Prefrontal parvalbumin interneurons require juvenile social experience to establish adult social behavior

The study investigates how juvenile social experience shapes the maturation of dorsal medial prefrontal cortex (dmPFC) parvalbumin-positive interneurons (PVIs) and, in turn, adult social behavior. Prior work indicates a critical juvenile window (postnatal day p21–35) in mice where social isolation yields lasting behavioral disruptions. The medial PFC, particularly dmPFC (anterior cingulate and prelimbic regions), is implicated in social behavior across species and undergoes prolonged development into the juvenile period characterized by strengthening inhibitory neurotransmission. PVIs, key regulators of cortical development and gamma oscillations, mature late and are implicated in neurodevelopmental disorders with social deficits. The authors hypothesize that dmPFC-PVI activity patterns are linked to specific components of social behavior (notably active social approach), and that juvenile social experience is necessary for dmPFC-PVI maturation that supports normal adult sociability.

• Human and rodent studies implicate mPFC/dmPFC in social behavior regulation. Inhibitory interneuron maturation, especially PVIs, is protracted and experience-dependent in sensory cortices. PVIs and gamma oscillation abnormalities are reported in autism and schizophrenia. Prior work shows that enhancing PVI activity in adulthood can mitigate social deficits in certain models, but the endogenous timing and role of dmPFC-PVI activity in natural social behaviors, and its developmental sensitivity to social experience, remained unclear. The authors position their work to define precise behavioral contingencies of dmPFC-PVI activation and its experience-dependent maturation.

• Animals: Male C57BL/6 wild type, PV-Cre, and PV-GFP (PV-Cre x Cre-dependent eGFP-L10a) mice. Standard housing on 12:12 light/dark cycle. Ethical approval by Icahn School of Medicine at Mount Sinai IACUC.
• Juvenile social isolation (jSI): Isolation from weaning (p21) for 2 weeks (to p35), then regrouped with age/sex/strain-matched males for 1 month; behavioral testing in adulthood (2–4 months). Controls: group housed (GH); 'shuffled' (group housed then regrouped at p35 without isolation); adult isolation (2 weeks in adulthood followed by rehousing) to test window specificity.
• Viral injections (dmPFC targeting): Stereotaxic bilateral injections (AP +1.7, +1.1, +0.4 mm; ML ±0.2 mm; DV −1.0/−0.8/−0.75 mm). Vectors: AAV8-hSyn-DIO-hM4Di-mCherry (iDREADD), AAV8-hSyn-DIO-hM3Dq-mCherry (eDREADD), AAV1-Syn-FLEX-GCaMP6f-WPRE-SV40 for photometry, AAV1-EF1-DIO-hChR2-mCherry for optogenetics. Fiber ferrules or wireless LED implants placed over dmPFC.
• Fiber photometry: GCaMP6f in dmPFC PVIs of adult PV-Cre mice; dual-excitation (465 nm calcium-dependent, 405 nm isosbestic) and demodulation; z-scoring against baseline. Tasks: reciprocal social interaction (novel mouse vs object; behavior epoch scoring into active, passive, orient) and three-chamber sociability (alignment via TTLs). Analysis included comparing activity 3 s pre- and post-bout initiation for active and passive bouts.
• Wireless optogenetics (PVIs): ChR2 activation with 40 Hz, 1 ms pulses for 3 s; randomized pulses (8–15 s apart) during 5-min reciprocal interaction; three-chamber test with center-triggered 3 s, 40 Hz pulses; open field velocity control. In vivo validation: multiunit recordings under isoflurane during 40 Hz light confirmed PVI spiking.
• Chemogenetics: iDREADD (hM4Di) to suppress dmPFC PVIs; eDREADD (hM3Dq) to activate dmPFC PVIs. CNO i.p.: 10 mg/kg for iDREADD; 1 mg/kg for eDREADD, 30 min pre-test. Verified CNO had no behavioral effects without DREADD. Validation: iDREADD reduced PVI membrane potential ex vivo; eDREADD increased PVI egr-1 expression in vivo.
• Behavioral assays: Reciprocal interactions (manual scoring of eight behaviors; grouped categories; transition matrix analysis); three-chamber sociability (time in social/object chambers; interaction per entry; entries after opto pulses); open field (locomotion/anxiety), light-dark box, elevated-plus maze.
• Electrophysiology (dmPFC PVIs): Whole-cell recordings at p35 and adult (p60–65) in PV-GFP mice (GH vs jSI). Intrinsic excitability (current-clamp; synaptic blockers present), sEPSC/sIPSC frequencies and amplitudes; miniature EPSCs/IPSCs with TTX. Computed sEPSC/sIPSC frequency ratio as input drive index.
• Statistics: Parametric/non-parametric tests as appropriate; repeated-measures ANOVA, mixed models for optogenetics, permutation tests for transition matrices; significance levels reported with post hocs.

• dmPFC-PVIs preferentially respond to social vs object introduction: Mean GCaMP6f z-score increased after introducing a novel mouse (Wilcoxon signed-rank p = 0.03) but not a novel object (p = 0.25).
• Pre-bout activation specificity: During reciprocal interaction, dmPFC-PVI activity transiently increased immediately before the first active bout initiation (one-way repeated measures ANOVA p = 0.02; Tukey baseline vs pre p < 0.05), with no sustained change during the bout; no change observed for the first passive bout.
• Causal promotion of active social approach by brief PVI activation (optogenetics): 3 s, 40 Hz dmPFC-PVI stimulation increased duration of active behaviors per pulse in reciprocal interactions (linear mixed model effect of light p = 0.04; active on vs off p = 0.01; passive p = 0.30; orient p = 0.40). Specific increases in nose-to-nose (paired t, p = 0.01) and approach (p = 0.05). No effect on open field velocity (p = 0.10). In the three-chamber test, light triggered from center increased social chamber entries (paired t, p = 0.03) but not object entries (p = 0.89); eYFP controls showed no effect.
• Necessity of PVI activity (chemogenetic suppression): iDREADD-mediated suppression reduced social interaction per social chamber entry (two-way repeated measures ANOVA drug effect p = 0.005) and trended to reduce time in social chamber (p = 0.07), without affecting locomotion or anxiety measures.
• Juvenile social isolation (jSI) induces adult social deficits and alters PVI-behavior coupling: jSI males showed decreased active social behavior duration in reciprocal interactions (two-way mixed model ANOVA; Bonferroni active p < 0.05) and distinct behavior transition structure (joint-distribution p < 0.05). jSI mice lacked the pre-active PVI activity increase (baseline vs pre, ns) and instead showed increased PVI activity after the first passive encounter (pre vs post p < 0.001). GH but not jSI mice showed significant pre-active increase from baseline; jSI showed significant post-passive increase. In three-chamber photometry, GH showed greater pre-entry PVI activity than jSI before social entries.
• Developmental electrophysiology indicates a maturation 'freeze' in jSI PVIs:
  • Intrinsic excitability: GH PVIs increased spike frequency from p35 to adult (age factor p = 0.0035; age x current p < 0.0001). jSI showed no developmental change (age p = 0.15). In adulthood, GH > jSI spike frequency (housing p = 0.04; age p = 0.0004; Bonferroni p60 p < 0.05).
  • Synaptic drive: sEPSC frequency decreased with age and was lower in jSI overall (age p < 0.001; housing p = 0.01). sIPSC frequency decreased with age (age p < 0.0001) with a trend for age x housing (p = 0.07). sEPSC/sIPSC frequency ratio increased from p35 to adult in GH but not in jSI (age x housing interaction p = 0.0005; adult GH > jSI p < 0.0001). mEPSC and mIPSC frequencies decreased with age independent of housing. Findings indicate network-level, action potential-dependent alterations and reduced excitatory drive onto adult jSI PVIs.
• Rescue: Chemogenetic activation (eDREADD) of dmPFC-PVIs in adult jSI mice restored sociability. jSI: significant drug x stimulus interaction in three-chamber (p = 0.03); SAL: no social preference (ns), CNO: robust preference (p < 0.001). Social investigation increased in first 2 min with CNO vs SAL in jSI (paired t, p = 0.02); no effect in GH (drug x stimulus p = 0.73). No significant effects on anxiety measures (open field, EPM, light-dark).

The findings reveal a precise temporal coupling between brief dmPFC-PVI activation and initiation of active social approach in adult mice, indicating that dmPFC-PVIs contribute to initiating, rather than sustaining, social interactions. Juvenile social experience is necessary to establish this coupling: isolation during a defined juvenile window (p21–35) disrupts adult dmPFC-PVI pre-active bout activation and reduces active social engagement. Electrophysiological data suggest that, in GH mice, dmPFC-PVIs continue to mature from adolescence into adulthood, increasing intrinsic excitability and receiving relatively greater excitatory versus inhibitory synaptic drive. jSI prevents this late maturation, producing a developmental 'freeze' phenotype with reduced adult PVI excitability and drive, which likely underlies the disrupted pre-active timing and social behavior deficits. Brief optogenetic PVI activation mimics the endogenous pre-active signal and biases behavior toward social approach, while chemogenetic suppression impairs sociability without altering general locomotion or anxiety, establishing necessity. Importantly, acute chemogenetic activation of dmPFC-PVIs in adulthood rescues sociability in jSI mice, highlighting dmPFC-PVIs as a potential therapeutic target. These insights align with clinical observations of PVI/gamma oscillation abnormalities in neurodevelopmental disorders with social deficits and emphasize the importance of developmental timing for circuit maturation underlying complex social behaviors.

This work identifies dmPFC parvalbumin interneurons as critical, experience-dependent regulators of adult social behavior. Key contributions include: (1) defining a brief, pre-active bout activation pattern in dmPFC-PVIs that predicts social approach; (2) demonstrating causality via optogenetic promotion and chemogenetic suppression; (3) showing that juvenile social isolation disrupts adult PVI-behavior coupling and arrests maturation of PVI excitability and synaptic drive; and (4) rescuing social deficits in jSI adults by chemogenetically activating dmPFC-PVIs. Future directions include elucidating molecular and epigenetic mechanisms by which juvenile social experience programs PVI maturation, mapping downstream projection circuits modulated by PVIs during social approach, testing sex differences and broader behavioral domains, and exploring translational strategies (e.g., rhythm-specific neuromodulation, pharmacological enhancers of PVI function) to restore social behavior.

• Sex specificity: Experiments focused on male mice; jSI may not induce persistent social deficits in females. Findings may not generalize across sexes.
• Circuit scope: Work centered on dmPFC PVIs; contributions from other interneuron subtypes and regions were not examined.
• Chemogenetic ligand considerations: While CNO controls showed no behavioral effects in this study, potential conversion to clozapine and off-target effects are general concerns.
• Photometry resolution: Calcium signals from PVI populations provide bulk activity and timing but do not capture single-cell spiking or precise microcircuit interactions.
• Causality for downstream targets: The specific excitatory neuron subpopulations and projection pathways gated by PVI pre-active activity remain to be identified.
• Developmental mechanism: The study infers a developmental 'freeze' without identifying the underlying molecular changes; longitudinal within-subject tracking was not performed.