Downregulation of parvalbumin expression in the prefrontal cortex during adolescence causes enduring prefrontal disinhibition in adulthood

Abstract

The expression of the calcium binding protein parvalbumin (PV) has been observed in several cortical regions during development in a temporal pattern consistent with increased afferent-dependent activity. In the prefrontal cortex (PFC), PV expression appears last and continues to substantially increase throughout adolescence, yet the significance of this increase remains unclear. Because of the expression of PV in fast-spiking GABAergic interneurons, we hypothesized that PV upregulation during adolescence is necessary to sustain the increase in GABAergic activity observed in the PFC during this period. To test this hypothesis, we utilized an RNAi strategy to directly downregulate PV levels in the PFC during adolescence and examined its impact on prefrontal GABAergic function, plasticity, and associated behaviors during adulthood. The data indicate that a mere 25% reduction of adult PV levels in the PFC was sufficient to reduce local GABAergic transmission onto pyramidal neurons, disrupt prefrontal excitatory-inhibitory balance, and alter processing of afferent information from the ventral hippocampus. Accordingly, these animals displayed an impairment in the level of extinction learning of a trace fear conditioning response, a behavioral paradigm that requires intact PFC-ventral hippocampus connectivity. These results indicate the PV upregulation observed in the PFC during adolescence is necessary for refinement of prefrontal GABAergic function, the absence of which results in immature afferent processing and a hypofunctional state. Importantly, these results suggest there is a critical window of plasticity during which PV upregulation supports the acquisition of mature GABAergic phenotype necessary to sustain adult PFC functions.

Fig. 1. PV downregulation modifies baskets and reduces GABAergic transmission in the PFC.

a Diagram of experimental design for quantification of PV downregulation after unilateral, intra-PFC delivery of scrambled (Scr; n = 5) or PV shRNA (n = 7) during postnatal days (P) 34–38. Anti-PV staining was performed when rats reached ~P65 in 50 µm-thick sections 300 µm anterior and posterior to the injection site. b Relative to the uninjected side, a ~25% reduction of PV immunoreactivity can be measured in the PFC 30 days after PV shRNA injection (***p < 0.0005, unpaired t-test). c Representative images of Scr and PV shRNA-treated PFC displaying a pronounced reduction of PV expression in the neuropil and deep layer “baskets” (scale bar: 100 µm). Magnified insets (C1–C4) showing the degree of PV downregulation in deep layer baskets. Right panels: intensity heat plots of individually magnified baskets. d Bar-graph summarizing the effect of PV shRNA treatment on spontaneous inhibitory postsynaptic current (IPSC) recorded in the PFC at P65–85. Whole-cell patch-clamp recordings from layer V pyramidal neurons revealed a marked reduction of IPSC frequency (events/min) in the PV shRNA group (12 cells, 6 rats) compared with Scr controls (11 cells, 6 rats; ***p < 0.0001, unpaired t-test). Inset are examples traces of spontaneous IPSC recorded from layer V pyramidal neurons illustrating the effect of PV shRNA in the PFC (calibration: 15pA, 1s). e Summary of the data obtained from layer V pyramidal neurons using a paired-pulse protocol of minimal stimulation at 50 ms interval. Note that the intensity of stimulation was titrated to elicit monosynaptic IPSC responses at ~50% failure rate in both groups to enable the detection of any changes in the probability of GABA release. While pyramidal neurons recorded from Scr controls (10 cells, 5 rats) exhibited IPSC₂/IPSC₁ ratios <1.0, all neurons recorded from the PV-shRNA group (13 cells, 7 rats) showed IPSC₂/IPSC₁ ratios >1.0 (***p < 0.001 vs. Scr shRNA, unpaired t-test), indicating that the probability of GABA release is decreased following PV downregulation.

Fig. 2. Downregulation of PV increases the ratio of excitatory–inhibitory synaptic activity onto layer V pyramidal neurons and reduces the frequency of excitatory transmission onto fast-spiking interneurons.

a Changes in excitatory (E)–inhibitory (I) balance of synaptic activity following intra-PFC delivery of Scr or PV shRNA at P35 were determined at P45–55 and P65–85. Data from the P45–55 group were collected to assess whether the functional impact of PV shRNA is already detectable at 10–15 days post-delivery. b All recordings were obtained from layer V pyramidal neurons in the PFC using a low-chloride-based internal solution that enables concurrent acquisition of inhibitory (PSC_{+15 mV}) and excitatory (PSC_{−60 mV}) postsynaptic currents (see Materials & Methods). Relative to Scr controls (P45–55: gray circles, 4 rats; P65–85: black circles, 5 rats), PV shRNA delivery did not alter the frequency of PSC_{−60 mV} events (PSC/min) at P45–55 (yellow circles, 4 rats) or P65–85 (orange circles, 5 rats). In contrast, intra-PFC delivery of PV shRNA diminished the frequency of PSC_{+15 mV} in layer V pyramidal neurons, an effect that can be detected at P45 (***p < 0.0005, unpaired t-test). c Calculation of the E/I ratio for each individual neuron recorded from P45–55 and P65–85. Data from both age groups were pooled. Relative to Scr controls, treatment with PV shRNA markedly increased the E/I ratio in layer V pyramidal neurons by more than 30% (***p < 0.0005, unpaired t-test). d Further analysis revealed a significant correlation (p < 0.001) between the E/I ratio and the frequency of PSC_{+15 mV} (i.e., IPSC) events. e Example traces of PSC_{+15 mV} and PSC_{−60 mV} recorded from layer V pyramidal neurons illustrating the effect of intra-PFC delivery of PV shRNA shown in b (calibration: 25 pA/0.5 s for PSC_{−60 mV} and 40 pA/0.5 s for PSC_{+15 mV}). f Impact of intra-PFC delivery of PV shRNA (~P35) on fast-spiking interneurons’ (FSI) excitatory postsynaptic (EPSC) events recorded at P45–55 and P65–85. g Relative to the Scr shRNA control group (P45–55: 4 rats; P65–85: 5 rats), a marked reduction in EPSC frequency was observed in FSI following adolescent PV downregulation (P45–55: 4 rats; P65–85: 5 rats; ***p < 0.0001, unpaired t-test). h Example traces of spontaneous EPSC recorded from FSI illustrating the impact of adolescent delivery of PV shRNA into the PFC shown in (f) (calibration: 10 pA, 500 ms).

Fig. 3. Decreased levels of PV in the prefrontal cortex confer juvenile-like pattern of hippocampal-evoked response.

a The pattern of ventral hippocampal-evoked (10 Hz) facilitation of local field potential (LFP) responses in the PFC was indistinguishable between Scr (n = 9) and PV shRNA (n = 13) groups. b Following hippocampal stimulation at 20 Hz, a normal transient suppression of LFP response was recorded in the PFC of Scr controls (P45–55: gray lines, 4 rats; P65–85: black lines, 5 rats) whereas a pattern of prefrontal LFP facilitation emerged in PV shRNA-treated animals (P45–55: yellow lines, 6 rats; P65–85: orange lines, 7 rats; treatment x pulse interaction, F₉₂₀₀ = 6.1, ***p < 0.0001; main treatment effect, F₁₂₀₀ = 349.9, p < 0.0001, two-way ANOVA). Note the abnormal facilitation of LFP response in the PFC of PV shRNA-treated animals is already apparent at P45 (yellow lines). c At 40 Hz, both Scr and PV shRNA groups exhibited similar patterns of LFP suppression in the PFC. However, the magnitude of prefrontal LFP suppression was markedly reduced following PV shRNA delivery (treatment × pulse interaction, F₉₂₀₀ = 2.6, **p < 0.005; main treatment effect, F₁₂₀₀ = 147.1, p < 0.0001, two-way ANOVA). Such a disruption was also apparent at P45 (yellow lines). d Diagram of PFC (top) and hippocampal (bottom) coronal sections showing the placement for all recording and stimulating electrodes, respectively (black: Scr shRNA group; orange: PV shRNA group). e Bar-graph summarizing the impact of PV shRNA on prefrontal LFP responses presented in panels (a–c). Data from a cohort of naïve P30–40 rats (n = 7) were included for comparison. Note that the levels of LFP facilitation (at 20 Hz) and LFP suppression (at 40 Hz) recorded in the PFC of PV shRNA-treated group are equivalent to those observed in P30–40 animals (***p < 0.0005 vs. P30–40 or PV shRNA, Tukey post-hoc test after significant one-way ANOVA; F₂₂₆ = 40.3, p < 0.0001 for 20 Hz and F₂₂₆ = 37.1, p < 0.0001 for 40 Hz). f Example traces of prefrontal LFP response to hippocampal train stimulation at 10, 20, and 40 Hz utilized for the analysis in panels (a–c) (calibration: 3 mV/200 ms at 10 Hz; 3 mV/100 ms at 20 Hz; 6 mV/50 ms at 40 Hz).

Fig. 4. PV downregulation impairs the inhibitory control of amygdalar inputs to the prefrontal cortex by the ventral hippocampus.

a Timeline of the experimental design and diagrams of brain coronal sections showing the location for all PFC recording sites and placement of ventral hippocampal (vHIP) and basolateral amygdalar (BLA) stimulating electrodes used to collect the data shown in (b) (black: Scr shRNA group; orange: PV shRNA group). b PFC downregulation of PV did not alter the typical potentiation of BLA-evoked LFP induced by a protocol of high-frequency stimulation in the BLA (HFS; see Materials & Methods). However, only the Scr shRNA group showed the normal response to vHIP HFS applied 40 min post-BLA potentiation. While HFS of the vHIP resets the LFP facilitation driven by the BLA in Scr controls (n = 6 rats), the suppression of BLA-evoked LFP response is no longer observed in PV shRNA-treated animals (n = 7 rats). c Summary of the mean LFP response obtained from the last 10 min post-HFS of the BLA (a) and vHIP (b) shown in (b) (area marked in gray). Two-way ANOVA revealed main effects of input stimulation (F₁₂₂ = 43.6, p < 0.0001), PFC shRNA treatment (F₁₂₂ = 29.7, p < 0.0005), and input × treatment interaction (F₁₂₂ = 39.9, p < 0.0001; ***p < 0.0005 vs. Scr shRNA, Tukey post-hoc test). d Traces of BLA-evoked LFP responses recorded from the PFC illustrating the impact of PV shRNA shown in (b) (−5’: baseline; (a): post-BLA HFS; (b) post-vHIP HFS; calibration: 5 mV, 20 ms).

Fig. 5. Downregulation of PV in the prefrontal cortex increases the level of freezing response during extinction testing.

a A “trace” fear conditioning paradigm in which the cue (tone) and the shock (1 s, 0.4 mA) are separated by 20 s was utilized to measure intact hippocampal-PFC connectivity. Following habituation (H) on day 1, both Scr-shRNA (n = 7 rats) and PV-shRNA (n = 9 rats) groups were conditioned to the tone as evidenced by increasing levels of freezing over five trials, with no differences in the rate of acquisition of the fear response. b Twenty-four hours later (day 2), extinction of the cue-induced freezing response was measured in both experimental groups. Relative to Scr controls, PV shRNA-treated rats exhibited a slower extinction rate (main effect of treatment, F₁₁₉₆ = 192.2, ***p < 0.0005; treatment × trial interaction, F₁₃₁₉₆ = 2.0, p = 0.024, two-way ANOVA).