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.
...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.