deletion in the mPFC reversed ES-induced reduction of PVI activity. Furthermore, discrimination index in the TOM task significantly correlated with PVI activity in the mPFC across all the animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Together, these results support our hypothesis that early-life stress may first upregulate CRH-CRHR1 signaling in prefrontal PNs, which reduces PN activity, inhibits excitatory inputs to PVIs, and in turn downregulates PVI activity and leads to cognitive impairments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMotivated by the recently established association between the CRH-CRHR1 system and cognition in the literature [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], we then carried out two pharmacological intervention experiments using the CRHR1 antagonist, antalarmin. In the first experiment, intraperitoneal injection of antalarmin was performed daily during stress procedure (PND2-8, Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). The TOM task and the activity of PVIs were then assessed in adolescent mice. For TOM, two-way ANOVA revealed a significant stress \u0026times; drug interaction, as antalarmin restored the ES-induced reduction of discrimination index (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). No significant group differences were observed in the test stage (Fig. S11D). For PVI activity in the mPFC, immunofluorescence staining for c-fos and PV showed significant main effects of stress and antalarmin, without stress \u0026times; drug interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003eI-J). That is, the percentage of PVIs co-labeled with c-fos was significantly reduced by ES and upregulated by antalarmin. Similar ES and antalarmin effects were observed for the density of neurogranin\u003csup\u003e+\u003c/sup\u003e neurons co-labeled with c-fos (Fig. S11E-F). Furthermore, discrimination index in the TOM task significantly correlated with PVI (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003eK) and PN (Fig. S11G) activity in the mPFC. In the second experiment, to examine the acute pharmacological effects of antalarmin, the drug was given 30 minutes prior to the TOM test in adolescent stressed and control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003eL). Similar stress \u0026times; drug interactions were observed for TOM and mPFC PVIs. Acute antalarmin injection blocked ES-induced reduction of discrimination index (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003eM and \u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003eS11\u003c/span\u003eH) and of mPFC PVIs (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003eN-O). Significant correlation between the discrimination index and PVI activity in the mPFC was also observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003eP).\u003c/p\u003e \u003cp\u003eTo further test the hypothesis that the cognition-improving effects of antalarmin may be mediated by PVI activity in the mPFC, we carried out an experiment to examine whether PVI inhibition could block the reversal effects of acute antalarmin treatment on ES-induced cognitive deficits (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003eQ-R). For PVI activity in the mPFC in stressed mice receiving antalarmin treatment, immunofluorescence staining for c-fos and PV showed that the percentage of PVIs co-labeled with c-fos (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003eO) was significantly reduced by DREADDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003eS). In the TOM task, compared with the stressed mice that received antalarmin treatment and exhibited intact temporal order memory, the CNO mice could not discriminate the \u0026ldquo;remote\u0026rdquo; object from the \u0026ldquo;recent\u0026rdquo; object and had significantly lower percentage time exploring the \u0026ldquo;remote\u0026rdquo; object (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003eT), indicating that the reversal effect of antalarmin on ES-induced TOM impairment (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003eM) was blocked by mPFC PVI inhibition. Together, these pharmacological experiments indicate that CRHR1 blockade could successfully reverse ES-induced temporal order memory deficits by restoring mPFC PVI activity, supporting the therapeutic potentials of antalarmin on ES-related cognitive impairments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEnvironmental Enrichment Alleviates Early-life Stress-induced Cognitive Deficits through Activation of Prefrontal PNs and PVIs in Adolescent Mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBesides pharmacological intervention, here we also tested whether EE, a commonly used non-pharmacological intervention for animals exposed to ES, could reverse ES-induced negative effects in adolescent mice. Stressed and control mice were exposed to three-week enriched or standard housing environments after weaning (PND21-42) and were then tested in the TOM task (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, despite lack of significant main effects and interactions in the two-way ANOVA, only the stressed mice kept in a standard environment failed to distinguish the \u0026ldquo;remote\u0026rdquo; object from the \u0026ldquo;recent\u0026rdquo; object, while mice in the other three groups exhibited intact recognition memory, which indicates that EE partially reversed ES-induced temporal order memory deficits.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then investigated the neural correlates underlying EE cognition-improving effects by measuring the activity of PNs and PVIs in mPFC. Regarding the density of neurogranin\u003csup\u003e+\u003c/sup\u003e cells that are c-fos\u003csup\u003e+\u003c/sup\u003e (indicative of the PN activity), we found a significant stress \u0026times; environment interaction in the mPFC (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-E). Specifically, ES significantly reduced neurogranin\u003csup\u003e+\u003c/sup\u003e neuron activity, which was reversed by environment enrichment. Similar with the results of PNs, for the density of PVIs that are c-fos\u003csup\u003e+\u003c/sup\u003e (indicative of the PVI activity), we also observed a significant stress \u0026times; environment interaction (6G-H). ES significantly reduced PVI activity and EE significantly upregulated the activity of PVIs in stressed mice. Importantly, the discrimination indices in the temporal order memory test were significantly correlated with both the density of activated PNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003eF) and PVIs (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003eI) in the mPFC.\u003c/p\u003e \u003cp\u003eThese data support the beneficial effects of EE in alleviating ES-induced temporal order memory deficits and reduced activity of PNs and PVIs.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we examined whether and how mPFC PVIs causally mediate ES-induced cognitive deficits in adolescent mice. We first demonstrated that exposure to resource scarcity environment in early-life led to selective reduction of PVI activity in the mPFC in adolescent male mice, along with cognitive deficits in temporal order memory and spatial working memory. Ablating or inhibiting PVIs in the mPFC in adolescent mice phenocopied the deficits observed in ES-exposed mice, and activating PVIs rescued ES-induced cognitive deficits, supporting the causal relationship between PVI activity and cognitive deficits in mice exposed to ES. We further demonstrated that ES also reduced excitatory inputs onto PVIs and the PN activity in the mPFC and that PN activity causally contributed to cognitive deficits, which required the activation of PVIs. In addition, genetic knockout of CRHR1 (mainly expressed in PNs) in the mPFC in adolescent mice improved ES-induced cognitive impairment and PVI activity reduction. These results collectively support the prefrontal CRHR1\u0026rarr;PN\u0026rarr;PVI pathway in mediating the adverse effects ES on cognition in adolescent mice. Finally, our intervention experiments revealed the beneficial effects of pharmacological (antalarmin) and non-pharmacological (EE) treatment on ES-induced cognitive deficits and mPFC PVI and PN activity, providing insight into early treatment and prevention of cognitive impairments in stress-related psychiatric diseases.\u003c/p\u003e \u003cp\u003eWhile the effects of ES on cognition have been extensively studied in adult animals in our own and other research groups [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], it is worth highlighting that several recent studies have started to examine this issue in adolescent rodents. Studies with maternal separation or deprivation models reported that ES impaired spatial learning and memory in morris water maze test[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], novel object recognition memory [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], and temporary order memory [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] in adolescent rodents. To our knowledge, only one study has used the LBN paradigm and found that exposure to LBN during PND4-11 led to spatial object recognition memory loss in adolescent male mice [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Different from these studies adopting one cognitive task for a given model, we tested the effects of the LBN paradigm in adolescent mice on a larger battery of cognitive tasks, including temporary order memory, spatial working memory, novel object recognition, and spatial object recognition. We found that ES-induced cognitive impairments we observed in adult mice [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] are already present in adolescence. Intriguingly, unlike previous studies showing both cognitive and emotional deficits following ES [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], here we observed cognitive deficits only, without significant alterations of anxiety-like, depression-like or social behaviors, which are consistent with a previous study [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Some LBN studies did report emotional alterations in adolescent animals, such as reduced sucrose preference [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], increased immobility time in FST test [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], and increased anxiety-like behaviors [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The inconsistency among these studies may be related to species (mouse or rat), stress mode, and stress exposure time. Together, our observation of ES-induced cognitive deficits in adolescent mice provides further evidence that the LBN paradigm is a valid model for the early onset of cognitive impairments, one of the core symptoms of psychiatric disorders, and for studying the underlying neural mechanisms.\u003c/p\u003e \u003cp\u003eConsidering the pivotal role of PVIs in mPFC-dependent cognitive behaviors and their prolonged maturation during adolescence, we hypothesized that PVIs may causally mediate ES-induced cognitive deficits we observed. We first examined the effects of ES on mPFC PVI activity. As mentioned in Introduction, current studies have largely focused on the effects of ES on the number (density) of PVIs or the expression of \u003cem\u003ePV\u003c/em\u003e mRNA or PV protein, some of which reported downregulation in adolescent animals [\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. To our knowledge, direct evidence is still lacking regarding how ES affects the functional activity of prefrontal PVIs. One recent study found that ES (maternal separation and early weaning) downregulated evoked action potentials of mPFC GABAergic neurons in adult mice [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], yet it remains unknown which type of interneurons was affected by ES and whether such effects emerge in adolescence. In this study, by the immediate early gene c-fos colocalization technique we demonstrated that ES significantly decreased the number of activated PVIs, not SST-INs, during TOM test in adolescent mice, which was validated by electrophysiological findings of the lower frequency of evoked action potentials in PVIs of stressed mice. These results indicate for the first time that ES specifically reduces PV-expressing (not SST-expressing) interneuron activity in mPFC in adolescent male mice.\u003c/p\u003e \u003cp\u003eNext, regarding the link between reduced mPFC PVI activity and cognitive deficits in adolescent mice exposed to ES, previous studies in adult animals (not adopting the ES models) have provided supporting evidence. For example, inactivation of prefrontal PVIs impaired prefrontal-dependent cognitive abilities, including spatial working memory, reversal learning[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], rule-shift learning[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], whereas enhancing the activity of PVIs in the mPFC could alleviate these cognitive deficits in various animal models [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Two recent studies in adolescent animals also suggest the crucial involvement of mPFC PVIs in cognitive behaviors. One study found that sustained inhibition of prefrontal PVIs activity during adolescence disrupted cognitive flexibility in adult mice [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The other study reported that selective activation of prefrontal PVIs during adolescence rescued deficits in novel object recognition induced by chronic MK801 treatment [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Here, by manipulating mPFC PVI functional activity through chemogenetic and optogenetic methods, we found that ablating or inhibiting PVIs in the mPFC in adolescent mice mimicked the deficits observed in ES-exposed mice, and activating PVIs rescued ES-induced cognitive deficits. These results provide causal evidence that mPFC PVI activity mediates the cognitive deficits induced by ES.\u003c/p\u003e \u003cp\u003eFinally, to understand the mechanisms by which ES modulates functional activity changes in PVIs during adolescence, we targeted at the CRH-CRHR1 system and PNs. As mentioned in Introduction, the association between the CRH-CRHR1 system and PFC-dependent cognitive dysfunction has been highlighted in recent clinical and animal studies [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. For PNs, numerous studies have reported that in adult animals, ES reduces their functional activity (measured as the action potential frequency, amplitude and frequency of excitatory postsynaptic currents [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]) and impairs their structural plasticity (e.g., dendritic retraction and spine loss [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]). Considering that CRHR1 is mainly expressed in excitatory neurons rather than inhibitory neurons in mPFC[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], we speculate that ES may first act on CRHR1 on PNs, reducing PN neuronal activity and their excitatory inputs to PVIs, leading to decreased activity of PVIs and ultimately leading to cognitive impairment in adolescent mice. This speculation was supported by the following experimental results: 1) we observed decreased excitatory inputs onto PVIs (measured by reduced VGluT1 expression levels) and decreased activation of mPFC PNs during the TOM task. 2) Inhibition of mPFC PNs mimicked ES-induced TOM damage and activation of mPFC PNs reversed ES-induced cognitive impairments; this reversal could be blocked by inhibiting mPFC PVI activity, indicating that PVIs play indispensable roles in the pyramidal neuron modulation. 3) ES-induced cognitive impairment and PVI activity reduction could be restored by genetic knockout of CRHR1 in the mPFC in adolescent mice. Together, these results indicate that the prefrontal CRHR1\u0026rarr;PN\u0026rarr;PVI pathway may underlie the cognitive deficits in adolescent mice exposed to ES.\u003c/p\u003e \u003cp\u003eFor cognitive impairment, the efficacy of current first-line therapeutic drugs and non-pharmacological treatments such as psychotherapy and physical therapy (e.g., rTMS, DBS) are limited [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and the effective measures for early intervention are still lacking in clinical practice[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Here targeting at ES-induced cognitive deficits and mPFC PVI activity reduction, we examined the efficacy of pharmacological (i.e., antalarmin, a CRHR1 antagonist) and non-pharmacological (i.e., environmental enrichment) interventions. There were several clinical trials on drugs targeting the CRH-CRHR1 system; they have largely focused on improving symptoms of depression and anxiety, not cognition, and have ended in failure [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Our series of studies have consistently shown the cognition-improving effects of antalarmin in animal models of early-life stress in adulthood, such as PFC-dependent temporary order memory and spatial working memory [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], and hippocampus-dependent cognitive changes [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In this study, we found that either co-administration or acute intraperitoneal injection of the CRHR1 antagonist antalarmin could reverse ES-induced cognitive impairment and mPFC PVI activity downregulation in adolescent mice. These results highlight the therapeutic potential of CRHR1 antagonism on cognitive dysfunctions (compared with emotional symptoms) of stress-related psychiatric disorders and also support its potential in early intervention.\u003c/p\u003e \u003cp\u003eCompared with medications, non-pharmacological interventions for psychiatric disorders have advantages in terms of fewer adverse effects and better acceptability. EE is a commonly used non-pharmacological intervention for animals exposed to ES and has beneficial effects on ES-induced behavioral and neural changes [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. For cognitive impairment, two studies using maternal separation have reported the beneficial effects of EE during adolescence (i.e., reversal learning[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], spatial working memory [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]). Our study found that cognitive impairment caused by LBN can also be reversed by EE. More importantly, EE significantly upregulated mPFC PVI activity, similar to antalarmin intervention, which may underlie its cognitive improvement effect. Previous studies have shown that inhibition of PVIs could block the improvement of EE on cognitive deficits [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e], supporting the hypothesis that PVIs mediate the cognitive improvement of EE. Taken together, these intervention experiments indicate that mPFC PVIs could serve as a potential target for early intervention in both pharmacological and non-pharmacological treatments of cognitive impairment.\u003c/p\u003e \u003cp\u003eIn summary, our study uncovers the crucial role of prefrontal PVIs, along with PNs and CRHR1, in mediating cognitive deficits induced by early-life stress in adolescent male mice. Our findings also suggest that interventions to enhance mPFC PVI activity could ameliorate the cognitive impairment of stress-related psychiatric disorders, which should be highlighted in developing early treatment and prevention strategies.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eAdult C57BL/6 (10\u0026ndash;12 weeks old) male and female mice were purchased from Vital River Laboratories (Beijing, China). The mice were transferred to individually ventilated cages (IVC) and 3\u0026ndash;4 mice were housed normally. The Pvalb\u003csup\u003e\u003cem\u003etm1(cre)Arbr\u003c/em\u003e\u003c/sup\u003e/J (PV-Cre) mice express Cre recombinase in parvalbumin-expressing interneurons (PVI) [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. The Gt (ROSA)26Sor\u003csup\u003e\u003cem\u003etm14(CAG\u0026minus;tdTomato)Hze\u003c/em\u003e\u003c/sup\u003e/J (Ai14) mice express robust tdTomato fluorescence following Cre-mediated recombination [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. The Tg (Camk2a-cre)\u003csup\u003e\u003cem\u003eT29\u0026minus;1Stl\u003c/em\u003e\u003c/sup\u003e/J (CamKIIa-Cre) mice express Cre recombinase in pyramidal neurons (PNs) in the forebrain [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. All transgenic mice were purchased from the Jackson Laboratory (CA, USA) and maintained fully back-crossed onto C57BL/6 mice, and adult male heterozygous mice were used.\u003c/p\u003e \u003cp\u003eFor breeding, male and female mice were mated 1:2 for 2 weeks and separated. Pregnant females were monitored daily for pup delivery, and the day of parturition was defined as postnatal day 0 (PND0). Only male offspring were used in the following experiments. All mice were housed under a 12-h light/dark cycle (lights on at 8:00 a.m.) and constant temperature (23\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃) conditions with plenty of food and water. All experiments were approved by the Peking University Committee on Animal Care, and they were performed in compliance with the NIH\u0026rsquo;s Guide for the Use and Care of Laboratory Animals.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eEarly-life stress paradigm\u003c/h2\u003e \u003cp\u003eThe limited nesting and bedding paradigm was used as an early-life stressor and was conducted as previously described [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The procedure was as follows: At PND2, the number of pups per cage was adjusted to ensure 6\u0026ndash;8 pups with a male-to-female ratio of 1:1. Control dams received 500 ml of sawdust bedding and 4.8 g of nesting material (2 squares of Nestlets, Ancare, New York, USA), while in the \"stress\" cages, dams were provided with a fine-gauge aluminum mesh platform (McNichols, Tampa, FL, USA) with 200 mL of corncob bedding on the bottom to collect droppings. A limited amount of nesting material [1/2 square (1.2 g) nestlets] was put on top of the mesh. After one week of stress treatment (PND2-9), all mice were returned to standard environment. Male offspring were weaned on PND21 and housed in groups of 3\u0026ndash;4 per cage for further study.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral Assays\u003c/h2\u003e \u003cp\u003eA battery of behavioral tests, including cognitive behaviors, anxiety-like behaviors, depressive-like behaviors and social approach, were performed in adolescent mice (PND35-42) between 09:00 and 15:00 as described previously [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Mice were handled for at least 3 days prior to behavioral testing. Behavioral data in the open field, elevated plus maze, and light-dark box tasks were automatically analyzed by ANY-maze 7.0 (Stoelting, Wood Dale, IL, USA). The remaining behavioral tests were scored by an experimenter blind to experimental conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTemporary Order Memory Test\u003c/h2\u003e \u003cp\u003eThe temporary order memory (TOM) test was to assess animals\u0026rsquo; ability to differentiate between two familiar objects presented at different time intervals, which relies on the medial prefrontal cortex (mPFC) [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. The test was carried out in the open field arena illuminated at 10 lux, including 3 trials with 1-hour intertrial interval (ITI). The first two trials were the acquisition trials where two identical triangular prisms or triangular pyramids were placed in the arena and the animals were allowed to freely explore the arena for 10 min. In the test phase, a triangular prism (the \u0026ldquo;remote\u0026rdquo; object) and a triangular pyramid (the \u0026ldquo;recent\u0026rdquo; object) were placed in the arena. During the 10 min test phase, the discrimination index was calculated as: 100% \u0026times; time probing the \u0026ldquo;remote\u0026rdquo; object /time probing both objects.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eNovel Object Recognition Test\u003c/h2\u003e \u003cp\u003eThe novel object recognition (NOR) task was designed to assess recognition memory based on the familiarity with the object itself, which depends on several brain regions, including the perirhinal cortex, the hippocampus, and mPFC [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. The NOR was also performed in the open field arena, illuminated at 10 lux, and consisted of 2 trials separated by an ITI of 1 hour. During the acquisition phase, two identical cubes were presented. In the test phase, one of the cubes (the \u0026ldquo;familiar\u0026rdquo; object) was replaced by a hexagonal column (the \u0026ldquo;novel\u0026rdquo; object). During the 10 min test phase, the discrimination index was calculated as: 100% \u0026times; time probing the \u0026ldquo;novel\u0026rdquo; object /time probing both objects.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSpatial Object Recognition Test\u003c/h2\u003e \u003cp\u003eThe spatial object recognition (SOR) task was designed to assess the animals\u0026rsquo; ability to detect the location changes of familiar objects, which depends on the hippocampus [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. The test was conducted in the open field arena with a 10-lux illumination and consisted of two acquisition trials and one test trial with 1-hour ITI. During the first two acquisition trials, two identical objects (cylinders) were placed approximately 15 cm apart. One hour later, one object was moved diagonally (the \u0026ldquo;displaced\u0026rdquo; object), and the other object was left in its original position (the \u0026ldquo;stationary\u0026rdquo; object). During the 10-min test phase, the discrimination index was calculated as: 100% \u0026times; time probing the \u0026ldquo;displaced\u0026rdquo; object /time probing both objects.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eY-maze Spontaneous Alternation Test\u003c/h2\u003e \u003cp\u003eY-maze spontaneous alternation task was designed to assess spatial working memory [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. The Y-maze apparatus consisted of gray polyvinyl chloride with three symmetrical arms (30 \u0026times; 10 \u0026times; 15 cm\u003csup\u003e3\u003c/sup\u003e, 10 lux) with spatial cues surrounding the maze. Mice were placed in the end of one arm and were allowed to explore freely for 8 min. The percentage of spontaneous alternations (SA: A\u0026rarr;B\u0026rarr;C), alternative arm returns (AAR: A\u0026rarr;B\u0026rarr;A) and same arm returns (SAR: A\u0026rarr;A) were recorded manually.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eOpen Field Test\u003c/h2\u003e \u003cp\u003eOpen field test was performed in a gray polyvinyl chloride chamber (50 cm \u0026times; 50 cm \u0026times; 50 cm) with smooth interior walls and evenly illuminated at 60 lux. During the test, mice were placed in one corner, facing the wall and permitted to explore the environment freely for 10 min. Time spent in the center area of the open field (20 cm in diameter), the latency and the number of entries to the center area were measured to reflect animals\u0026rsquo; anxiety levels. The total distance traveled was also quantified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eElevated plus maze test\u003c/h2\u003e \u003cp\u003eElevated plus maze test was carried out in an elevated plus maze, consisting of a central platform (5 \u0026times; 5 cm\u003csup\u003e2\u003c/sup\u003e) with two opposing open arms (30 \u0026times; 5 \u0026times; 0.5 cm\u003csup\u003e3\u003c/sup\u003e, 40 lux) and two opposing closed arms (30 \u0026times; 5 \u0026times; 15 cm\u003csup\u003e3\u003c/sup\u003e, 10 lux) extending from it in a plus shape. The maze was elevated 50 cm above the floor. Mice were individually placed in the center with their heads facing a closed arm and allowed to explore for 5 min. Time spent in open arms, the latency and the number of entries to open arms were recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eLight-dark box test\u003c/h2\u003e \u003cp\u003eLight-dark box test was performed in a plastic box containing a dark chamber (15 \u0026times; 20 \u0026times; 25 cm\u003csup\u003e3\u003c/sup\u003e, 10 lux) and a brightly illuminated chamber (30 \u0026times; 20 \u0026times; 25 cm\u003csup\u003e3\u003c/sup\u003e, 700 lux), connected by a 4-cm long tunnel. Mice were placed in the dark chamber, facing the other chamber. The time spent in the light chamber during 5 min, the latency and the number of entries to the light chamber were measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTail Suspension Test\u003c/h2\u003e \u003cp\u003eThe test was carried out in a plastic enclosure (15 cm \u0026times; 17 cm \u0026times; 50 cm). Mice were suspended by the distal end of the tail for 6 min using a suspension hook. Mice were considered immobile when they were passively suspended and completely immobile. The total time spent immobile and the latency of immobilization were manually scored for each animal.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eForced Swimming Test\u003c/h2\u003e \u003cp\u003eMice were individually placed for 6 min in a transparent cylinder (25 cm high, 10 cm diameter) filled with water to a depth of 18 cm and maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃. After the test, the mouse was wiped dry with a towel and returned to its home cage. Mice were considered immobile if they did not make any active movements. The total immobility time was recorded and analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eSucrose Preference Test\u003c/h2\u003e \u003cp\u003eFour days prior to the test, the mice were individually housed in home cage with two bottles of tap water to avoid place preference bias. On the fourth day, two bottles of 1% sucrose solution were given. The test began at 20:00, during which the mice were given one bottle of 1% sucrose solution and one bottle of water, and the weight of each bottle was recorded. The positions of the two bottles were changed every 12 hours, and the bottle weights were recorded at 24, 48, and 72 hours later. Sucrose preference index (%)\u0026thinsp;=\u0026thinsp;100 \u0026times; (sucrose solution consumption) / (sucrose solution consumption\u0026thinsp;+\u0026thinsp;water consumption).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eSocial Approach Test\u003c/h2\u003e \u003cp\u003eSocial approach test was performed in a gray polyvinyl chloride chamber (50 cm \u0026times; 50 cm \u0026times; 50 cm) illuminated at 10 lux. Mice were acclimated to the chamber with an empty wire mesh cage in the center for 10 min. One hour later, a strange mouse of the same sex and age (defined as \u0026ldquo;tool\u0026rdquo; mice) was placed in the empty wire mesh cage and then testing mouse was placed in one corner, facing the wall and allowed to explore freely for 10 min. The time spent interacting with the \u0026ldquo;tool\u0026rdquo; mouse was measured manually.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eImmunohistochemistry and Image Analysis\u003c/h2\u003e \u003cp\u003eMice were anesthetized with 2,2,2-Tribromoethanol (250 mg/kg, i.p., T48402, Sigma-Aldrich) and transcardially perfused with 0.9% saline followed by 4% buffered paraformaldehyde. Then, mouse brains were dissected, post-fixed for 10\u0026ndash;12 h at 4 ℃ in 4% buffered paraformaldehyde, and dehydrated in a 30% sucrose solution for 72 h at 4 ℃. Using a cryostat (Leica, Wetzlar, Germany), serial coronal sections through the mPFC (1.98 mm-1.54 mm from bregma) were obtained at 30-um by a 180-um interval.\u003c/p\u003e \u003cp\u003e For immunofluorescence, sections were washed into 0.1 M PBS three times (10 min each time), and permeabilized with 0.3% Triton X-100 in 0.1 M PB three times for 10 min each time, followed by 1% normal donkey serum blocking solution at room temperature for 1 h. The following primary antibodies were used: goat anti-somatostatin (1:100, sc-7819, SANTA Cruz), goat anti-parvalbumin (1:2000, PVG-213, SWANT), mouse anti-CamKIIa (1:1000, 22609, Abcam), mouse anti-parvalbumin (1:2000, PV-235, SWANT), mouse anti-VGluT1 (1:1000, 135511, Synaptic Systems), rabbit anti-parvalbumin (1:2000, PV-25, SWANT), rabbit anti-c-fos (1:1000, 2250S, Cell Signaling), rabbit anti-neurogranin (1:1000, 217672, Abcam), pig anti-c-fos (1:1000, 226005, Synaptic Systems). After rinsing in 0.1 M PB containing 0.3% Triton X-100 three times (10 min each time), sections were incubated with secondary antibody (Alexa Fluor 488-, 594- and or 647-conjugated donkey) diluted 1:500 in 1% normal donkey serum blocking solution for 2 h at room temperature. After rinsing in 0.1 M PB three times (10 min each time), sections were mounted on slides and covered with Vectashield containing 40,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA).\u003c/p\u003e \u003cp\u003eFor immunohistochemistry, sections were washed in 0.1 M PBS three times (5 min each time) and treated with 3% hydrogen peroxide (10 min), followed by 1% normal goat serum (1 h). They were labeled with rabbit anti-parvalbumin (1:20000, PV-25, SWANT) or goat anti somatostatin (1:200, sc-7819, SANTA Cruz) antibody at 4\u0026deg;C (overnight). The next day, sections were rinsed and incubated with a biotinylated goat anti-rabbit or rabbit anti-goat secondary antibody (Zhongshan Golden Bridge Biotechnology, Beijing, China) at room temperature (2 h). After rinsing, sections were stained with the 3,3\u0026rsquo;-Diaminobenzidine Horseradish Peroxidase Color Development Kit (Zhongshan Golden Bridge, ZLI-9019, Beijing, China), transferred onto slides and coverslipped with mounting medium (Zhongshan Golden Bridge, ZLI-9516, Beijing, China).\u003c/p\u003e \u003cp\u003eFor image analysis, brain sections were assigned random numbers so that investigators were blind to the experimental conditions. To count the number of PVIs and somatostatin-expressing interneurons (SST-INs) in the mPFC, images were obtained from 3 sections per animal at 10\u0026times; objective using an Olympus VS120-S6-W automated slide scanner (Olympus, Tokyo, Japan) and three subregions of mPFC (Cg, PrL, IL) were defined as ROI (Region of Interest) for analysis using NIH ImageJ software. For colocalization analysis, brain sections were captured at 20\u0026times; objective from 3 sections per animal using an Olympus FV1000 laser-scanning confocal microscope (Olympus, Tokyo, Japan). To examine the optical density of VGluT1 at different distances from the soma of PVIs, sections were acquired at 20\u0026times; objective from 3 sections per animal using an Olympus FV3000 laser-scanning confocal microscope (Olympus, Tokyo, Japan); for each group, 30\u0026ndash;40 PVIs were chosen and sholl analysis was performed using NIH ImageJ software at different distances (step by 1.0 um) from the soma of PVIs. To validate the virus- or optical fiber-target regions, images were photographed at 10\u0026times; objective using an Olympus VS200-S6-W automated slide scanner (Olympus, Tokyo, Japan). Images were adjusted for optimal brightness and contrast using FV10-ASW 4.2 software or OlyVIA 3.4.1 (Olympus, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eBrain-slice Preparation and Electrophysiological recordings\u003c/h2\u003e \u003cp\u003eAdolescent male PV::Ai14 mice (PND35-42) were anesthetized with isoflurane rapidly and decapitated. The brain slices (200 \u0026micro;m) containing mPFC were obtained using standard techniques and incubated in artificial cerebrospinal fluid (ACSF)at room temperature (20\u0026ndash;24℃) for 1 hour. The slices were then transferred to a recording chamber at room temperature (20\u0026ndash;24℃) and continuously perfused with oxygenated-standard and ACSF heated at 37℃ containing (in mM) 10 glucose, 125 NaCl, 5 KCl, 2 NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 2.6 CaCl\u003csub\u003e2\u003c/sub\u003e, 1.3 MgCl\u003csub\u003e2\u003c/sub\u003e, 26 NaHCO\u003csub\u003e3\u003c/sub\u003e (pH: 7.3\u0026ndash;7.4, osmolarity: 300\u0026ndash;310 mOsm/kg), at a rate of 2 mL/min. Target mPFC PVIs were identified by tdTomato fluorescence using an Olympus BX-51 microscope equipped with DIC optics, a water-immersion objective (\u0026times;60 NA 1.1). To measure the evoked firing of PVIs, incremental current steps (0.5 s duration, 50 pA step size) were injected through the recording pipette. Recordings were obtained with an EPC-10 amplifier and Patchmaster software (HEKA Elektronik, Lambrecht /Pfalz, Germany). Signals were analyzed with Clampfit 10.3 (Molecular Devices, Union City, CA, USA).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eStereotaxic Surgery and Viral Microinjection\u003c/h2\u003e \u003cp\u003eAdolescent mice (PND22-23) were anesthetized with isoflurane (induction 2.5%, maintenance 1-1.5%) with perioperative meloxicam analgesia (3 mg/kg, i.p.) and placed in a stereotaxic frame (RWD Life Science Co., LTD, Shenzhen, China). Mice received viral microinjections into the mPFC (250 nL per side, 30 nL/min) through a glass micropipette. The injection coordinates (relative to bregma) were anterior\u0026thinsp;+\u0026thinsp;1.9 mm, lateral\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 mm, and ventral-1.8 mm. The micropipette was left in the site for another 5 min. Mice were allowed to recover until the beginning of behavioral tests (PND35). For manipulation of PVIs in mPFC, an adeno-associated virus (AAV) of DIO-hM3Dq-mCherry (1.14 \u0026times; 10\u003csup\u003e13\u003c/sup\u003e genome copies/mL, Vigene Biotechnology, China), DIO-hM4Di-mCherry (3.00 \u0026times; 10\u003csup\u003e13\u003c/sup\u003e genome copies/mL, Vigene Biotechnology, China), DIO-eNpHR3.0-EYFP (1.81 \u0026times; 10\u003csup\u003e14\u003c/sup\u003e genome copies/mL, Vigene Biotechnology, China), flex-taCasp3-TEVp (3.04 \u0026times; 10\u003csup\u003e12\u003c/sup\u003e genome copies/mL, ObioTechnology, China), or ACSF was delivered bilaterally to the mPFC of male PV-Cre mice. For manipulation of pyramidal neurons in mPFC, an AAV virus of DIO-hM4Di-mCherry was bilaterally injected into the mPFC of male CamkIIa-Cre mice or AAV virus of CamkIIa-hM3Dq-mCherry (7.07 \u0026times; 10\u003csup\u003e13\u003c/sup\u003e genome copies/mL, Vigene Biotechnology, China) was bilaterally injected into the mPFC of male C57BL/6 mice. For simultaneous manipulation of mPFC PNs and PVIs, a mixture of CamkIIa-hM3Dq-mCherry and DIO-hM4Di-mCherry or a mixture of CamkIIa-hM4Di-mCherry and DIO-hM3Dq-mCherry were bilaterally injected into the mPFC of male PV-Cre mice. To achieve CRISPR-mediated deletion of \u003cem\u003eCrhr1\u003c/em\u003e, an AAV vector carrying sgRNA targeting \u003cem\u003eCrhr1\u003c/em\u003e (AAV2/9-CMV-SaCas9-U6-crhr1.gRNA, 7.22 \u0026times; 10\u003csup\u003e13\u003c/sup\u003e genome copies/mL, Vigene Biotechnology, China) and scrambled sgRNA (AAV2/9-CMV-SaCas9-U6-scrambled. gRNA, 4.13 \u0026times; 10\u003csup\u003e13\u003c/sup\u003e genome copies/mL, Vigene Biotechnology, China) was bilaterally injected into the mPFC of male C57BL/6 mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eChemogenetic Manipulation of Neurons\u003c/h2\u003e \u003cp\u003eClozapine-N-oxide (CNO, HY-17366, MEC) was dissolved in 100% DMSO to a storage concentration of 20 mg/mL and stored at -20℃. For chemogenetic inactivation of PNs or PVIs, storage solution of CNO was diluted into a working solution (0.3 mg/mL) and then the working solution of CNO was delivered intraperitoneally (i.p. 3 mg/kg body weight) to hM4Di transfected mice 30 min prior to each behavioral test. For chemogenetic activation of PNs or PVIs storage solution of CNO (20 mg/mL) was diluted into a working solution (0.1 mg/mL) and then the working solution of CNO was administered intraperitoneally (i.p. 1 mg/kg) to hM3Dq transfected mice 30 min prior to each behavioral test. To simultaneously manipulate PNs and PVIs, the working solution of CNO (0.3 mg/mL) was administered intraperitoneally (i.p. 3 mg/kg) 30 min prior to each behavioral test. For vehicle groups, 3 mg/kg body weight of 1.5% DMSO or 1 mg/kg body weight of 0.5% DMSO were injected intraperitoneally in the inactivation and activation experiments, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eOptogenetic Inactivation of PV-expressing Interneurons\u003c/h2\u003e \u003cp\u003eFor optogenetic inactivation of prefrontal PVIs, an AAV carrying a DIO-eNpHR3.0-EYFP or DIO-EGFP was injected bilaterally into the mPFC and optic-fiber cannula (200-um-diameter; 0.37 NA) was implanted above the mPFC with \u0026minus;\u0026thinsp;1.55-mm DV coordinate. Light was provided by a 594 nm laser diode (ThinkerTech, Nanjing, China). The light intensity at the fiber tip was measured with a light sensor (Thorlabs, Newton, NJ, USA). A 4\u0026ndash;5 mW laser pulse (ON-OFF-ON-OFF, 2 min/section) was delivered by a Master-8 pulse stimulator (AMPI, Jerusalem, Israel) through the optical fiber embedded in the mPFC.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eRNAscope in Situ Hybridization\u003c/h2\u003e \u003cp\u003eThe mPFC mRNA expression was visualized using RNAscope (Advanced Cell Diagnostics). Following the manufacturer's instructions, mouse brains were rapidly dissected, dehydrated, and frozen. After cryoprotection, serial coronal sections (bregma 1.98\u0026ndash;1.54 mm) of the mPFC at 15 \u0026micro;m thickness and 180 \u0026micro;m interval were obtained using a cryostat (Leica), which were dried at -20 ℃ for 1 h and then stored at -80 ℃ for up to one week. Then, slices were processed following the RNAscope protocol using a fluorescent multiplex reagent kit (ACD: 323100) and probes for \u003cem\u003eCrhr1\u003c/em\u003e (Mm-Crhr1-C1; ACD, catalog #418011), \u003cem\u003eSlc32a1\u003c/em\u003e (Mm-Slc32a1-C2; ACD, catalog #319198-C2), and \u003cem\u003eSlc17a7\u003c/em\u003e (Mm-Slc17a7-C3; ACD, catalog #416631).\u003c/p\u003e \u003cp\u003eFor co-localization analysis, images (1024 \u0026times; 1024 pixel\u003csup\u003e2\u003c/sup\u003e) were captured at 20 \u0026times; objective using an Olympus FV3000 laser-scanning confocal microscope (Olympus, Tokyo, Japan). Images were then separated into multiple color channels and cell nuclei were identified in the DAPI channel. Signals in the red, green, and magenta channels were thresholded, identified, and filtered by the locations of nuclei. If a signal was found in a nucleus, the cell was defined as \u0026lsquo;\u0026lsquo;positive\u0026rsquo;\u0026rsquo; for the respective RNA species. Nuclei positive for \u003cem\u003eSlc32a1\u003c/em\u003e or \u003cem\u003eSlc17a7\u003c/em\u003e were finally filtered to determine whether they co-expressed \u003cem\u003eCrhr1\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eCRHR1 Antagonist Antalarmin Treatment\u003c/h2\u003e \u003cp\u003eTo investigate the intervention effects of CRHR1 blockade on the ELS negative effects, we intraperitoneally administered the CRHR1 antagonist antalarmin (20 \u0026micro;g/g of body weight; Sigma-Aldrich, USA) or vehicle (15% β-cyclodextrin in sterile normal saline, Solarbio, Beijing, China) in two treatment strategies. First, the concurrent blockade of CRHR1 receptors during early-life stress exposure was achieved by daily intraperitoneal injections of antalarmin or vehicle at 09:00\u0026ndash;12:00 during PND2-8. The entire injection procedure did not exceed 10 min per cage to avoid maternal separation stress. Second, the acute pharmacological effects of antalarmin were tested in adolescent mice. That is, antalarmin or vehicle was injected 30 min before behavioral tests.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEnvironmental Enrichment\u003c/h3\u003e\n\u003cp\u003eStressed and control mice were exposed to environmental enrichment (EE) with 3\u0026ndash;4 mice per cage from PND 22\u0026ndash;42. EE cage is an acrylic cage with a volume of 36 cm \u0026times; 25 cm \u0026times; 60 cm, divided into 3 layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Each layer is connected by a tunnel, with facilities such as a running wheel, a swing, a pipe and a house, and toys of different shapes and colors. To ensure the novelty of the objects for the mice, the type, number, and location of the toys were changed every three days.\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eQuantification and Statistical Analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism 9.0 (GraphPad Software, Inc., USA) was used for the statistical analyses and graphing. Signals obtained from electrophysiological recordings were analyzed with Clampfit 10.3 (Molecular Devices, Union City, CA, USA). Kolmogorov-Smirnov test was used to checked for normality before \u003cem\u003et\u003c/em\u003e test, ANOVA and descriptive statistics. Comparisons between two groups were analyzed by unpaired \u003cem\u003et\u003c/em\u003e test (with same variance) or unpaired \u003cem\u003et\u003c/em\u003e test with Welch's correction (with unequal variance). Paired \u003cem\u003et\u003c/em\u003e test was used to compare the percentage of time probing two objects in the NOR, SOR and TOM test. For multiple group comparisons, data were analyzed by two-way analysis of variance (ANOVA) followed by Bonferroni\u0026rsquo;s \u003cem\u003epost hoc\u003c/em\u003e test when yielded a significant interaction. Repeated measures ANOVA was used for detecting the effect of ES on the frequency of evoked action potential and VGluT1 expression on PVIs. Data are shown as individual values or expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, and significance levels are indicated as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and not significant (n.s.). All sample size, statistical methods and results are specified in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Table S2.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTS, JL, CZ designed research; YM, CY, Y-XS, C-CZ, XL, and HW performed research; YM, CY, TW, Y-AS and X-XL analyzed data; JT, TS, Y-AS and YM wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Beijing National Science Foundation (grant No., 7222236), the National Natural Science Foundation of China (grant No., 82271569, 82171529, 82071528, 82001145, and 82001418), the Capital Medical Development Research Fund (2022-1-4111). The funders have no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Graphical abstract and cartoons in Figures 1(A, C) were created with BioRender.com\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u0026nbsp;\u003c/strong\u003eThe authors report no conflict of interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSolmi M, Radua J, Olivola M, Croce E, Soardo L, de Pablo GS \u003cem\u003eet al.\u003c/em\u003e Age at onset of mental disorders worldwide: large-scale meta-analysis of 192 epidemiological studies. Mol Psychiatr 2022; 27(1): 281\u0026ndash;295.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTom\u0026aacute;š Paus MK, Jay N. Giedd. Why do many psychiatric disorders emerge during adolescence. Nature reviews neuroscience 2008; 9: 947\u0026ndash;957.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeMoult J, Humphreys KL, Tracy A, Hoffmeister JA, Ip E, Gotlib IH. 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Neurosci Biobehav R 2002; 26(1): 91\u0026ndash;104.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"mp","sideBox":"Learn more about [Molecular Psychiatry](http://www.nature.com/mp/)","snPcode":"41380","submissionUrl":"https://mts-mp.nature.com/cgi-bin/main.plex","title":"Molecular Psychiatry","twitterHandle":"@molpsychiatry","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3572074/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3572074/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCognitive impairment, one core symptom of psychiatric disorders, is frequently observed in adolescents exposed to early-life stress (ES). However, the underlying neural mechanisms are unclear and the therapeutic efficacy is limited. Targeting at parvalbumin-expressing interneurons (PVIs) in the medial prefrontal cortex (mPFC), we report that mPFC PVI activity was reduced by ES and causally mediated ES-induced cognitive deficits in adolescent mice through chemogenetic or optogenetic experiments. We then demonstrate that ES reduced the excitatory inputs onto PVIs and pyramidal neuron (PN) activity and that ES negative effects were reversed by the knockout of corticotropin-releasing hormone receptor 1 (CRHR1, mainly expressed in PNs) in mouse mPFC, supporting the prefrontal CRHR1\u0026rarr;PN\u0026rarr;PVI pathway in mediating ES-induced cognitive deficits. Finally, antalarmin (a CRHR1 antagonist) treatment and environmental enrichment successfully restored PVI activity and cognitive deficits induced by ES. These findings highlight the critical role of PVIs in mediating and preventing ES-induced cognitive deficits in adolescent mice.\u003c/p\u003e","manuscriptTitle":"The CRHR1→PN→PVI Pathway in Medial Prefrontal Cortex Mediates Early-life Stress-induced Cognitive Deficits in Adolescent Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-12 15:17:39","doi":"10.21203/rs.3.rs-3572074/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-02-27T14:46:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-01-24T03:11:29+00:00","index":4,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-01-22T11:34:49+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-01-21T01:24:27+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-01-19T15:52:13+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-01-12T02:36:09+00:00","index":4,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-01-11T14:16:11+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-01-11T13:28:00+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-01-11T00:00:14+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-01-10T21:40:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-11-07T13:46:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Psychiatry","date":"2023-11-07T05:55:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-11-07T05:55:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"mp","sideBox":"Learn more about [Molecular Psychiatry](http://www.nature.com/mp/)","snPcode":"41380","submissionUrl":"https://mts-mp.nature.com/cgi-bin/main.plex","title":"Molecular Psychiatry","twitterHandle":"@molpsychiatry","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0b4b0730-5c44-48ff-94da-67e6ceb7b8d0","owner":[],"postedDate":"January 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28071963,"name":"Biological sciences/Psychology"},{"id":28071964,"name":"Biological sciences/Neuroscience"},{"id":28071965,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2024-11-23T08:07:17+00:00","versionOfRecord":{"articleIdentity":"rs-3572074","link":"https://doi.org/10.1038/s41380-024-02845-6","journal":{"identity":"molecular-psychiatry","isVorOnly":false,"title":"Molecular Psychiatry"},"publishedOn":"2024-11-22 05:00:00","publishedOnDateReadable":"November 22nd, 2024"},"versionCreatedAt":"2024-01-12 15:17:39","video":"","vorDoi":"10.1038/s41380-024-02845-6","vorDoiUrl":"https://doi.org/10.1038/s41380-024-02845-6","workflowStages":[]},"version":"v1","identity":"rs-3572074","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3572074","identity":"rs-3572074","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}