Developmental window of vulnerability to methylphenidate: Selective reduction of prelimbic PV+ interneurons impairs adult attention

Developmental window of vulnerability to methylphenidate: Selective reduction of prelimbic PV+ interneurons impairs adult attention | 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 Research Article Developmental window of vulnerability to methylphenidate: Selective reduction of prelimbic PV+ interneurons impairs adult attention Antonio Pérez-Colorado, Reyes Martínez-Marín, Juan Carlos López, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7176694/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Adolescence is a critical period for the maturation of the medial prefrontal cortex (mPFC), particularly involving the reorganization of the GABAergic network mediated by parvalbumin-expressing (PV+) interneurons. Methylphenidate (MPH), the most commonly prescribed treatment for attention-deficit/hyperactivity disorder (ADHD), is often administered over prolonged periods starting in childhood. However, sustained dopaminergic stimulation during adolescence may interfere with mPFC development by altering dopamine-dependent excitability of PV+ interneurons. In this study, we investigated whether chronic MPH exposure during adolescence affects the acquisition of PV+ interneurons and whether such alterations in GABAergic activity lead to long-lasting impairments in mPFC-dependent functions such as sustained attention. Male and female Wistar rats received 5 mg/kg MPH (a therapeutically relevant dose) for 20 days, beginning in early (PD35–55), middle (PD42–62), or late adolescence (PD49–69). From PD100 onward, animals were tested on a sustained attention task requiring lever presses in response to signal (hit) or non-signal (correct rejection, CR) trials. Once baseline performance reached >75% correct, stimulus durations were reduced to 500 ms, 100 ms, and 25 ms to increase attentional demand. Our results revealed a selective reduction in PV+ interneuron density in the prelimbic, but not infralimbic, cortex—predominantly in animals exposed to MPH during PD49–69. This reduction was associated with persistent deficits in attentional performance in adulthood. These findings highlight the critical role of PV+ interneurons in attentional processes and identify late adolescence as a window of heightened vulnerability during mPFC maturation. parvalbumin adolescence methylphenidate attention prelimbic Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Dopamine (DA) is essential for neuronal pathways involved in attention, reward processing, and motivation. Its actions are particularly critical within the prefrontal cortex (PFC), which mediates higher-order cognitive functions, as well as in subcortical structures such as the striatum and the thalamus [ 1 , 2 , 3 , 4 ]. Optimal DA function is critically dependent on a precise interplay with other major neurotransmitter systems, including the excitatory glutamate and inhibitory gamma-aminobutyric acid (GABA) systems [ 5 , 6 ]. GABAergic neurons maintain neurochemical balance by inhibiting neuronal circuits, preventing excessive excitability, and tuning the excitatory/inhibitory (E/I) balance. Their bidirectional interaction with dopaminergic pathways in cortical and subcortical regions supports a dynamic network essential for normal brain function. Consequently, any alteration in DA receptors, transporters, or synthesis implies a molecular dysregulation that contributes to the manifestation of symptoms observed in many mental disorders [ 7 , 8 ]. Dysregulation of the E/I balance during neurodevelopment has received attention recently, since it may contribute to the etiology of psychiatric disorders such as schizophrenia, bipolar disorder, and ADHD [ 9 ]. Adolescence represents a particularly vulnerable window for brain maturation, especially within the PFC, involving a reorganization and integration of local inhibitory circuit inputs from the ventral hippocampus and basolateral amygdala [ 10 ]. Preclinical studies in animal models have indicated that proper functional mPFC maturation relies on the appropriate recruitment of DA-mediated outputs to GABAergic interneurons, which modulate their excitability, synaptic plasticity, and gene expression through specific DA receptor subtypes located on these cells [ 11 , 12 , 13 , 14 ]. These interneurons, classified by their expression of calcium-binding proteins –parvalbumin (PV), calretinin (CR), and calbindin (CB)– constitute over 80% of GABAergic cells in the mPFC of rats and primates [ 15 , 16 ]. During developmental transition from adolescence to adulthood, their protein expression profile changes: CR expression decreases, PV increases (peaking at PD45-55), while CB levels remain stable [ 17 ]. The expression of fast-spiking PV + cells in adulthood is critically linked to enhanced prefrontal GABAergic function and the maturation of higher-order cognitive abilities, including working memory, decision-making, sustained attention, and impulse control [ 18 ]. This underscores that adolescence is not merely a period of general development, but a precisely timed "critical refinement period" for the PFC and its inhibitory circuits. Disruptions to the developmental trajectory of PV + interneurons during adolescence, caused by repeated drug exposure, could have lasting adverse effects on PFC function and associated behaviors. This establishes a clear vulnerability window where external or pharmacological challenges can lead to persistent deficiencies in prefrontal maturation. Pharmacological interventions, particularly with psychostimulants like methylphenidate (MPH), are a common cause of brain disturbance during childhood and adolescence. MPH is widely used to treat inattention and impulsivity symptoms, often observed in neurodevelopmental disorders like ADHD [ 19 , 20 , 21 ]. As symptoms often appear in adolescence and may persist across the lifespan, medications like MPH are frequently prescribed from an early age and for extended periods [ 22 , 23 ]. Its therapeutic efficacy is attributed to its ability to enhance DA release in the PFC by inhibiting DA and norepinephrine reuptake [ 24 ]. Since DA-D1 receptors on PV + interneurons regulate their firing properties and synaptic integration, increased synaptic DA concentrations induced by MPH may specifically impact the developmental trajectory and function of these critical inhibitory cells, affecting E/I balance. This specific DA-PV interaction under chronic MPH exposure during development represents a novel aspect of inquiry for the study. We hypothesize that chronic exposure to MPH during different adolescent developmental windows will disturb the normative maturation of PV interneurons in the PFC, leading to an imbalance in E/I tuning and subsequent persistent impairments in PFC-dependent cognitive functions, such as sustained attention. To test this hypothesis, we analyzed whether chronic administration of a therapeutic dose of MPH (5mg/kg over 20 days) in Wistar rats alters the expression and maturation of PV + interneurons in the PFC and, whether these changes correlate with impairments in PFC-dependent cognitive functions, specifically sustained attention. Materials And Methods Animals Forty male (300-500g) and thirty female (200-300g) Wistar rats were obtained from the University of Seville Animal Production Center at 3 weeks of age. They were subsequently used for behavioral testing starting at 16 weeks of age. Animals were housed in pairs in type IIIH cages (820 cm2) in a 12-hour dark/light cycle with ambient temperatures ranging from 22–24 ºC. Each cage was furnished with wood shavings as bedding, and pieces of fabric, cardboard, and wood for enrichment. Additional animal welfare details during procedure are included in Supplementary Materials and Methods. All experimental procedures were approved by the Ethics Committee for Animal Research of the University of Seville (approval code: CEEA-US2020-11) and were conducted in accordance with the guidelines outlined in EU Directive 2010/63/EU for animal experiments and the Spanish R.D. 53/2013. Drug administration Animals were administered MPH (Sigma Aldrich Laboratories, UK) dissolved in tap water at a dose of 5mg/kg. This solution was provided ad libitum in home cages for 20 consecutive days in three different groups according to its age: PD35-55, PD42-62 and PD49-69. The control group only received tap water. Sustained attention procedure (SAT) Rats were trained in operant chambers equipped with levers, stimulus lights, and a pellet dispenser to discriminate between signaled (light) and non-signaled trials. Performance was assessed using hits, misses, correct rejections, and false alarms applying the Vigilance Index proposed by McGaughy and Sarter [ 25 ]. Training involved five stages, increasing in complexity, with a final phase adding a flashing house light to heighten attentional demands. Each session comprised 120 trials, requiring over 75% accuracy to advance. Detailed descriptions of the sustained attention apparatus, different procedural testing phases and methodology used for data collection are provided in the Supplementary Materials and Methods. Histology The details of histological analysis are described in Supplementary Materials and Methods. Statistical analysis Data are presented as means ± SEM. Statistical analyses were conducted using one or two-way and mixed analysis of variance (ANOVA) followed by Bonferroni’s post hoc test, depending on whether neurochemical or behavioral data were analyzed. All analyses were performed using IBM SPSS Statistics 21 (SPSS, Inc.) and differences with p < 0.05 were considered statistically significant. Detailed descriptions of statistical analyses, F and p values for each figure are provided in the Supplementary Table S1. Results PV + interneurons density after chronic MPH exposure during adolescence Two areas of the mPFC (Fig. 1 a) corresponding to prelimbic and infralimbic were analyzed to quantify the number of PV + cells. Two-way ANOVAs were conducted with drug treatment (MPH vs control) and sex (male vs female) as factors for both regions. A significant reduction of PV + cells in both sexes was found only in the PrL (p < 0.001) (Fig. 1 . c-e). As no sex effects were found, data were pooled comparing control animals to the three MPH-treated groups: PD35-55, PD42-62 and P49-69 (Fig. 1 f-h) Although all treated groups showed reduced mean values compared to controls, only the PD49–69 group exhibited a statistically significant decrease (p < 0.001). Normalized data confirmed a marked reduction in PV + cells in the PrL exclusively in this group. No changes were observed in the IL. Spatial distribution of PV + changes across rostro-caudal levels Six rostro-caudal serial sections were counted in both PrL and IL to map spatial effects and grouped into three levels: L1 (rostral), L2 (central) and L3 (caudal) (Fig. 2 a). In controls, prelimbic PV + density increased from L1 to L3, forming a significant rostro-caudal gradient, while IL density remained stable (Fig. 2 b). Significant main effects of rostro-caudal level and treatment timing were found in the PrL. The PD49–69 group showed a significant reduction in PV + density at L2 and L3 compared to controls, but not at L1. IL showed no significant changes. Behavioral performance: sustained attention analysis MPH did not influence attentional performance during Acquisition phase. A mixed ANOVA with Signal Length (3) × Sessions (3) × Blocks (4) × Week of Treatment (4) × Sex (2) on mean SATs, with Signal Length, Sessions, and Blocks as within-group factors, showed that Sex variable was not significant. Therefore, data were pooled across sexes to characterize Acquisition performance. The animals' ability to discriminate between signal and non-signal events, as measured by SAT, was dependent on signal length (Fig. 3 a). Signal detection remained stable across Acquisition sessions and was similar between Control and MPH groups (Fig. 3 d-f). Animals did not maintain performance within sessions, as evidenced by a significant effect of Blocks. Performance improved in intermediate blocks within sessions (Fig. 4 a, c, e). Overall, MPH did not affect SAT performance during this phase. Neither the effect of the week of treatment nor any interactions involving this variable were significant. Similar results were obtained for hit rates data. MPH impairs attentional performance during TEST phase, varying by signal duration Mixed ANOVA revealed a significant main effect for all variables and a four-way interaction. All groups showed reduced signal discrimination as signal duration decreased. MPH affected these discriminations. The ability to discriminate 500ms signals from non-signals significantly decreased in all drug-treated groups, with SAT-500 being significantly greater in the Control group than in all MPH groups. Compared to the Control group, the PD49-69 group showed a reduced ability to discriminate shorter (100 and 25ms) signals from non-signals (Fig. 3 a). Across sessions, only the Control group showed a (non-significant) upward trend in performance. In contrast, MPH-treated groups, especially PD49-69, remained consistently impaired (Fig. 3 d-f). SAT-500 scores were lower in PD49-69 across all sessions; PD42-62 and PD35-55 groups also showed deficits in Sessions 1 and 3, respectively. No vigilance decrement over time was observed (no Blocks × Treatment interaction), but MPH treatment during PD49-69 led to lower SAT-500 performance across Blocks 1–3, with additional deficits in Block 2 for PD42-62 and PD35-55 groups. For SAT-100, PD49-69 underperformed relative to Control across all blocks, and for SAT-25, in Block 1(Fig. 4 a, c, e). The effects on the vigilance index appear to be primarily driven by disruptions in signal detection rather than by deficits in false alarm responses. A visual inspection of Fig. 3 b-c suggests a reduction in the proportion of hits in the MPH groups but no change in the number of CR. PD35-55 and PD49-69 groups showed significantly fewer hits than Control. Block-by-block analysis revealed that for 500ms signals, PD49-69 group had lower hit rates in Blocks 1–3, while PD35-55 and PD42-62 groups showed reduced hits in Block 2. For 100ms signals, differences emerged across all blocks for PD49-69. For 25ms signals, only Block 1 showed significantly reduced hits in this group (Fig. 4 b, d, f). The training effect, measured by changes in hit rates over the three sessions, was also influenced by methylphenidate. As shown in Fig. 3 g-i, performance in the control group improved across sessions for all signal durations, although not significantly. However, this trend was not observed in the drug-treated groups, particularly in the PD35-55 and PD49-69 groups. This pattern of behaviour led to more pronounced differences between the control and drug-treated groups as the sessions progressed. Effects of distractor flashing houselight (dSAT). Compared to the Test phase, the introduction of a flashing houselight significantly reduced animals’ ability to distinguish signal from non-signal events (Fig. 5 a–c). This increased ‘background’ noise caused a decline in SAT scores for both Control and P49–69 groups, regardless of signal length, block, or session. In Control animals, comparisons between the Test and Flash phases revealed that this impairment was session-dependent. During the Flash phase, SAT scores dropped across all blocks in Session 1, but performance gradually improved. Thus, in Session 2, impairments were limited to Block 1, and by Session 3, only Blocks 3 and 4 showed significant reductions, indicating progressive adaptation. In contrast, MPH-treated animals showed persistent impairment, with no improvement across blocks or sessions. Only a main effect of Phase was found, suggesting a generalized negative effect of the flashing houselight on SAT performance. Analysis within the Flash phase showed a significant main effect of signal length, with poorer discrimination at shorter durations. The Control group improved across sessions, while MPH-treated animals did not. Further analysis showed that SAT impairments were primarily due to reduced hit rates. CR remained stable across sessions and was unaffected by drug treatment. The percentage of hits dropped significantly from Test to Flash phases across all durations, blocks, and sessions, and this decline was evident in both groups. However, intra-phase analysis confirmed that the Control group exhibited progressive recovery across sessions, while MPH-treated animals did not. Across all sessions, the Control group achieved consistently higher hit rates than the MPH group. Discussion Our findings consistently demonstrate that MPH consumption during adolescence disrupts PFC maturational processes, with consequences persisting into adulthood. Specifically, our data show that the magnitude of this impact varies depending on the timing of drug exposure within the developmental period. Although a reduction in PV + interneuron density was observed across all MPH-treated groups, only animals exposed to MPH during the PD49–69 period exhibited a significant disruption in the normal acquisition of PV + interneurons in the PrL. Importantly, this reduction in GABAergic transmission within the PFC was associated with attentional impairments, reflected by poorer performance on a sustained attention task—particulary under conditions of high attentional demand. MPH effects on PFC GABAergic interneurons varied across developmental stages The differing effects of chronic MPH exposure across distinct postnatal periods—despite some overlap in treatment windows—suggest that adolescence is not a uniform developmental phase but rather a period marked by dynamic neurodevelopmental events. Our results align with previous studies emphasizing that the timing of psychostimulant exposure critically shapes drug-induced plasticity, leading to neurobehavioral abnormalities. For instance, MPH treatment during adolescence (PD35-42) has been shown to sensitize responses to rewards [ 26 ], whereas animals treated at earlier stages (PD20-35) exhibited reduced sensitivity to natural or drug rewards [ 27 , 28 ]. In our study, chronic administration of a therapeutically relevant dose of MPH during three adolescent windows (PD35–55, PD42–62, and PD49–69) resulted in reduced PV + interneurons density in the PrL, although this effect reached significance only in the PD49–69 group. Consistent with our findings, previous research has shown that exposure to other psychostimulants during adolescence, such as amphetamine or cocaine, reduces inhibitory transmission in the mPFC [ 29 , 30 , 31 ]. Previous studies suggest that PFC maturation during adolescence relies on remodeling local inhibitory circuits, driven by glutamatergic inputs from the ventral hippocampus and dopaminergic recruitment, which together facilitate GABAergic interneurons activity, believed to underlie the maturation of cognitive abilities [ 10 ]. In line with this framework, our results indicate that the most pronounced alterations in PV + expression—and, by extension, in GABAergic transmission within the mPFC—occurred in animals exposed to MPH during the PD49–69 window, corresponding to late adolescence. Therefore, it is plausible that disruptions to the normal developmental trajectory of inhibitory signaling in the PFC during this critical period contribute to long-lasting deficits in PFC-dependent cognitive functions. MPH affects GABAergic interneurons in the caudal prelimbic cortex Our results also indicate that chronic MPH consumption specifically alters the density of PV + GABAergic interneurons in the caudal portion of the PrL. These interneurons are key regulators of the E/I balance in the PFC, playing a crucial role in optimizing the representation and processing of supramodal information. A reduction in the number of PV + interneurons in the PrL may disrupt this balance, potentially leading to increased circuit excitability. Since PV + interneurons are essential for both tonic and phasic inhibition of pyramidal neurons, their loss could result in decreased inhibitory control and heightened pyramidal neuron excitability. While this hypothesis requires further investigation, it is supported by previous electrophysiological studies showing that in adult rats, an acute dose of MPH exerts excitatory effects on PFC pyramidal neurons, particularly in prelimbic, cingulate, and medial regions [ 32 , 33 ]. Consistent with our findings, Di Micelli et al. [ 34 ] reported that chronic MPH administration during late adolescence (PD42) resulted in a persistent increase in PFC pyramidal neurons firing rates into adulthood, without affecting NMDA-mediated neurotransmission. Other studies have demonstrated that chronic MPH effects on PFC neuronal excitability are age-dependent, with adult rats showing increased spike activity in layer V pyramidal neurons, whereas juveniles exhibited a transient suppression of excitability [ 35 ]. Similarly, Morshedi and Meredith [ 36 ] found a significant reduction in PV + density in layer V of the PrL -but not in the IL- following repeated amphetamine treatment, an effect absent with acute administration. This region-specific reduction in PV immunoreactivity suggests that PrL neurophysiology is particularly vulnerable to the long-term effects of repeated psychostimulant exposure. There are several potential mechanisms that may underlie the observed increases in PFC-pyramidal neuron firing rates following chronic MPH exposure. Some studies have suggested that these effects could be attributable to altered GABAergic interneurons activity. For instance, a single 1 mg/kg dose of MPH in juvenile rats enhances the excitation of GABAergic interneurons [ 37 ]. Moreover, studies with other psychostimulants corroborate the impact on GABAergic transmission. Repeated cocaine exposure during early adolescence, for example, induces mPFC disinhibition through long-lasting impairments in the local GABAergic networks [ 29 ]. Similarly, repeated amphetamine exposure has been associated with altered interneuron physiology, including decreased sensitivity to D1 receptor stimulation and reduced inhibitory output [ 36 ]. MPH effects on PFC-PV + interneurons are independent of sex In contrast to studies suggesting that estrogen modulates PV + interneuron activity and influences GABAergic signaling reorganization [ 38 , 39 , 40 ], our findings revealed no significant sex differences in PV + interneuron expression following repeated MPH exposure. This is particularly notable since our study included both males and females subjects at pubertal onset, when sex-dependent differences were anticipated. The absence of such differences may indicate that the mechanisms regulating PV + interneuron acquisition are hormonally independent or, as proposed by Torres-Reveron et al [ 41 ], the estrous cycle and estrogen levels may not affect PV + cell numbers but rather their availability for inhibitory transmission. PV neurons, characterized by fast-spiking activity and high metabolic demands, are particularly susceptible to oxidative stress [ 42 ], despite partial protected by perineuronal nets (PNNs) [ 43 ]. Considering that repeated amphetamine exposure induces oxidative damage in brain regions such as the mPFC [ 44 , 45 ], we hypothesize that persistent oxidative stress caused by MPH –a sex-independent insult– may disrupt normal neuronal activity. This disruption could lead to cell death or, more likely, selectively impair the mechanism by which calretinin is replaced by parvalbumin, resulting in deficient PV + upregulation rather than a loss of PV + cells. Reduced GABAergic activity in the caudal prelimbic cortex contributes to impairments in sustained attention. Our findings show that the decrease in PV + interneurons in the PrL induced by early exposure to MPH significantly impairs performance on a sustained attention task in adulthood. The degree of impairment depended on the developmental window of MPH administration. Specifically, MPH exposure from PD42–62, which caused a minor reduction in PV + neuron density, resulted in minimal alterations in task performance compared to the Control group. In contrast, MPH exposure during PD35-55 resulted in a slightly greater reduction in PV + interneuron density, along with more pronounced impairments in task performance. The most severe impairments were observed in animals exposed to MPH during PD49-69, the only group exhibiting a statistically significant reduction in PV + density compared to controls. This group showed consistently poorer performance than the Control group on both overall SAT scores and hit rates. However, CR rates remained close to 80% across all MPH groups and did not differ from controls. Exposure to a flashing houselight increased the demands on top-down attentional control, reducing the animals' ability to discriminate signal from non-signal events in both control and MPH-treated groups. Analyses revealed that this attention impairment was primarily driven by decreased hit rates, as CR performance remained stable from Test to dSAT phases, with no group differences observed during the distractor condition. However, control animals adapted to this increased attentional load, displaying performance deficits only in early blocks of the first session. In contrast, animals treated from PD49-69 did not recover performance across sessions or blocks, indicating an inability to enhance top-down attentional control over time. These findings support the notion that GABAergic activity in the PFC is critical for sustaining attention, as its reduction increases failures to detect signal events. We hypothesize that reduced PV + interneuron density in the PrL cortex could lead to a lower vigilance index and fewer hits, while leaving false alarm rates unaffected. This pattern indicates that attentional deficits from reduced GABAergic function in the PrL arise mainly from impaired signal detection rather than impulsivity, deficits in rule comprehension, or bottom-up sensory processing. In this regard, our results align with Kim et al. [ 46 ], who showed that FS-PV interneuron activity in the mPFC is essential for detecting relevant stimuli, but not for inhibiting responses to non-relevant events. Similarly, Fisher et al. [ 47 ] demonstrated that specific prelimbic lesions produce persistent deficits in sustained attention, especially under high attentional load or distraction, impairing signal discrimination sensitivity (d') and hit rates without significantly altering inhibitory control. Besides maintaining the E/I balance in the PFC, PV + interneurons also play a key role in maintaining a high signal-to-noise ratio during information processing, facilitating the selection of relevant stimuli for cognitive operations such as attentional engagement [ 47 – 50 ]. The errors observed in our study likely stem from disruptions in the cognitive processing of signals and their translation into goal-directed behavior, or may result from interference generated by irrelevant cognitive activity [ 51 ]. Specifically, the hit deficit may reflect an inability to maintain sufficient attentional engagement at signal onset due to the loss of precise inhibitory modulation by PV + interneurons in the PrL. In contrast, preserved performance in non-signal trials suggests that tonic or baseline inhibitory control remains intact, indicating a selective disruption of attentional processing of external stimuli, as proposed in cortical GABAergic interneuron-deficient models [ 52 ]. This pattern points to a deficit in top-down attentional control, a function critically dependent on the integrity of the prelimbic cortex and its PV + interneurons. Supporting this possibility, evidence indicates that FS-PV interneuron activity in the mPFC is essential for maintaining attention on relevant stimuli, especially in the presence of distractors [ 46 ]. Disruption of this region through pharmacological manipulation or lesions has also been shown to impair attentional accuracy and reduce the capacity to adapt under increasing task demands such as persistent distraction [ 47 , 53 ]. The absence of recovery in the PD49–69 group suggests that PV + interneuron reduction compromises the attentional system’s ability to adapt and filter out irrelevant information, preventing improvement with practice in high-demand conditions. Taken together, these results demonstrate significant attentional impairment in the PD49–69 group, underscoring the importance of maintaining appropriate PV + neuron density in the prelimbic cortex for accurate visual stimulus detection and the development of attentional processes. Notably, the reduction in PV + neuron density resulting from prolonged exposure to moderate doses of MPH was relatively modest; nevertheless, measurable attentional deficits still emerged. The absence of more severe outcomes may reflect compensatory mechanisms in the brain that help mitigate such damage or fluctuations. These findings have important implications, suggesting that misuse of drugs such as MPH during adolescence—whether due to ADHD misdiagnosis or recreational use—can lead to maturational delays and adverse effects on attentional function. Overall, these results highlight the vulnerability of attentional mechanisms to changes in PV + neuron density; and underscore the need for further research to clarify long-term implications across other brain regions and behavioral outcomes to fully assess the relevance of these findings in humans. Conclusion In conclusion, our findings demonstrate that chronic administration of 5 mg/kg methylphenidate during adolescence—specifically during the PD49–69 period—blocks the normal gain of PV + interneurons in the prelimbic cortex, but not in the infralimbic region, in both male and female Wistar rats. This alteration disrupts attentional maintenance and visual discrimination abilities, with effects persisting into adulthood. These results underscore the critical role of PV + interneurons in sustained attention and suggest that there are specific windows of vulnerability during the functional maturation of the medial prefrontal cortex. Declarations Author contributions Pérez-Colorado, A.: Writing – original draft, Visualization, Investigation, Formal analysis, Conceptualization. Martínez-Marín, R.: Writing – review & editing, Visualization, Investigation, Formal analysis. López, J.C.: Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition, Resources, Formal analysis, Conceptualization. Vargas, J.P.: Writing – review & editing, Supervision, Project administration, Funding acquisition, Formal analysis, Conceptualization. Díaz, E.: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization. Funding This research was funded by Agencia Estatal de Investigación (AEI) of Spain PID2019- 110739GB-I00/AEI/10.13039/501100011033 and PID2023-149901NB-I00 funded by MICIU/AEI/ 10.13039/501100011033 and FEDER/EU. Competing Interests The authors have nothing to disclose. References Blum, K., Chen, A. L., Braverman, E. R., Comings, D. E., Chen, T. J., Arcuri, V., Blum, S. H., Downs, B. W., Waite, R. L., Notaro, A., Lubar, J., Williams, L., Prihoda, T. J., Palomo, T., & Oscar-Berman, M. (2008). Attention-deficit-hyperactivity disorder and reward deficiency syndrome. Neuropsychiatric disease and treatment , 4 (5), 893–918. https://doi.org/10.2147/ndt.s2627 Brennan, A. R., & Arnsten, A. F. (2008). Neuronal mechanisms underlying attention deficit hyperactivity disorder: the influence of arousal on prefrontal cortical function. Annals of the New York Academy of Sciences , 1129 , 236–245. https://doi.org/10.1196/annals.1417.007 Díaz, E., Vargas, J. 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Sign and goal tracker rats process differently the incentive salience of a conditioned stimulus. PloS one , 14 (9), e0223109. https://doi.org/10.1371/journal.pone.0223109 Pérez-Díaz, F., Díaz, E., Sánchez, N., Vargas, J. P., Pearce, J. M., & López, J. C. (2017). Different involvement of medial prefrontal cortex and dorso-lateral striatum in automatic and controlled processing of a future conditioned stimulus. PloS one , 12 (12), e0189630. https://doi.org/10.1371/journal.pone.0189630 Lustig, C., Kozak, R., Sarter, M., Young, J. W., & Robbins, T. W. (2013). CNTRICS final animal model task selection: control of attention. Neuroscience and biobehavioral reviews , 37 (9 Pt B), 2099–2110. https://doi.org/10.1016/j.neubiorev.2012.05.009 Bissonette, G. B., Bae, M. H., Suresh, T., Jaffe, D. E., & Powell, E. M. (2014). Prefrontal cognitive deficits in mice with altered cerebral cortical GABAergic interneurons. Behavioural brain research , 259 , 143–151. https://doi.org/10.1016/j.bbr.2013.10.051 Auger, M. L., & Floresco, S. B. (2017). Prefrontal cortical GABAergic and NMDA glutamatergic regulation of delayed responding. Neuropharmacology , 113 (Pt A), 10–20. https://doi.org/10.1016/j.neuropharm.2016.09.022 Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryMaterialsandMethods.doc Supplementary Materials and Methods Suplementarytable.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7176694","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":488519832,"identity":"f5c37b78-8c65-464f-bed4-9853f1f7b089","order_by":0,"name":"Antonio Pérez-Colorado","email":"","orcid":"https://orcid.org/0000-0002-5363-693X","institution":"Universidad de Sevilla","correspondingAuthor":false,"prefix":"","firstName":"Antonio","middleName":"","lastName":"Pérez-Colorado","suffix":""},{"id":488519833,"identity":"651a1fa1-5e48-4ba9-9bfb-10bff37584f7","order_by":1,"name":"Reyes Martínez-Marín","email":"","orcid":"https://orcid.org/0000-0001-5422-4067","institution":"Universidad de Sevilla","correspondingAuthor":false,"prefix":"","firstName":"Reyes","middleName":"","lastName":"Martínez-Marín","suffix":""},{"id":488519834,"identity":"b64e3062-3917-4c71-808a-1ea0c7111b16","order_by":2,"name":"Juan Carlos López","email":"","orcid":"https://orcid.org/0000-0001-8106-5006","institution":"Universidad de Sevilla","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"Carlos","lastName":"López","suffix":""},{"id":488519835,"identity":"e592c0f6-59d0-4e8d-9469-bd972c1f289d","order_by":3,"name":"Juan Pedro Vargas","email":"","orcid":"https://orcid.org/0000-0002-4358-5737","institution":"Universidad de Sevilla","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"Pedro","lastName":"Vargas","suffix":""},{"id":488519836,"identity":"845c3ca2-6e60-4900-9ea0-4e764acea5f9","order_by":4,"name":"Estrella Díaz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqElEQVRIiWNgGAWjYNACNgk5UpQzg7UYk6yFIbGBaA38/OePffhQZpG+4fwBxoc/iNEiOSOZeeaMcxK5G24kMBvzEKPF4AYzMzNvG0gLA5s0UQ6zP3+Ymflvm0S6wfkD7D+JcpgBQzIzM2ObRILBgQQ2BqIcJnEj2Zix55yE4cwbic3SRGnh7z/4mOFHWZ083/nDBz8S5TAkwNhAooZRMApGwSgYBTgBADnCLRvrHl0rAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-9465-9460","institution":"Universidad de Sevilla","correspondingAuthor":true,"prefix":"","firstName":"Estrella","middleName":"","lastName":"Díaz","suffix":""}],"badges":[],"createdAt":"2025-07-21 11:08:25","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-7176694/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7176694/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87419623,"identity":"a5a3b97d-49d5-48bc-9bf5-efa96456048d","added_by":"auto","created_at":"2025-07-23 15:19:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1523687,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantification of PV+ interneurons in mPFC of animals chronically treated with MPH (a) \u003c/strong\u003eSchematic representation of a coronal section of the mPFC, including PrL and IL regions, with the defined ROIs imaged for each slice. \u003cstrong\u003e(b)\u003c/strong\u003e Treatment and behavioral experimental timeline \u003cstrong\u003e(c-e) \u003c/strong\u003eTwo-way ANOVA analysis of PV+ cell counts revealed a significant effect of MPH in both males and females in PrL (F(1,46)=14,334, p\u0026lt;0.001, η\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e = 0.238) but not in IL or overall mPFC. \u003cstrong\u003e(f)\u003c/strong\u003e Overall density of PV+ cells was altered following MPH administration across different developmental stages: early adolescence (PD35-55, n=9) middle adolescence (PD42-62, n=9) and late adolescence (PD49-69, n=13). Two-way ANOVA analysis showed a significant effect of MPH in PrL (F(3,46)=7,497, p\u0026lt;0,001, η\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e= 0,328) with a notable reduction observed only in the PD49-69 group (p\u0026lt;0,001) \u003cstrong\u003e(g)\u003c/strong\u003e Normalized PV+ number relative to controls confirmed a significant reduction in the prelimbic of the PD49-69 group (p\u0026lt;0,001).\u003c/p\u003e","description":"","filename":"figure1v2.png","url":"https://assets-eu.researchsquare.com/files/rs-7176694/v1/a14402c189787597357b4109.png"},{"id":87421026,"identity":"5d55973b-878d-4006-b06e-d9b786d1fc9d","added_by":"auto","created_at":"2025-07-23 15:35:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7420890,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePV+ distribution is reduced at different levels across the mPFC after MPH consumption (a) \u003c/strong\u003eDiagram illustrating the consecutive rostro-caudal sections (80μm per level, 40μm per slice) and the regions sampled for PV+ quantification \u003cstrong\u003e(b)\u003c/strong\u003e Summary of PV+ cells distribution across the 3 levels (L1: rostral, L2: medial, L3: caudal) in prelimbic and infralimbic cortices. Mixed ANOVA analysis revealed a significant main effect of levels (F(2, 86)=16.964, p\u0026lt;0.001,η\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e= 0.283) and timing of MPH (F(3, 43)= 5.84, p=0.002, η\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e=0.29) in PrL but not in IL. Specifically, PV+ density was significantly reduced in PrL at medial (F(3,43)=7.449, p\u0026lt;0.001, η\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e= 0.342) and caudal levels (F(3,43)=6.207, p=0.001, η\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e= 0.302) compared to Control group. No significant effects were observed at L1 in PrL or across any level in the IL \u003cstrong\u003e(c) \u003c/strong\u003eExamples of PV inmunohistochemical staining from different MPH-treated groups across the 3 rostro-caudal levels (L1, L2, L3) of the PrL, illustrating the observed at 10X. Scale bar: 400 μm.\u003c/p\u003e","description":"","filename":"figure2v2.png","url":"https://assets-eu.researchsquare.com/files/rs-7176694/v1/35073bd4768f44e4f571e4f6.png"},{"id":87419619,"identity":"66e1f738-38f9-43d5-aa33-012f872c9f87","added_by":"auto","created_at":"2025-07-23 15:19:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2006328,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMacroanalysis of attentional performance, SAT scores, and hit rates across sessions. (a)\u003c/strong\u003ePlot illustrating global SAT score index for MPH-treated and Control groups during ACQUISITION and TEST phases at 500ms, 100ms, and 25ms stimulus durations. Significant MPH effects occurred only in TEST phase. At 500ms, controls outperformed PD35-55, PD42-62 and PD49-69. At 100ms and 25ms, controls outperformed PD49-69, and PD35-55. \u003cstrong\u003e(b)\u003c/strong\u003e Hit rates during ACQUISITION and TEST. MPH effects appeared only in TEST; controls outperformed MPH-treated groups at 500ms and 100ms. At 25ms, only PD35-55 performed worse than controls. \u003cstrong\u003e(c)\u003c/strong\u003e No significant effects in correct rejection rates during ACQUISITION or TEST. \u003cstrong\u003e(d, g)\u003c/strong\u003e SAT score and hit rate plots for 500ms. MPH-treated groups performed below controls across sessions, especially in TEST. Controls outperformed PD49-69 in all sessions, PD42-62 in S-1, and PD35-55 in S-3. Hit rates dropped significantly for PD49-69 and PD35-55 in S-2 and S-3. \u003cstrong\u003e(e, h)\u003c/strong\u003eAt 100ms, most significant differences were in TEST phase S-3; controls outperformed PD49-69 and PD35-55. Hit rates decreased for PD49-69, PD35-55, and PD42-62 in S-3. \u003cstrong\u003e(f, i)\u003c/strong\u003e At 25ms, in S-3; PD49-69 and PD35-55 performed below controls, with reduced hit rates.\u003c/p\u003e","description":"","filename":"figure3v21.png","url":"https://assets-eu.researchsquare.com/files/rs-7176694/v1/646ff902a099ae3428a5398e.png"},{"id":87420654,"identity":"5cf7f5a4-598c-4852-8107-a1c7b557ab74","added_by":"auto","created_at":"2025-07-23 15:27:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1607823,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSAT score and hit rates performance across blocks (a, b) \u003c/strong\u003ePlots illustrating SAT score index and hit rate performance across blocks for 500ms signal duration. MPH-treated groups performed below the Control group across all blocks, with notable differences during TEST phase. Control group significantly outperformed the PD49-69 in the first 3 blocks and both PD42-62 and PD35-55 groups in B2. Analysis of hit rates revealed significant reductions in PD49-69 in B1, B2 and B3. A marginally reduction in PD35-55 groups during B4 was also observed \u003cstrong\u003e(c, d)\u003c/strong\u003e For 100ms, only significant differences were observed between Control and PD49-69 during B1 and B2 and B3. This only corresponded to a significant reduction in hit rates for PD49-69 during B3 and B4 and PD35-55 during B3, but no significant differences were observed in previous blocks.\u003cstrong\u003e(e ,f)\u003c/strong\u003e For 25ms, only a significant reduction was found between Control and PD49-69 groups in B1. Further analysis of hit rates revealed a reduction in B4 of PD35-55 group.\u003c/p\u003e","description":"","filename":"figure4v21.png","url":"https://assets-eu.researchsquare.com/files/rs-7176694/v1/2149e2a485b3d8176e80ea86.png"},{"id":87419625,"identity":"59bd973d-8959-4794-b79f-1fd9d239cc50","added_by":"auto","created_at":"2025-07-23 15:19:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":978870,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSAT score index performance of Control and PD49-69 cohorts across blocks and sessions comparing TEST and FLASH phases. (a-c) \u003c/strong\u003eGraphs depict SAT score index performance for 500, 100 and 25ms signal duration across blocks, comparing Control (blue) and PD49-69 (orange) during TEST phase (solid line) and FLASH phase (dashed line). Statistical comparisons: between phases (TEST vs FLASH; blue and orange asterisks) and between treated and non-treated group within the same phase (Control vs PD49-69; red and green asterisks, respectively). \u003cstrong\u003e(a)\u003c/strong\u003e \u003cstrong\u003e500ms\u003c/strong\u003e: Control performance dropped during first two blocks of S1 (both p\u0026lt;0.001), recovering to almost basal levels in subsequent sessions. PD49-69 performed below TEST rates during the three FLASH sessions: S1 (S1-B2, p\u0026lt;0.001, S1-B4, p\u0026lt;0.05), S2 (S2-B2, S2-B4, both p\u0026lt;0.01, S2-B3, p\u0026lt;0.05), and S3 (S3-B1, S3-B2, both p\u0026lt;0.01, S3-B4, p\u0026lt;0.001). Drug effects produced notable differences during S2 (S2-B1 and S2-B2 both p\u0026lt;0.01) and S3 of FLASH phase (S3-B1, p\u0026lt;0.001 S3-B2 and S3-B4, both p\u0026lt;0.01). Less differences were found in TEST, with differences in S3 (S3-B1, S3-B2 and S3-B3 all p\u0026lt;0.05) and marginal reductions in S1-B2 and S2-B1 (both p\u0026lt;0.05) \u003cstrong\u003e(b) 100ms: \u003c/strong\u003eOnly significant differences in Control were observed during the first two blocks of S1 (both p\u0026lt;0.01) and S2-B1 (p\u0026lt;0.05). For PD49-69, marginally significances were found during S1-B1 (p\u0026lt;0.01) and S2-B2 (p\u0026lt;0.05) with more consistent deficits during S3-B3 (p\u0026lt;0.001) and S3-B4 (p\u0026lt;0.05). Most drug effects were evident during TEST phase in S3, with S3-B1 (p\u0026lt;0.01), S3-B2 and S3-B4 (both p\u0026lt;0.05), with a marginal reduction during S1-B2 (p\u0026lt;0.05). According with 500ms results, Control group outperformed across sessions, only finding consistent differences with PD49-69 in S3 (S3-B1 and S3-B3, both p\u0026lt;0.01, S3-B2 and S3-B4, both p\u0026lt;0.05). Marginal differences were found in S1-B3 and S2-B2 (both p\u0026lt;0.05) \u003cstrong\u003e(c) 25ms: \u003c/strong\u003eIn Control group differences between phases were limited to S1 (S1-B1, S1-B2, both p\u0026lt;0.01) and S1-B1 (p\u0026lt;0.05), with a small difference in S3-B4 (p\u0026lt;0.05). PD49-69 performance dropped consistently in S1-B2, S1-B3 (both p\u0026lt;0.01) and S3-B4 (p\u0026lt;0.001), with a marginal reduction in S2-B2 (p\u0026lt;0.01). Drug effects were inconsistent across blocks due to floor effect, with marginal reductions during TEST in S1-B1, S2-B4 (both p\u0026lt;0.05), and S1-B1 (p\u0026lt;0.05) during FLASH. However, performance reductions were more consistent in S3 during TEST (S3-B1, p\u0026lt;0.01, and S3-B3, p\u0026lt;0.05) and FLASH (S3-B1, S3-B3, S3-B4, all p\u0026lt;0.05). *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"figure51.png","url":"https://assets-eu.researchsquare.com/files/rs-7176694/v1/464a987e94747a78133b2a08.png"},{"id":87422105,"identity":"2e56a590-7a90-4877-a73e-4a62cd3f6964","added_by":"auto","created_at":"2025-07-23 15:43:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13861091,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7176694/v1/074f1053-c61d-4891-935f-cdceef623558.pdf"},{"id":87419630,"identity":"efb6d0ba-aa0f-4c9d-bcd0-fea5a075829a","added_by":"auto","created_at":"2025-07-23 15:19:35","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":98314240,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Materials and Methods\u003c/p\u003e","description":"","filename":"SupplementaryMaterialsandMethods.doc","url":"https://assets-eu.researchsquare.com/files/rs-7176694/v1/51d1edbba4ecefe2ec5fcef4.doc"},{"id":87419618,"identity":"81a5a553-f8c1-4cc0-9b5e-4c8506fd38f6","added_by":"auto","created_at":"2025-07-23 15:19:34","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17377,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Suplementarytable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7176694/v1/233525603abc76e844ae099b.xlsx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eDevelopmental window of vulnerability to methylphenidate: Selective reduction of prelimbic PV+ interneurons impairs adult attention\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDopamine (DA) is essential for neuronal pathways involved in attention, reward processing, and motivation. Its actions are particularly critical within the prefrontal cortex (PFC), which mediates higher-order cognitive functions, as well as in subcortical structures such as the striatum and the thalamus [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Optimal DA function is critically dependent on a precise interplay with other major neurotransmitter systems, including the excitatory glutamate and inhibitory gamma-aminobutyric acid (GABA) systems [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. GABAergic neurons maintain neurochemical balance by inhibiting neuronal circuits, preventing excessive excitability, and tuning the excitatory/inhibitory (E/I) balance. Their bidirectional interaction with dopaminergic pathways in cortical and subcortical regions supports a dynamic network essential for normal brain function. Consequently, any alteration in DA receptors, transporters, or synthesis implies a molecular dysregulation that contributes to the manifestation of symptoms observed in many mental disorders [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDysregulation of the E/I balance during neurodevelopment has received attention recently, since it may contribute to the etiology of psychiatric disorders such as schizophrenia, bipolar disorder, and ADHD [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Adolescence represents a particularly vulnerable window for brain maturation, especially within the PFC, involving a reorganization and integration of local inhibitory circuit inputs from the ventral hippocampus and basolateral amygdala [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Preclinical studies in animal models have indicated that proper functional mPFC maturation relies on the appropriate recruitment of DA-mediated outputs to GABAergic interneurons, which modulate their excitability, synaptic plasticity, and gene expression through specific DA receptor subtypes located on these cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These interneurons, classified by their expression of calcium-binding proteins \u0026ndash;parvalbumin (PV), calretinin (CR), and calbindin (CB)\u0026ndash; constitute over 80% of GABAergic cells in the mPFC of rats and primates [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. During developmental transition from adolescence to adulthood, their protein expression profile changes: CR expression decreases, PV increases (peaking at PD45-55), while CB levels remain stable [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe expression of fast-spiking PV\u0026thinsp;+\u0026thinsp;cells in adulthood is critically linked to enhanced prefrontal GABAergic function and the maturation of higher-order cognitive abilities, including working memory, decision-making, sustained attention, and impulse control [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This underscores that adolescence is not merely a period of general development, but a precisely timed \"critical refinement period\" for the PFC and its inhibitory circuits. Disruptions to the developmental trajectory of PV\u0026thinsp;+\u0026thinsp;interneurons during adolescence, caused by repeated drug exposure, could have lasting adverse effects on PFC function and associated behaviors. This establishes a clear vulnerability window where external or pharmacological challenges can lead to persistent deficiencies in prefrontal maturation.\u003c/p\u003e\u003cp\u003ePharmacological interventions, particularly with psychostimulants like methylphenidate (MPH), are a common cause of brain disturbance during childhood and adolescence. MPH is widely used to treat inattention and impulsivity symptoms, often observed in neurodevelopmental disorders like ADHD [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. As symptoms often appear in adolescence and may persist across the lifespan, medications like MPH are frequently prescribed from an early age and for extended periods [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Its therapeutic efficacy is attributed to its ability to enhance DA release in the PFC by inhibiting DA and norepinephrine reuptake [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Since DA-D1 receptors on PV\u0026thinsp;+\u0026thinsp;interneurons regulate their firing properties and synaptic integration, increased synaptic DA concentrations induced by MPH may specifically impact the developmental trajectory and function of these critical inhibitory cells, affecting E/I balance. This specific DA-PV interaction under chronic MPH exposure during development represents a novel aspect of inquiry for the study.\u003c/p\u003e\u003cp\u003eWe hypothesize that chronic exposure to MPH during different adolescent developmental windows will disturb the normative maturation of PV interneurons in the PFC, leading to an imbalance in E/I tuning and subsequent persistent impairments in PFC-dependent cognitive functions, such as sustained attention. To test this hypothesis, we analyzed whether chronic administration of a therapeutic dose of MPH (5mg/kg over 20 days) in Wistar rats alters the expression and maturation of PV\u0026thinsp;+\u0026thinsp;interneurons in the PFC and, whether these changes correlate with impairments in PFC-dependent cognitive functions, specifically sustained attention.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eAnimals\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eForty male (300-500g) and thirty female (200-300g) Wistar rats were obtained from the University of Seville Animal Production Center at 3 weeks of age. They were subsequently used for behavioral testing starting at 16 weeks of age. Animals were housed in pairs in type IIIH cages (820 cm2) in a 12-hour dark/light cycle with ambient temperatures ranging from 22\u0026ndash;24 \u0026ordm;C. Each cage was furnished with wood shavings as bedding, and pieces of fabric, cardboard, and wood for enrichment. Additional animal welfare details during procedure are included in Supplementary Materials and Methods.\u003c/p\u003e\u003cp\u003e All experimental procedures were approved by the Ethics Committee for Animal Research of the University of Seville (approval code: CEEA-US2020-11) and were conducted in accordance with the guidelines outlined in EU Directive 2010/63/EU for animal experiments and the Spanish R.D. 53/2013.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eDrug administration\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAnimals were administered MPH (Sigma Aldrich Laboratories, UK) dissolved in tap water at a dose of 5mg/kg. This solution was provided \u003cem\u003ead libitum\u003c/em\u003e in home cages for 20 consecutive days in three different groups according to its age: PD35-55, PD42-62 and PD49-69. The control group only received tap water.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eSustained attention procedure (SAT)\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eRats were trained in operant chambers equipped with levers, stimulus lights, and a pellet dispenser to discriminate between signaled (light) and non-signaled trials. Performance was assessed using hits, misses, correct rejections, and false alarms applying the Vigilance Index proposed by McGaughy and Sarter [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Training involved five stages, increasing in complexity, with a final phase adding a flashing house light to heighten attentional demands. Each session comprised 120 trials, requiring over 75% accuracy to advance.\u003c/p\u003e\u003cp\u003eDetailed descriptions of the sustained attention apparatus, different procedural testing phases and methodology used for data collection are provided in the Supplementary Materials and Methods.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eHistology\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe details of histological analysis are described in Supplementary Materials and Methods.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical analyses were conducted using one or two-way and mixed analysis of variance (ANOVA) followed by Bonferroni\u0026rsquo;s post hoc test, depending on whether neurochemical or behavioral data were analyzed. All analyses were performed using IBM SPSS Statistics 21 (SPSS, Inc.) and differences with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. Detailed descriptions of statistical analyses, \u003cem\u003eF\u003c/em\u003e and \u003cem\u003ep\u003c/em\u003e values for each figure are provided in the Supplementary Table S1.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003ePV\u0026thinsp;+\u0026thinsp;interneurons density after chronic MPH exposure during adolescence\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTwo areas of the mPFC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) corresponding to prelimbic and infralimbic were analyzed to quantify the number of PV\u0026thinsp;+\u0026thinsp;cells. Two-way ANOVAs were conducted with drug treatment (MPH vs control) and sex (male vs female) as factors for both regions. A significant reduction of PV\u0026thinsp;+\u0026thinsp;cells in both sexes was found only in the PrL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. c-e). As no sex effects were found, data were pooled comparing control animals to the three MPH-treated groups: PD35-55, PD42-62 and P49-69 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef-h)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAlthough all treated groups showed reduced mean values compared to controls, only the PD49\u0026ndash;69 group exhibited a statistically significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Normalized data confirmed a marked reduction in PV\u0026thinsp;+\u0026thinsp;cells in the PrL exclusively in this group. No changes were observed in the IL.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eSpatial distribution of PV\u0026thinsp;+\u0026thinsp;changes across rostro-caudal levels\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eSix rostro-caudal serial sections were counted in both PrL and IL to map spatial effects and grouped into three levels: L1 (rostral), L2 (central) and L3 (caudal) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn controls, prelimbic PV\u0026thinsp;+\u0026thinsp;density increased from L1 to L3, forming a significant rostro-caudal gradient, while IL density remained stable (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Significant main effects of rostro-caudal level and treatment timing were found in the PrL. The PD49\u0026ndash;69 group showed a significant reduction in PV\u0026thinsp;+\u0026thinsp;density at L2 and L3 compared to controls, but not at L1. IL showed no significant changes.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eBehavioral performance: sustained attention analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMPH did not influence attentional performance during Acquisition phase.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA mixed ANOVA with Signal Length (3) \u0026times; Sessions (3) \u0026times; Blocks (4) \u0026times; Week of Treatment (4) \u0026times; Sex (2) on mean SATs, with Signal Length, Sessions, and Blocks as within-group factors, showed that Sex variable was not significant. Therefore, data were pooled across sexes to characterize Acquisition performance. The animals' ability to discriminate between signal and non-signal events, as measured by SAT, was dependent on signal length (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSignal detection remained stable across Acquisition sessions and was similar between Control and MPH groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-f). Animals did not maintain performance within sessions, as evidenced by a significant effect of Blocks. Performance improved in intermediate blocks within sessions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, c, e). Overall, MPH did not affect SAT performance during this phase. Neither the effect of the week of treatment nor any interactions involving this variable were significant. Similar results were obtained for hit rates data.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eMPH impairs attentional performance during TEST phase, varying by signal duration\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eMixed ANOVA revealed a significant main effect for all variables and a four-way interaction. All groups showed reduced signal discrimination as signal duration decreased. MPH affected these discriminations. The ability to discriminate 500ms signals from non-signals significantly decreased in all drug-treated groups, with SAT-500 being significantly greater in the Control group than in all MPH groups. Compared to the Control group, the PD49-69 group showed a reduced ability to discriminate shorter (100 and 25ms) signals from non-signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eAcross sessions, only the Control group showed a (non-significant) upward trend in performance. In contrast, MPH-treated groups, especially PD49-69, remained consistently impaired (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-f). SAT-500 scores were lower in PD49-69 across all sessions; PD42-62 and PD35-55 groups also showed deficits in Sessions 1 and 3, respectively.\u003c/p\u003e\u003cp\u003eNo vigilance decrement over time was observed (no Blocks \u0026times; Treatment interaction), but MPH treatment during PD49-69 led to lower SAT-500 performance across Blocks 1\u0026ndash;3, with additional deficits in Block 2 for PD42-62 and PD35-55 groups. For SAT-100, PD49-69 underperformed relative to Control across all blocks, and for SAT-25, in Block 1(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, c, e).\u003c/p\u003e\u003cp\u003eThe effects on the vigilance index appear to be primarily driven by disruptions in signal detection rather than by deficits in false alarm responses. A visual inspection of Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-c suggests a reduction in the proportion of hits in the MPH groups but no change in the number of CR. PD35-55 and PD49-69 groups showed significantly fewer hits than Control.\u003c/p\u003e\u003cp\u003eBlock-by-block analysis revealed that for 500ms signals, PD49-69 group had lower hit rates in Blocks 1\u0026ndash;3, while PD35-55 and PD42-62 groups showed reduced hits in Block 2. For 100ms signals, differences emerged across all blocks for PD49-69. For 25ms signals, only Block 1 showed significantly reduced hits in this group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, d, f).\u003c/p\u003e\u003cp\u003eThe training effect, measured by changes in hit rates over the three sessions, was also influenced by methylphenidate. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-i, performance in the control group improved across sessions for all signal durations, although not significantly. However, this trend was not observed in the drug-treated groups, particularly in the PD35-55 and PD49-69 groups. This pattern of behaviour led to more pronounced differences between the control and drug-treated groups as the sessions progressed.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eEffects of distractor flashing houselight (dSAT).\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eCompared to the Test phase, the introduction of a flashing houselight significantly reduced animals\u0026rsquo; ability to distinguish signal from non-signal events (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u0026ndash;c). This increased \u0026lsquo;background\u0026rsquo; noise caused a decline in SAT scores for both Control and P49\u0026ndash;69 groups, regardless of signal length, block, or session.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn Control animals, comparisons between the Test and Flash phases revealed that this impairment was session-dependent. During the Flash phase, SAT scores dropped across all blocks in Session 1, but performance gradually improved. Thus, in Session 2, impairments were limited to Block 1, and by Session 3, only Blocks 3 and 4 showed significant reductions, indicating progressive adaptation. In contrast, MPH-treated animals showed persistent impairment, with no improvement across blocks or sessions. Only a main effect of Phase was found, suggesting a generalized negative effect of the flashing houselight on SAT performance.\u003c/p\u003e\u003cp\u003eAnalysis within the Flash phase showed a significant main effect of signal length, with poorer discrimination at shorter durations. The Control group improved across sessions, while MPH-treated animals did not.\u003c/p\u003e\u003cp\u003eFurther analysis showed that SAT impairments were primarily due to reduced hit rates. CR remained stable across sessions and was unaffected by drug treatment. The percentage of hits dropped significantly from Test to Flash phases across all durations, blocks, and sessions, and this decline was evident in both groups. However, intra-phase analysis confirmed that the Control group exhibited progressive recovery across sessions, while MPH-treated animals did not. Across all sessions, the Control group achieved consistently higher hit rates than the MPH group.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur findings consistently demonstrate that MPH consumption during adolescence disrupts PFC maturational processes, with consequences persisting into adulthood. Specifically, our data show that the magnitude of this impact varies depending on the timing of drug exposure within the developmental period. Although a reduction in PV\u0026thinsp;+\u0026thinsp;interneuron density was observed across all MPH-treated groups, only animals exposed to MPH during the PD49\u0026ndash;69 period exhibited a significant disruption in the normal acquisition of PV\u0026thinsp;+\u0026thinsp;interneurons in the PrL. Importantly, this reduction in GABAergic transmission within the PFC was associated with attentional impairments, reflected by poorer performance on a sustained attention task\u0026mdash;particulary under conditions of high attentional demand.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMPH effects on PFC GABAergic interneurons varied across developmental stages\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe differing effects of chronic MPH exposure across distinct postnatal periods\u0026mdash;despite some overlap in treatment windows\u0026mdash;suggest that adolescence is not a uniform developmental phase but rather a period marked by dynamic neurodevelopmental events. Our results align with previous studies emphasizing that the timing of psychostimulant exposure critically shapes drug-induced plasticity, leading to neurobehavioral abnormalities. For instance, MPH treatment during adolescence (PD35-42) has been shown to sensitize responses to rewards [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], whereas animals treated at earlier stages (PD20-35) exhibited reduced sensitivity to natural or drug rewards [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In our study, chronic administration of a therapeutically relevant dose of MPH during three adolescent windows (PD35\u0026ndash;55, PD42\u0026ndash;62, and PD49\u0026ndash;69) resulted in reduced PV\u0026thinsp;+\u0026thinsp;interneurons density in the PrL, although this effect reached significance only in the PD49\u0026ndash;69 group. Consistent with our findings, previous research has shown that exposure to other psychostimulants during adolescence, such as amphetamine or cocaine, reduces inhibitory transmission in the mPFC [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePrevious studies suggest that PFC maturation during adolescence relies on remodeling local inhibitory circuits, driven by glutamatergic inputs from the ventral hippocampus and dopaminergic recruitment, which together facilitate GABAergic interneurons activity, believed to underlie the maturation of cognitive abilities [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In line with this framework, our results indicate that the most pronounced alterations in PV\u0026thinsp;+\u0026thinsp;expression\u0026mdash;and, by extension, in GABAergic transmission within the mPFC\u0026mdash;occurred in animals exposed to MPH during the PD49\u0026ndash;69 window, corresponding to late adolescence. Therefore, it is plausible that disruptions to the normal developmental trajectory of inhibitory signaling in the PFC during this critical period contribute to long-lasting deficits in PFC-dependent cognitive functions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMPH affects GABAergic interneurons in the caudal prelimbic cortex\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur results also indicate that chronic MPH consumption specifically alters the density of PV\u0026thinsp;+\u0026thinsp;GABAergic interneurons in the caudal portion of the PrL. These interneurons are key regulators of the E/I balance in the PFC, playing a crucial role in optimizing the representation and processing of supramodal information. A reduction in the number of PV\u0026thinsp;+\u0026thinsp;interneurons in the PrL may disrupt this balance, potentially leading to increased circuit excitability. Since PV\u0026thinsp;+\u0026thinsp;interneurons are essential for both tonic and phasic inhibition of pyramidal neurons, their loss could result in decreased inhibitory control and heightened pyramidal neuron excitability.\u003c/p\u003e\u003cp\u003eWhile this hypothesis requires further investigation, it is supported by previous electrophysiological studies showing that in adult rats, an acute dose of MPH exerts excitatory effects on PFC pyramidal neurons, particularly in prelimbic, cingulate, and medial regions [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Consistent with our findings, Di Micelli et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] reported that chronic MPH administration during late adolescence (PD42) resulted in a persistent increase in PFC pyramidal neurons firing rates into adulthood, without affecting NMDA-mediated neurotransmission. Other studies have demonstrated that chronic MPH effects on PFC neuronal excitability are age-dependent, with adult rats showing increased spike activity in layer V pyramidal neurons, whereas juveniles exhibited a transient suppression of excitability [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Similarly, Morshedi and Meredith [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] found a significant reduction in PV\u0026thinsp;+\u0026thinsp;density in layer V of the PrL -but not in the IL- following repeated amphetamine treatment, an effect absent with acute administration. This region-specific reduction in PV immunoreactivity suggests that PrL neurophysiology is particularly vulnerable to the long-term effects of repeated psychostimulant exposure.\u003c/p\u003e\u003cp\u003eThere are several potential mechanisms that may underlie the observed increases in PFC-pyramidal neuron firing rates following chronic MPH exposure. Some studies have suggested that these effects could be attributable to altered GABAergic interneurons activity. For instance, a single 1 mg/kg dose of MPH in juvenile rats enhances the excitation of GABAergic interneurons [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Moreover, studies with other psychostimulants corroborate the impact on GABAergic transmission. Repeated cocaine exposure during early adolescence, for example, induces mPFC disinhibition through long-lasting impairments in the local GABAergic networks [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Similarly, repeated amphetamine exposure has been associated with altered interneuron physiology, including decreased sensitivity to D1 receptor stimulation and reduced inhibitory output [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eMPH effects on PFC-PV\u0026thinsp;+\u0026thinsp;interneurons are independent of sex\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn contrast to studies suggesting that estrogen modulates PV\u0026thinsp;+\u0026thinsp;interneuron activity and influences GABAergic signaling reorganization [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], our findings revealed no significant sex differences in PV\u0026thinsp;+\u0026thinsp;interneuron expression following repeated MPH exposure. This is particularly notable since our study included both males and females subjects at pubertal onset, when sex-dependent differences were anticipated. The absence of such differences may indicate that the mechanisms regulating PV\u0026thinsp;+\u0026thinsp;interneuron acquisition are hormonally independent or, as proposed by Torres-Reveron et al [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], the estrous cycle and estrogen levels may not affect PV\u0026thinsp;+\u0026thinsp;cell numbers but rather their availability for inhibitory transmission. PV neurons, characterized by fast-spiking activity and high metabolic demands, are particularly susceptible to oxidative stress [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], despite partial protected by perineuronal nets (PNNs) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Considering that repeated amphetamine exposure induces oxidative damage in brain regions such as the mPFC [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], we hypothesize that persistent oxidative stress caused by MPH \u0026ndash;a sex-independent insult\u0026ndash; may disrupt normal neuronal activity. This disruption could lead to cell death or, more likely, selectively impair the mechanism by which calretinin is replaced by parvalbumin, resulting in deficient PV\u0026thinsp;+\u0026thinsp;upregulation rather than a loss of PV\u0026thinsp;+\u0026thinsp;cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eReduced GABAergic activity in the caudal prelimbic cortex contributes to impairments in sustained attention.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur findings show that the decrease in PV\u0026thinsp;+\u0026thinsp;interneurons in the PrL induced by early exposure to MPH significantly impairs performance on a sustained attention task in adulthood. The degree of impairment depended on the developmental window of MPH administration.\u003c/p\u003e\u003cp\u003eSpecifically, MPH exposure from PD42\u0026ndash;62, which caused a minor reduction in PV\u0026thinsp;+\u0026thinsp;neuron density, resulted in minimal alterations in task performance compared to the Control group. In contrast, MPH exposure during PD35-55 resulted in a slightly greater reduction in PV\u0026thinsp;+\u0026thinsp;interneuron density, along with more pronounced impairments in task performance. The most severe impairments were observed in animals exposed to MPH during PD49-69, the only group exhibiting a statistically significant reduction in PV\u0026thinsp;+\u0026thinsp;density compared to controls. This group showed consistently poorer performance than the Control group on both overall SAT scores and hit rates. However, CR rates remained close to 80% across all MPH groups and did not differ from controls.\u003c/p\u003e\u003cp\u003eExposure to a flashing houselight increased the demands on top-down attentional control, reducing the animals' ability to discriminate signal from non-signal events in both control and MPH-treated groups. Analyses revealed that this attention impairment was primarily driven by decreased hit rates, as CR performance remained stable from Test to dSAT phases, with no group differences observed during the distractor condition. However, control animals adapted to this increased attentional load, displaying performance deficits only in early blocks of the first session. In contrast, animals treated from PD49-69 did not recover performance across sessions or blocks, indicating an inability to enhance top-down attentional control over time.\u003c/p\u003e\u003cp\u003eThese findings support the notion that GABAergic activity in the PFC is critical for sustaining attention, as its reduction increases failures to detect signal events. We hypothesize that reduced PV\u0026thinsp;+\u0026thinsp;interneuron density in the PrL cortex could lead to a lower vigilance index and fewer hits, while leaving false alarm rates unaffected. This pattern indicates that attentional deficits from reduced GABAergic function in the PrL arise mainly from impaired signal detection rather than impulsivity, deficits in rule comprehension, or bottom-up sensory processing. In this regard, our results align with Kim et al. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], who showed that FS-PV interneuron activity in the mPFC is essential for detecting relevant stimuli, but not for inhibiting responses to non-relevant events. Similarly, Fisher et al. [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] demonstrated that specific prelimbic lesions produce persistent deficits in sustained attention, especially under high attentional load or distraction, impairing signal discrimination sensitivity (d') and hit rates without significantly altering inhibitory control.\u003c/p\u003e\u003cp\u003eBesides maintaining the E/I balance in the PFC, PV\u0026thinsp;+\u0026thinsp;interneurons also play a key role in maintaining a high signal-to-noise ratio during information processing, facilitating the selection of relevant stimuli for cognitive operations such as attentional engagement [\u003cspan additionalcitationids=\"CR48 CR49\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The errors observed in our study likely stem from disruptions in the cognitive processing of signals and their translation into goal-directed behavior, or may result from interference generated by irrelevant cognitive activity [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Specifically, the hit deficit may reflect an inability to maintain sufficient attentional engagement at signal onset due to the loss of precise inhibitory modulation by PV\u0026thinsp;+\u0026thinsp;interneurons in the PrL. In contrast, preserved performance in non-signal trials suggests that tonic or baseline inhibitory control remains intact, indicating a selective disruption of attentional processing of external stimuli, as proposed in cortical GABAergic interneuron-deficient models [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis pattern points to a deficit in top-down attentional control, a function critically dependent on the integrity of the prelimbic cortex and its PV\u0026thinsp;+\u0026thinsp;interneurons. Supporting this possibility, evidence indicates that FS-PV interneuron activity in the mPFC is essential for maintaining attention on relevant stimuli, especially in the presence of distractors [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Disruption of this region through pharmacological manipulation or lesions has also been shown to impair attentional accuracy and reduce the capacity to adapt under increasing task demands such as persistent distraction [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The absence of recovery in the PD49\u0026ndash;69 group suggests that PV\u0026thinsp;+\u0026thinsp;interneuron reduction compromises the attentional system\u0026rsquo;s ability to adapt and filter out irrelevant information, preventing improvement with practice in high-demand conditions.\u003c/p\u003e\u003cp\u003eTaken together, these results demonstrate significant attentional impairment in the PD49\u0026ndash;69 group, underscoring the importance of maintaining appropriate PV\u0026thinsp;+\u0026thinsp;neuron density in the prelimbic cortex for accurate visual stimulus detection and the development of attentional processes. Notably, the reduction in PV\u0026thinsp;+\u0026thinsp;neuron density\u003c/p\u003e\u003cp\u003eresulting from prolonged exposure to moderate doses of MPH was relatively modest; nevertheless, measurable attentional deficits still emerged. The absence of more severe outcomes may reflect compensatory mechanisms in the brain that help mitigate such damage or fluctuations. These findings have important implications, suggesting that misuse of drugs such as MPH during adolescence\u0026mdash;whether due to ADHD misdiagnosis or recreational use\u0026mdash;can lead to maturational delays and adverse effects on attentional function. Overall, these results highlight the vulnerability of attentional mechanisms to changes in PV\u0026thinsp;+\u0026thinsp;neuron density; and underscore the need for further research to clarify long-term implications across other brain regions and behavioral outcomes to fully assess the relevance of these findings in humans.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our findings demonstrate that chronic administration of 5 mg/kg methylphenidate during adolescence\u0026mdash;specifically during the PD49\u0026ndash;69 period\u0026mdash;blocks the normal gain of PV\u0026thinsp;+\u0026thinsp;interneurons in the prelimbic cortex, but not in the infralimbic region, in both male and female Wistar rats. This alteration disrupts attentional maintenance and visual discrimination abilities, with effects persisting into adulthood. These results underscore the critical role of PV\u0026thinsp;+\u0026thinsp;interneurons in sustained attention and suggest that there are specific windows of vulnerability during the functional maturation of the medial prefrontal cortex.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP\u0026eacute;rez-Colorado, A.: Writing \u0026ndash; original draft, Visualization, Investigation, Formal analysis, Conceptualization. Mart\u0026iacute;nez-Mar\u0026iacute;n, R.: Writing \u0026ndash; review \u0026amp; editing, Visualization, Investigation, Formal analysis. L\u0026oacute;pez, J.C.: Writing \u0026ndash; review \u0026amp; editing, Visualization, Supervision, Project administration, Funding acquisition, Resources, Formal analysis, Conceptualization. Vargas, J.P.: Writing \u0026ndash; review \u0026amp; editing, Supervision, Project administration, Funding acquisition, Formal analysis, Conceptualization. D\u0026iacute;az, E.: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization.\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThis research was funded by Agencia Estatal de Investigación (AEI) of Spain PID2019- 110739GB-I00/AEI/10.13039/501100011033 and PID2023-149901NB-I00 funded by MICIU/AEI/ 10.13039/501100011033 and FEDER/EU.\u003c/p\u003e\n\u003col start=\"2\"\u003e\n \u003cli\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThe authors have nothing to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBlum, K., Chen, A. L., Braverman, E. R., Comings, D. E., Chen, T. J., Arcuri, V., Blum, S. H., Downs, B. W., Waite, R. 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Prefrontal cortical GABAergic and NMDA glutamatergic regulation of delayed responding. \u003cem\u003eNeuropharmacology\u003c/em\u003e, \u003cstrong\u003e113\u003c/strong\u003e(Pt A), 10\u0026ndash;20. https://doi.org/10.1016/j.neuropharm.2016.09.022\u003cu\u003e\u003c/u\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"c146a9ae-d54b-4f48-8389-de85ad0c26b4","identifier":"10.13039/100014440","name":"Ministerio de Ciencia, Innovación y Universidades","awardNumber":"PID2023-149901NB-I00/MICIU/AEI/ 10.13039/501100011033 and FEDER/EU","order_by":0},{"identity":"338923e7-5e0a-41d8-b3e8-f466579c4ef0","identifier":"10.13039/100014440","name":"Ministerio de Ciencia, Innovación y Universidades","awardNumber":"PID2019- 110739GB-I00/AEI/10.13039/501100011033","order_by":1}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Seville","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"parvalbumin, adolescence, methylphenidate, attention, prelimbic","lastPublishedDoi":"10.21203/rs.3.rs-7176694/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7176694/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAdolescence is a critical period for the maturation of the medial prefrontal cortex (mPFC), particularly involving the reorganization of the GABAergic network mediated by parvalbumin-expressing (PV+) interneurons. Methylphenidate (MPH), the most commonly prescribed treatment for attention-deficit/hyperactivity disorder (ADHD), is often administered over prolonged periods starting in childhood. However, sustained dopaminergic stimulation during adolescence may interfere with mPFC development by altering dopamine-dependent excitability of PV+ interneurons. In this study, we investigated whether chronic MPH exposure during adolescence affects the acquisition of PV+ interneurons and whether such alterations in GABAergic activity lead to long-lasting impairments in mPFC-dependent functions such as sustained attention. Male and female Wistar rats received 5 mg/kg MPH (a therapeutically relevant dose) for 20 days, beginning in early (PD35–55), middle (PD42–62), or late adolescence (PD49–69). From PD100 onward, animals were tested on a sustained attention task requiring lever presses in response to signal (hit) or non-signal (correct rejection, CR) trials. Once baseline performance reached \u0026gt;75% correct, stimulus durations were reduced to 500 ms, 100 ms, and 25 ms to increase attentional demand. Our results revealed a selective reduction in PV+ interneuron density in the prelimbic, but not infralimbic, cortex—predominantly in animals exposed to MPH during PD49–69. This reduction was associated with persistent deficits in attentional performance in adulthood. These findings highlight the critical role of PV+ interneurons in attentional processes and identify late adolescence as a window of heightened vulnerability during mPFC maturation.\u003c/p\u003e","manuscriptTitle":"Developmental window of vulnerability to methylphenidate: Selective reduction of prelimbic PV+ interneurons impairs adult attention","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-23 15:19:29","doi":"10.21203/rs.3.rs-7176694/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"11909553-75c3-46c5-86dd-52fa9c793c0b","owner":[],"postedDate":"July 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-23T15:19:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-23 15:19:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7176694","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7176694","identity":"rs-7176694","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}