Diazepam modulates hippocampal CA1 functional connectivity in people at clinical high-risk for psychosis

schizophrenia; benzodiazepine; neuroimaging; pharmacological MRI; resTng- state; early intervenTon . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

Background: Preclinical evidence suggests that diazepam enhances hippocampal γ- aminobutyric acid (GABA) signalling and normalises a psychosis -relevant corTco -limbic- striatal circuit. Hippocampal network dysconnecTvity, parTcularly from the CA1 subfield, is evident in people at clinical high-risk for psychosis (CHR-P), represenTng a potenTal treatment target. This study aimed to forward-translate this preclinical evidence.

In this randomised, double-blind, placebo-controlled study, 18 CHR-P individuals underwent resTng-state funcTonal magneTc resonance imaging twice, once following a 5mg dose of diazepam and once following a placebo. They were compared to 20 healthy controls (HC) who did not receive diazepam/placebo . FuncTonal connecTvity (FC) between the hippocampal CA1 subfield and the nucleus accumbens (NAc), amygdala, and ventromedial prefrontal cortex (vmPFC) was calculated . Mixed-effects models invesTgated the effect of group (CHR-P placebo/diazepam vs. HC) and condiTon (CHR-P diazepam vs. placebo) on CA1- to-region FC.

In the placebo condiTon, CHR-P individuals showed significantly lower CA1-vmPFC (Z=3.17, PFWE=0.002) and CA1-NAc (Z=2.94, PFWE=0.005) FC compared to HC. In the diazepam compared to placebo condiTon , CA1 -vmPFC FC was significantly increased (Z=4.13, PFWE=0.008) in CHR-P individuals, and both CA1 -vmPFC and CA1-NAc FC w ere normalised to HC levels. In contrast, compared to HC, CA1-amygdala FC was significantly lower contralaterally and higher ipsilaterally in CHR-P individuals in both the placebo and diazepam condiTons (lower: placebo Z=3.46, PFWE=0.002, diazepam Z=3.33, PFWE=0.003; higher: placebo Z=4.48, PFWE<0.001, diazepam Z=4.22, PFWE<0.001).

This study demonstrates that diazepam can parTally restore hippocampal CA1 dysconnecTvity in CHR-P individuals, suggesTng that modulaTon of GABAergic funcTon might be useful in the treatment of this clinical group. . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint Introduc6on IdenTfying novel pharmacological intervenTons to reduce symptom severity and prevent transiTon to psychosis in individuals at clinical high -risk for psychosis (CHR-P) is a significant unmet clinical need1,2. Current neurobiological theories of psychosis development idenTfy the hippocampus as a central hub of pathophysiology47,59,107 and a promising pharmacological target6. Several neuroimaging studies in individuals at CHR -P have idenTfied increased hippocampal cerebral blood flow/volume compared to healthy controls (HC) 3–5. The cornu ammonis 1 (CA1) subfield is proposed to be the origin of hippocampal dysfuncTon in the CHR- P state, in terms of volume loss 7 and hyperacTvity8,9, which then spreads to the subiculum following psychosis onset 9. The CA1 and subiculum have a high number of glutamatergic efferent projecTons 10, and anterior projecTons innervate a corTco -limbic-striatal circuit encompassing the nucleus accumbens (NAc) of the striatum, amygdala, and the ventromedial prefrontal cortex (vmPFC)11. These regions are highly interconnected11–19 and are associated with posiTve, negaTve, and cogniTve symptoms of schizophrenia, respecTvely20–22. Therefore, hippocampal dysfuncTon preceding the onset of psychosis may disrupt downstream corTco- limbic-striatal regions, contribuTng to circuit dysfuncTon and the emergence of psychosis11. Circuit dysfuncTon can be invesTgated in terms of the funcTonal connecTvity (FC) between brain regions measured using resTng-state funcTonal magneTc resonance imaging (rs-fMRI)23. rs-fMRI studies have idenTfied altered hippocampal FC with the corTco -limbic-striatal circuit in individuals with a first episode of psychosis or chronic schizophrenia compared to HC. More specifically, these studies reported lower hippocampal FC with the striatum 24–30 and vmPFC24,26,28,29,29,31–40, and either lower 38,41, higher42, or unaltered42 hippocampal FC to the amygdala. The paFern is less clear in subclinical psychosis spectrum individuals (although there are far fewer studies) : lower hippocampal -striatal FC has been shown in healthy individuals with high schizotypy traits 43,44, while both lower 25,34 and normal 33,45,46 hippocampal-striatal and hippocampal -PFC FC ha ve been observed in individuals at CHR -P compared to HC. To our knowledge , no studies in CHR -P individuals have invesTgated hippocampal-amygdala FC, or FC alteraTons from specific hippocampal subfields to the corTco-limbic-striatal circuit. Given that hippocampal dysfuncTon may be localised to the CA1 subfield in the CHR -P stage 8, alteraTons in FC may may not be present across the whole hippocampus. . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint GABAergic dysfuncTon has been proposed as a key mechanism underlying hippocampal hyperacTvity in psychosis47. Studies in rats exposed to the mitotoxin methylazoxymethanol acetate (MAM) during neurodevelopment showed that reduced PV+ interneuron number in the hippocampus was associated with an increased firing rate of local excitatory neurons and excitatory/inhibitory imbalance48. This hyperacTvity is found to drive funcTonal alteraTons of downstream regions in MAM -treated rats, evidenced by experiments where chemical48 or pharmacological inacTvaTon of the hippocampus (with a nonspecific GABA A-enhancing benzodiazepine49 or an α5-GABAA specific compound49,50) normalised midbrain dopaminergic neuron firing. Furthermore, this mechanism is proposed to underlie the findings that repeated peripubertal diazepam administraTon in MAM -treated rats prevented the emergence of schizophrenia-related neurophysiological and behavioural phenotypes in adulthood . Such phenotypes included prevenTon of midbrain dopamine hyperacTvity and hyperlocomoTon response to amphetamine (posiTve symptoms), amygdala hyperacTvity (negaTve symptoms), and PFC dysfuncTon (cogniTve symptoms)51–53. This preclinical evidence suggests that GABA-enhancing compounds may be an effecTve strategy for psychosis prevenTon by downregulaTng hippocampal hyperacTvity and normalising downstream circuit dysfuncTon. In healthy individuals, prior rs-fMRI studies using an acute, non -sedaTng dose of a GABA -enhancing compound report increases in FC under benzodiazepine (or other GABA -enhancing drugs e.g., Z -drugs such as zopiclone/zolpidem ) compared to placebo across the hippocampal-amygdala-PFC circuit 54, the default mode network55,56, and a wider brain network including visual, auditory, sensorimotor , and prefrontal regions57. In CHR -P individuals, w e recently demonstrated that an acute, non - sedaTng dose of diazepam normalised elevated hippocampal and subfield cerebral blood flow towards levels seen in healthy controls 58. However, whether this is accompanied by a normalisaTon of the FC between the hippocampus and downstream corTco -limbic-striatal regions was not known . Therefore, the current study examined the effects of an acute dose of diazepam vs. placebo on FC between the hippocampus and this corTco -limbic-striatal circuit in the same cohort of CHR-P individuals58. Each condiTon was also compared to HC data collected on the same scanner. We focussed on the CA1 subfield as a seed, g iven its proposed role in psychosis . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint development59 and the substanTal number of anatomical connecTons to output regions of interest (NAc, amygdala, and vmPFC60,61). On the basis of previous findings in hippocampal FC across the psychosis spectrum 16–26,29–32,34–36,41,43, we hypothesised that individuals at CHR -P (in the placebo condiTon) would display lower CA1-NAc and CA1-vmPFC FC and altered CA1- amygdala FC compared to HC . Based on prior benzodiazepine challenge rs -fMRI studies in healthy individuals50–53, we hypothesise d that a single dose of diazepam would increase CA1 FC within this circuit, to the extent that it would no longer differ from HC. For completeness, the following supplementary analyses were included : i) using the anterior hippocampus as a seed (given it is specifically the anterior porTon of the CA1 implicated in psychosis development8,9) and ii) exploring broader effects of diazepam on CA1/anterior hippocampus FC with the rest of the brain. . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint

and Materials Study design, par0cipants, and procedure This experimental medicine study was conducted at King’s College London. The study received ethical approval from the NaTonal Health Service UK Research Ethics CommiFee (18/LO/0618), and each parTcipant gave wriFen informed consent. While the study received ethical clearance as ‘not a Clinical Trial of an InvesTgaTonal Medicinal Product’ by the EU direcTve 2001/20/EC, it was registered on clinicaltrials.gov (NCT06190483). Full study details, including inclusion/exclusion criteria, can be found in our recent publicaTon describing the hippocampal cerebral blood flow findings in the same parTcipants58. Briefly, this study used a randomised, double-blind, placebo-controlled, crossover design, whereby 24 anTpsychoTc - naïve individuals at CHR-P underwent MRI scanning on two occasions, once following a single oral dose of diazepam (5mg) and once following an oral placebo (50mg ascorbic acid). The diazepam/placebo capsule was administered 60 minutes before MRI scanning, and there was a minimum 3-week washout period between scans. Data from a group of 22 HC from a prior study (PSYAUD17/25) acquired with the same MRI scanner, scanning sequences, and acquisiTon parameters were used as a comparison group65. MRI acquisi0on MRI data were acquired on a General Electric MR750 3.0T MR scanner with an 8-channel head coil at the Centre for Neuroimaging Sciences, KCL. A 3D T1-weighted scan was acquired using a SPGR sequence and rs-fMRI data was acquired using a mulT -echo echo planar imaging sequence (full acquisiTon details in Supplementary Methods) . During the rs -fMRI scan, parTcipants were instructed to remain awake with their eyes open , while a fixaTon cross was displayed in the centre of the screen. Neuroimaging data processing Preprocessing The structural and rs -fMRI data were preprocessed using fMRIPrep (version 23. 1.3)66, SPM1267, CONN 68, and FSL 69. Structural images from both sessions were corrected for intensity non-uniformity using N4, skull-stripped, segmented, and averaged across sessions to generate a singular parTcipant structural image which was then normalised to MNI space . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint (1mm3 resoluTon)66. For the rs -fMRI data, volume re -alignment and slice -Tming correcTon parameters were calculated using the first echo and applied to all echoes66. ParTcipants were excluded if they moved >3mm on any translaTon/rotaTon parameter or had a mean framewise displacement of >0.5mm, as advised by prior methodological invesTgaTons70. The three echoes in naTve space underwent TE -dependent ICA -based denoising and were opTmally-combined using T2* weighted averaging via TEDANA71, before being normalised to MNI space (2mm 3 resoluTon) with transformaTons generated during fMRIPrep (see supplementary materials for full boiler plate) 66. The denoised, opTmally combined , normalised funcTonal data was then spaTally smoothed in SPM 1267 with a 6mm FWHM Gaussian kernel, and further denoised by removing white maFer and CSF signal using the first 5 components of aCompCor, despiking, scrubbing, and band -pass filtering (0.008-0.09 Hz) in CONN68. Genera/on of seed and region -of-interest masks Hippocampal and subfield seed masks were generated for each parTcipant from their preprocessed structural scan collected during their first scanning visit using the MAGeT Brain (mulTple automaTcally generated templates of different brains) toolbox72 (see previous publicaTon for further details58). Using all parTcipants’ CA1 segmentaTons, study-specific lew and right CA1 masks were generated by using majority vote (ANTs/2.5.0; Figure 1). ROI masks for the corTco -limbic-striatal circuit (NAc, amygdala, and vmPFC) were derived from Neurosynth ( hFps://www.neurosynth.org/ ) using the search terms ‘nucleus accumbens’, ‘amygdala’, and ‘vmPFC’ (uniformity tests). The resulTng images were thresholded, binarised, and dilated (MINC toolkit; hFps://bic -mni.github.io/). Neuroimaging data analysis To control for the number of models , FDR correcTon was performed on all FWE-corrected second-level analyses described below. First- and second-level analysis To generate parTcipant -level seed -to-voxel Z-maps, the mean funcTonal Tme series was extracted from the lew and right CA1 and used in first-level analysis models as regressors of interest in FSL. These first -level seed-to-voxel Z-maps were then entered into second -level analysis models using FLAME -1 (FMRIB’s Local Analysis of Mixed Effects) 69, which employs . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint Bayesian modelling and a weighted least -squares approach to perform a mixed-effects analysis. FLAME-1 was chosen as mixed-effects modelling is opTmal for within-subject designs (i.e., CHR-P diazepam vs. placebo) to account for within -subject correlaTons, and FLAME-1 is able to esTmate different variances for different groups of subjects within a model, which is advantageous for unpaired two -sample comparison (i.e., CHR -P vs. HC) 73. All models below use an FWE-corrected (P2.3. This threshold with FLAME -1 models has been shown to produce FWE rates lower than 5% , and is therefore similar to tradiTonal FSL ordinary least square analyses using a threshold of Z>3.174. Within-group CA1 res/ng -state FC analyses Before comparing differences between groups/condiTons, we first validated within -group resTng-state FC networks for the CA1 to the whole brain to ensure they matched expected networks (one-sample contrast for each group independently)75. Group and condi/on s eed-to-ROI analyses To invesTgate the effect of group (CHR -P placebo/diazepam vs. HC) and condiTon (CHR -P diazepam vs. placebo) on FC differences between CA1 and corTco -limbic-striatal circuit regions, we conducted seed -to-ROI analysis. Second-level models were run per seed -to-ROI per hemisphere for each group/condiTon comparison using a small volume adjustment approach by applying a pre-threshold ROI mask generated from Neurosynth as described above. Models were run both contralaterally (e.g., lew CA1 to the right amygdala) and ipsilaterally (e.g., lew CA1 to the lew amygdala), as disrupTons to both have been found across the psychosis spectrum within this circuit 76. Voxel-level thresholding was used ( Z>2.3) for inference, which was FWE-corrected (P<0.05) for mulTple comparisons. Again, this threshold has been demonstrated to be quite conservaTve when using voxel-level inference in FLAME- 1 models74. For CHR-P placebo/diazepam vs. HC models, age (mean centred) and sex were added in as covariates of no interest. For CHR-P diazepam vs. placebo, change in pre-post scan faTgue score from the Bodily Symptoms Scale77 for each condiTon was included as a covariate of no interest to control for drug effects of sedaTon/faTgue. . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint Supplementary / exploratory analyses For completeness, supplementary analyses explored the effect of group/condiTon on FC between 1) anterior hippocampus and the corTco-limbic-striatal circuit (seed-to-ROI), and 2) hippocampal seeds (CA1 and anterior hippocampus) and the rest of the brain on a voxel-wise basis. Anterior hippocampal masks were generated by masking the study -specific averaged whole hippocampus segmentaTon with a hippocampus head mask derived from the Allen human reference atlas 78, then thresholded, binarized, and dilated . IdenTcal second -level models were run as described above , and for the seed -to-voxel analyses an inclusive grey maFer mask was used during the pre-threshold masking. . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint

Demographics and Clinical Assessments Following data quality checks, 6 CHR-P parTcipants were excluded (n=2 missing rs-fMRI data, n=1 poor data quality, n=3 excessive moTon), along with 2 HC parTcipants (n=1 missing rs - fMRI data, n=1 poor data quality) . This resulted in a final sample of 18 CHR -P and 20 HC for analyses. ParTcipant details can be found in Table 1. CHR-P (n = 18) HC (n = 20) Comparison t/χ2 p Demographic Age (years; mean ±SD) 24.1 ±4.6 26.5 ±5.1 CHR-P vs. HC 1.5 0.136 Sex (male/female; n) 5/13 9/11 CHR-P vs. HC 1.2 0.272 Ethnicity (n) CHR-P vs. HC 11.2 0.024 Asian 1 6 - - - Black 4 0 - - - Mixed or multiple 2 0 - - - Other 1 0 - - - White 10 14 - - - IQ (WAIS-III short version81; mean ±SD) 96.0 ±22.1 122.9 ±13.9 CHR-P vs. HC 4.4 <0.001 Current daily cigarette use, n (%) 4 (22) 2 (10) CHR-P vs. HC 1.1 0.302 Current alcohol use, n (%) 14 (77) 18 (90) CHR-P vs. HC 1.1 0.302 Current cannabis use, n (%) 5 (28) 3 (15) CHR-P vs. HC 1.8 0.181 Clinical characteristics CAARMS83 score (mean ±SD) Positive symptoms 47.5 ±12.9 NA - - - Negative symptoms (n=21) 29.5 ±25.3 NA - - - Total (n=21) 77.9 ±28.2 NA - - - Global functioning score 85 (mean ±SD) Social 6.3 ±1.5 NA - - - Role 6.1 ±1.7 NA - - - Hamilton scale score (mean ±SD) Anxiety87 (n=22) 17.6 ±9.3 NA - - - Depression89 (n=21) 13.5 ±6.9 NA - - - Current antidepressant medication, n (%) 7 (38) NA - - - Current or prior antipsychotic medication, n (%) 0 (0) NA - - - Current benzodiazepine/hypnotic medication, n (%) 0 (0) NA - - - Head motion Fractional displacement in mm (mean ±SD) Total group 0.148 ±0.08 0.146 ±0.07 CHR-P vs. HC 0.05 0.957 Placebo condition 0.156 ±0.10 - CHR-P placebo vs. HC 0.36 0.724 Diazepam condition 0.139 ±0.07 - CHR-P diazepam vs. HC -0.35 0.722 - - CHR-P diazepam vs. placebo 0.62 0.544 Bodily Symptoms Scale Fatigue scores post-scan (mean ±SD) Placebo condition 0.944 ±0.93 - CHR-P diazepam vs. placebo 1.51 0.148 Diazepam condition 1.38 ±1.58 - - - - Table 1. Par;cipant demographic informa;on, clinical characteris;cs, head mo;on parameters and fa;gue scores CAARMS: comprehensive assessment of at -risk mental states; CHR -P: clinical high -risk for psychosis; HC: healthy control; IQ: intelligent quoBent; WAIS: Weschler adult intelligence scale . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint CHR-P individuals had a significantly lower IQ (as is owen observed in this clinical populaTon79) and differed in terms of ethnicity compared to the HC group (which was driven by a high proporTon of white ethnicity in the HC group ). There were no significant differences in head moTon parameters or change between pre - and post-scan Bodily Symptom Scale 77 scores between the placebo and diazepam condiTons. Res0ng-State Func0onal Connec0vity Within-group CA1 res/ng -state FC Within each group/condiTon, as expected75, the CA1 showed significant FC with the rest of the hippocampus, extending to the temporal lobe, amygdala, precuneus, posterior cingulate cortex, mPFC, and parieto-occipital regions (Z>2.3, PFWE<0.05; Figure 1). CA1-to-voxel funcOonal connecOvity networks averaged across each group independently (healthy controls, CHR-P placebo, and CHR-P diazepam) for the leX and right CA1 subfield using study-specific mask (Z > 2.3, PFWE < 0.05). CHR-P: clinical high-risk for psychosis Figure 1. Within-group CA1-to-voxel func8onal connec8vity networks CHR-P placebo CHR-P diazepam Right Left CA1 CHR-P placebo CHR-P diazepam Healthy controls Healthy controls . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint CA1-to-ROI Compared to HC, individuals at CHR-P in the placebo condiTon showed significantly lower FC between the lew CA1 and the right NAc (Figure 2A, Table 2), and between the right CA1 and the lew NAc, lew amygdala, and lew vmPFC (Figure 2B, Table 2). Figure 2. Region-of-interest func8onal connec8vity results for the CA1 Left CA1 A Right CA1 B Parameter esOmates of funcOonal connecOvity strength between leX (A) and right (B) CA1 and output regions (nucleus accumbens, amygdala, and ventromedial prefrontal cortex) displayed for healthy controls and individuals at clinical high -risk for psychosis (in the placebo and diazepam condiOons) at peak coordinate of significant effect of group/condiOon (Z > 2.3, PFWE < 0.05). CA1 (green), amygdala (red), nucleus accumbens (yellow), and vmPFC (purple) are visualised on the brain using masks. CHR-P clinical high -risk for psychosis; Amy: amygdala; NAc: nucleus accumbens; vmPFC: ventromedial prefrontal cortex; *** < 0.001; * < 0.05, ns not significant Left CA1 A Right CA1 B . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint AddiTonally, the right CA1 showed higher FC to the right amygdala (Figure 2B, Table 2). In the diazepam condiTon, these differences observed in the placebo condiTon compared to HC were ameliorated (no significant difference) , apart from the right CA1 to lew and right amygdala, which sTll showed significantly lower and higher FC compared to HC, respecTvely (Figure 2B, Table 2). We observed a significant drug effect on CA1-vmPFC FC, where diazepam (compared to placebo) significantly increased the FC strength from the lew CA1 to lew vmPFC and right CA1 to bilateral vmPFC (Figure 2, Table 2). Supplementary / exploratory analyses At the whole -brain level, compared to HC, individuals at CHR -P in the placebo condiTon showed significantly higher FC between the right CA1 and a right medial temporal network including the hippocampus, insula, and inferior/medial temporal gyri (Figure 3A, Supplementary Table 1). Conversely, lower FC was observed between the right CA1 and a lew medial temporal network that extended to include key regions of the default mode network (bilateral mPFC, anterior cingulate cortex , and posterior cingulate cortex) . In the diazepam condiTon, higher FC between right CA1 and a right medial temporal lobe network was also Contrast Seed ROI Peak Z x y z pFDR value # voxels CHR-P placebo > HC Right CA1 Right amygdala 4.48 30 -8 20 HC Right CA1 Right amygdala 4.22 28 -8 -22 CHR-P placebo Right CA1 Left amygdala 3.46 -20 -6 -24 CHR-P diazepam Right CA1 Left amygdala 3.33 -20 -6 -22 0.002 44 CHR-P diazepam > placebo Right CA1 Right vmPFC 4.42 10 46 -6 <0.001 79 Left vmPFC 3.25 -10 48 0 0.002 3 Left CA1 Left vmPFC 3.20 -4 38 -12 0.002 8 CHR-P: clinical high-risk for psychosis; FDR: false discovery rate; HC: healthy control; NAc: nucleus accumbens; vmPFC: ventromedial prefrontal cortex Table 2. Summary sta;s;cs of region-of-interest func;onal connec;vity results for the CA1 . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint Figure 3. Voxel-wise whole-brain func8onal connec8vity results for the CA1 Significant clusters showing differences (Z > 2.3, PFWE HC) are displayed in red colourbar, whilst areas displaying funcOonal hypoconnecOvity are displayed in blue. N.B., no significant differences were found for the anterior hippocampus, nor for any of the regions (CA1 or anterior hippocampus) when contrasOng CHR-P diazepam vs. CHR-P placebo. CHR-P clinical high-risk for psychosis; HC healthy controls observed compared to HC, and addiTonally extended to parieto-occipital regions such as the angular gyrus (Figure 3B, Supplementary Table 1 ). When comparing CHR -P diazepam vs. placebo condiTons directly, no significant differences in whole -brain FC were observed for either the right or lew CA1. Finally, there were no significant differences between groups (CHR- P placebo/diazepam vs. HC) or condiTons (CHR -P diazepam vs. placebo) in FC strength using the anterior hippocampus as a seed on a ROI or whole-brain level. . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint

The main finding of the current study was that a single, non -sedaTng dose of the GABA - enhancing drug diazepam parTally normalised CA1 dysconnecTvity to a corTco-limbic-striatal circuit in individuals at CHR-P. More specifically, CHR-P individuals in the placebo condiTon (compared to HC) showed lower CA1-vmPFC and CA1 -NAc FC. Diazepam significantly increased CA1-vmPFC FC compared to placebo , and the lower CA1-vmPFC and CA1-NAc FC observed in the placebo condiTon was normalised to HC levels. We observed more complex

for CA1-amygdala FC, as CHR-P individuals in the placebo condiTon showed lower and higher FC compared to HC, which were sTll present in the diazepam condiTon. Previously, we demonstrated that diazepam normalised increased hippocampal and subfield regional cerebral blood flow in the same CHR-P individuals, and here we extend this work by showing that diazepam can also parTally normalise CA1 dysconnecTvity to a downstream circuit. Taken together, these results indicate that GABA-enhancing compounds can rescue brain funcTon in a psychosis -relevant circuit in CHR -P individuals , and therefore show promise as a novel treatment strategy for clinical intervenTon in this group. Our finding of lower CA1-vmPFC and CA1-NAc FC contralaterally (but normal FC ipsilaterally) in CHR-P individuals in the placebo condiTon (vs. HC) is consistent with prior rs -fMRI reports of subtle dysconnecTvity in sub-clinical psychosis populaTons25,33,34,41,43–46. In contrast, studies in first-episode and chronic schizophrenia samples consistently report lower FC between these regions24–28,28–40. This may suggest that in psychosis vulnerability stages , as hippocampal hyperacTvity begins to drive glutamatergic input to the corTco -limbic-striatal circuit11,20,21, there is preserved temporal coherence (i.e., FC) between the hippocampus and these regions. As CHR-P symptoms persist, hippocampal hyperacTvity and dysrhythmia may lead to uncoupling between the hippocampus and downstream circuitry, which may further deteriorate following the onset of psychosis. For example, experiments in MAM-treated rats demonstrated that NAc hyperacTvity due to hippocampal dysfuncTon , drives a striatal - midbrain circuit loop48 which increases phasic dopamine efflux in the NAc itself80. Importantly, this increase in phasic dopamine can potenTate the hippocampal drive on the NAc 82, which may result in reduced hippocampal -NAc FC. This inverse relaTonship of hippocampal hyperacTvity and reduced hippocampal-striatal FC has been observed previously in individuals at CHR-P , as higher hippocampal glutamate levels (indicaTve of hyperacTvity) was associated . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint with reduced hippocampal-striatal FC45. In accordance with this, reduced CA1-NAc FC was the most robust finding in our sample of individuals at CHR -P (i.e., it was observed bilaterally in the CA1), in whom we have previously demonstrated hippocampal hyperacTvity 58. Beyond illness chronicity, the more pronounced reducTons observed in hippocampal FC in individuals with psychoTc disorders compared to those at CHR -P might be related to anTpsychoTc treatment. For instance, we observed reducTons in right, but not lew, CA1-vmPFC FC in our sample of anTpsychoTc-naïve individuals at CHR-P compared to the more robust observaTons in anTpsychoTc-treated individuals with schizophrenia 26,28,31–35. Whilst cogniTve symptoms which are present in the prodrome may worsen following the onset of psychosis84,86,88, chronic anTpsychoTc treatment may also play a role in further cogniTve impairment related to hippocampal-PFC FC uncoupling90,91. We found both higher and lower CA1-amygdala FC in individuals at CHR -P in the placebo condiTon compared to HC. Prior rs -fMRI studies have found lower 38,41 and normal 42 hippocampal-amygdala FC in individuals with psychoTc disorders. However, hippocampal - amygdala FC was increased in people with schizophrenia with paranoia vs. no paranoia42, and higher hippocampal-amygdala-PFC FC was associated with higher fear/anxiety in individuals with early psychosis92. Whilst amygdala dysfuncTon is associated with negaTve symptoms of schizophrenia21, it is also implicated in clinically disTnct comorbid anxiety /mood disorders, which are more common in those at CHR -P93,94. This increased affecTve component might explain the higher hippocampal-amygdala FC observed in our sample of individuals at CHR -P compared to HC. Furthermore, the findings in our study appeared hemisphere dependent (i.e., the right CA1 showed increased FC to the right amygdala and decreased FC to the lew amygdala). This was also observed at the whole -brain level, whereby the right CA1 showed hyperconnecTvity with a right medial temporal network, including the amygdala, but hypoconnecTvity with a lew hippocampal network and frontal regions of the lew default mode network. Increased hippocampal FC with the medial temporal lobe has been observed previously in the psychosis spectrum26,95,96, and could therefore be driving hyperconnecTvity to the amygdala given the close proximity and number of bidirecTonal connecTons 97. Furthermore, this paFern of intra -hemispheric hyperconnecTvity and inter -hemispheric hypoconnecTvity has been found previously in individuals with psychoTc disorders, indicaTng increased local network segregaTon and decreased remote network integraTon98. . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint The main effect of diazepam vs. placebo in CHR-P individuals on CA1 FC to the corTco-limbic- striatal network was a bilateral increase in CA1-vmPFC FC. Furthermore, all decreases in CA1- vmPFC and CA1-NAc FC in CHR-P individuals in the placebo condiTon compared to HC were not present in the diazepam condiTon. The general direcTon of the drug effect (that is, increasing FC) is in line with our predicTons and with prior pharmacological rs -fMRI studies using acute doses of GABA -enhancing drugs in healthy in dividuals54–57,64. GABA -enhancing drugs, such as diazepam, are posiTve allosteric modulators of the GABA A receptors via the benzodiazepine site 99. Most commonly, benzodiazepine binding leads to increased hyperpolarisaTon of post-synapTc glutamatergic pyramidal cells99, reducing their acTvity 100. The mechanism by which inhibiTon of neural acTvity in one brain region can result in increased FC to another has been recently elucidated by chemogeneTc fMRI study in mice. Rocchi and colleagues101 demonstrated that either acute or chronic inhibiTon of the PFC led to increases in FC with direct thalamo-corTcal output regions. The spiking acTvity was reduced but became more rhythmic and phase -locked to low -frequency oscillatory rhythms, leading to an increase in FC with connecTng regions. Therefore, through this mechanism, it is likely that downregulaTon of hippocampal hyperacTvity under diazepam (which we have demonstrated previously in this sample) led to increases in FC with connecTng output regions. InteresTngly, the effect of diazepam on CA1 -vmPFC FC showed the least inter -individual differences between people at CHR -P , whilst the effects in the amygdala and NAc were more varied. This may be due to the fact that the vmPFC , similar to the hippocampus , contains a high number of benzodiazepine receptors 102. ConsequenTally, similar local effects on neural acTvity in the hippocampus and vmPFC might have also contributed to a more robust increase in temporal coherence between them. Increases in hippocampal -PFC FC under benzodiazepine vs. placebo have previously been reported54, along with increases in FC to somatosensory and occipital regions 103,104 which also have high number of benzodiazepine binding sites 102. Furthermore, as noted earlier, the largest alteraTons in hippocampal FC observed in individuals at CHR -P in the placebo condiTon compared to HC were with the amygdala. This suggests that hippocampal-amygdala FC was the most perturbed out of the corTco-limbic-striatal regions. Given the proposed role of the amygdala in the iniTaTon of hippocampal hyperacTvity 105 and PV+ interneuron loss 106, and the high number of connecTons between these regions97, a single dose of diazepam may not have been sufficient . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint to regulate altered hippocampal -amygdala FC in individuals at CHR -P . In support of this, benzodiazepines have been shown to either increase 54 or decrease55 hippocampal-amygdala FC in healthy individuals. This suggests the pharmacological effects of GABA -enhancing compounds on this circuity are inherently complex, without the presence of potenTal alteraTons to the GABAergic system in individuals at CHR-P. Finally, we found no differences in FC strength between groups or drug condiTons for the anterior hippocampus to the corTco -limbic-striatal circuit. This was unexpected, based on preclinical evidence22 and current theories about the pathophysiology of psychosis 47,59,107. However, the anterior hippocampus contains subfields beyond the CA1 and subiculum, such as the CA2/3, which largely only have intra -hippocampal projecTons108. Therefore, inclusion of this signal may increase noise , making it difficult to detect subtle FC alteraTons between the anterior hippocampus and the corTco-limbic-striatal circuit within individuals at CHR-P . In line with this, whilst preclinical evidence focuses on the anterior hippocampus, it specifically idenTfies the anterior CA1 as the site of dysfuncTon109. This study had several strengths. We used a gold standard randomised, double-blind, placebo- controlled, crossover study design in a sample of anTpsychoTc naïve individuals at CHR-P. The hippocampus and CA1 subfield were segmented with a high degree of accuracy using novel computaTonal methods72, allowing the generaTon of study-specific hippocampal and subfield masks. We acquired rs -fMRI data using an advanced mulT -echo sequence, allowing robust data cleaning and removal of non -physiological noise with advanced methodological techniques such as TEDANA71. This led to high quality data as within-group/condiTon resTng- state FC networks for the CA1 to the rest of the brain replicated those found previously75. We were able to contextualise baseline differences and direcTon of drug effects in the CHR -P group by comparing them with data from a HC group. Finally, we used advanced staTsTcal mixed-effects modelling, which is opTmal for examining both inter-group differences without assuming uniform variance and also for invesTgaTng within -subject effects73. This study also had some limitaTons. Our sample size of CHR-P individuals was reduced from 24 down to 18 awer quality control , but retrospecTve power analysis demonstrated that the diazepam vs. placebo analyses (mean Cohen’s d=0.83) had an achieved power of 91%. AddiTonally, this study was not powered to invesTgate relaTonship between FC alteraTons and symptoms, . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint which would require a much larger CHR-P sample. Due to limitaTons with the resoluTon of rs- fMRI, we were not able to invesTgate differences in FC from specifically the anterior CA1 which is of parTcular relevance for psychosis22. In conclusion, this study provides evidence that a single dose of a non -specific GABA - enhancing drug, such as diazepam, can normalise CA1 FC alteraTons with the vmPFC and NAc in individuals at CHR-P. C o n v e r s e l y, C A 1-amygdala FC was greatly perturbed in people at CHR- P under placebo compared to HC and was largely unaffected by diazepam challenge. Given this mechanisTc evidence, future research is warranted with extended treatment duraTons to link these neurobiological changes to symptoms and clinical outc omes, including psychosis prevenTon. Acknowledgments This research was funded by the Wellcome Trust and The Royal Society (202397/Z/16/Z to GM) and the NaTonal InsTtute for Health and Care Research (NIHR) Maudsley Biomedical Research Centre (BRC). The views expressed are those of the authors and not necessarily those of the Welcome Trust, NIHR or the Department of Health and Social Care. For the purpose of open access, the author has applied a CC-BY public copyright licence to any Author Accepted Manuscript version arising from this submission. NRL was fund ed by a n MRC DTP PhD studentship at the Tme of data collecTon and analysis. PBL was in receipt of a PhD studentship funded by the NIHR Maudsley BRC at the Tme of data collecTon. OO is funded by the Maudsley BRC. LAJ was supported by an MRC Clinical Research Training Fellowship (MR/T028084/1) at the Tme of data collecTon. TJR is supported by an MRC Clinical Research Training Fellowship (MR/W015943/1). PFP is supported by the European Union funding within the MUR PNRR Extended Partnership iniTaTve on Neuroscience and Neuropharmacology (Project no. PE00000006 CUP H93C22000660006 “MNESYS, A mulTscale integrated approach to the study of the nervous system in health and disease”). AAG received funding from USPHS NIMH MH57440. MMC receives salary support from the Fonds de Recherche Québec – Santé and from a James McGill Professorship. MMC also receives research support from Canadian InsTtutes of Health Research, Natural Sciences and Engineering Research Council – Canada, . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint McGill University’s Health Brains for Health Lives (a Canada Research Excellence Fund IniaTve), and TRIDENT (a New FronTers in Research Fund program). Disclosures AAG has received consulTng fees from Alkermes, Lundbeck, Takeda, Roche, Lyra, Concert, Newron and SynAgile, and research funding from Newron and Merck. SCRW has recently received research funding from Boehringer Ingelheim and GE Healthcare to perform invesTgator-led research. AE has received consultancy fees from Leal TherapeuTcs. GM has received consulTng fees from Boehringer Ingelheim . The remaining authors have no disclosures to declare . CC-BY 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 21, 2024. ; https://doi.org/10.1101/2024.12.20.24319330doi: medRxiv preprint

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