PGC-1α knockout effects on PV+ interneuron maturation

PRIMARY OUTCOME

PV+ interneuron maturation defects

EXPECTED OUTCOMES

## Primary Outcomes **PV+ Interneuron Hyperexcitability**: PGC-1α cKO PV+ interneurons show increased firing frequency (+40%, p < 0.01) and reduced sIPSC frequency (-35%, p < 0.05) vs. controls, consistent with impaired mitochondrial energy metabolism and disrupted GABAergic output. **Circuit Dysfunction**: cKO mice show impaired working memory (T-maze alternation: 60-65% vs. 80-85% in controls, p < 0.01) and reduced prefrontal cortex gamma oscillations (30-80 Hz power: -25%, p < 0.05). ## Secondary Outcomes **Molecular Signature**: RNA-seq reveals downregulation of mitochondrial biogenesis genes (PGC-1α targets: NRF-1, NRF-2, TFAM, COX family) by ≥30% in cKO PV+ cells. GABAergic signaling genes also show altered expression (GAD1, GAD2, parvalbumin itself). **Sleep Architecture**: cKO mice show increased REM sleep fragmentation (+40% transitions, p < 0.05) and altered ultradian REM cycles, consistent with PV+ interneuron role in thalamocortical rhythm generation.

SUCCESS CRITERIA

## Primary Success Criteria **Molecular Confirmation**: PGC-1α protein must be reduced by ≥80% in PV+ interneurons (co-immunofluorescence quantification) while remaining unchanged in non-PV neurons (control for systemic effects of Cre). **Electrophysiological Phenotype**: PV-specific PGC-1α deletion must produce significant alteration in at least one PV+ intrinsic property: either (a) firing frequency change ≥30%, (b) sIPSC frequency change ≥30%, or (c) resting membrane potential shift ≥5 mV. ## Secondary Success Criteria **Behavioral Relevance**: Electrophysiological changes must correlate with measurable behavioral phenotype (working memory or social behavior deficit) at p < 0.05, establishing circuit-level consequences. **Control for Off-Target Effects**: Use tamoxifen-induced vs. constitutive Cre to confirm phenotype is not due to developmental compensation or off-target Cre effects.

PROTOCOL

# PGC-1α Knockout Effects on PV+ Interneuron Maturation Protocol ## Phase 1: Conditional Knockout Mouse Generation and Verification (Days 1-21) **Breeding Strategy**: Cross PGC-1α flox/flox mice (B6;129-Ppargc1a<tm1.1Dlb>/J, JAX #024119) with PV-Cre mice (B6;129P2-Pvalb<tm1(cre)Jpb>/J, JAX #017493) to generate PV-Cre;PGC-1α flox/flox cKO and Cre-negative flox/flox controls. Verify genotype via PCR (flox primers: 5'-GGAGCCAGTGAAGATGATGTTC-3', 3'-CCACAGAATCCAGAGGCAAC-5'; Cre: commercial primers). **Tamoxifen Induction** (if inducible model): Administer tamoxifen (75 mg/kg, i.p., 5 consecutive days) at P28-P35 to induce recombination in adult animals. Use corn oil vehicle for control injections. **Verification of Recombination**: At designated timepoints, collect cortex and verify PGC-1α deletion via qRT-PCR (primer: exon 2 excision junction, Applied Biosystems) and western blot (anti-PGC-1α, 1:500, Abcam #ab191695). Confirm PV+ cell-specificity via co-immunostaining. ## Phase 2: Electrophysiological Analysis (Days 22-42) **Brain Slice Preparation**: Prepare acute coronal brain slices (300 μm) from P42-P56 cKO and control mice. Decapitate under isoflurane anesthesia, dissect brain in ice-cold cutting solution (in mM: 210 sucrose, 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, 0.5 CaCl2, 26 NaHCO3, 10 glucose). Slice on Leica VT1200S vibratome. Recover in artificial CSF (aCSF, in mM: 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, 26 NaHCO3, 10 glucose) at 34°C for 30 min then RT. **PV+ Interneuron Recording**: Identify PV+ cells in layer 2/3 or layer 5 of prefrontal cortex via tdTomato (PV-Cre reporter) or post-hoc immunostaining. Perform whole-cell voltage-clamp recording (pipette: Cs-gluconate internal, 280 mOsm, 5-7 MΩ) at 32°C. Measure spontaneous excitatory postsynaptic currents (sEPSCs, holding at -70 mV) and inhibitory postsynaptic currents (sIPSCs, holding at 0 mV). **Action Potential Properties**: Current-clamp mode to measure resting membrane potential, input resistance, and action potential threshold/firing frequency. Inject step currents (-100 to +300 pA, 500 ms). Analyze action potential half-width, amplitude, and adaptation ratio. ## Phase 3: Circuit-Level and Behavioral Consequences (Days 43-63) **In Vivo Recordings**: For chronic assessment, implant multielectrode arrays (32-site, Cambridge NeuroTech) in prefrontal cortex of adult cKO and control mice. Record LFP and single-unit activity during REM and NREM sleep and during working memory tasks (T-maze, delayed non-match-to-sample). **Behavioral Phenotyping**: Test cKO and control mice at P60-P90 on: (a) working memory (T-maze alternation, 5-day acquisition), (b) social preference (3-chamber assay), (c) rotarod (motor coordination), (d) open field (anxiety). Compare via repeated measures ANOVA. **Molecular Phenotype**: At experiment endpoint, collect prefrontal cortex for RNA-seq (NovaSeq, 30M reads, 3 biological replicates per genotype). Pathway analysis: GABAergic signaling, mitochondrial function, and neuronal activity-dependent gene programs.

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