Exposure to prenatal stress (PS) and mutations in Gad1, which encodes GABA synthesizing enzyme glutamate decarboxylase (GAD) 67, are the primary risk factors for psychiatric disorders associated with abnormalities in parvalbumin (PV)-positive GABAergic interneurons in the medial prefrontal cortex (mPFC). Decreased expression of extracellular matrix (ECM) glycoproteins has also been reported in patients with these disorders, raising the possibility that ECM abnormalities may play a role in their pathogenesis. To elucidate pathophysiological changes in ECM induced by the gene–environment interaction, we examined heterozygous GAD67-GFP (Knock-In KI; GAD67+/GFP) mice subjected to PS from embryonic day 15.0 to 17.5. Consistent with our previous study, we confirmed a decrease in the density of PV neurons in the mPFC of postnatal GAD67+/GFP mice with PS, which was concurrent with a decrease in density of PV neurons surrounded by perineuronal nets (PNNs), a specialized ECM important for the maturation, synaptic stabilization and plasticity of PV neurons. Glycosylation of α-dystroglycan (α-DG) and its putative mediator fukutin (Fktn) in the ECM around inhibitory synapses has also been suggested to contribute to disease development. We found that both glycosylated α-DG and the mRNA level of Fktn were reduced in GAD67+/GFP mice with PS. None of these changes were detected in GAD67+/GFP naive mice or wild type (GAD67+/+) mice with PS, suggesting that both PS and reduced Gad1 gene expression are prerequisites for these changes. When assessing the function of interneurons in the mPFC of GAD67+/GFP mice with PS through evoked inhibitory post-synaptic currents (eIPSCs) in layer V pyramidal neurons, we found that the threshold stimulus intensity for eIPSC events was reduced and that the eIPSC amplitude was increased without changes in the paired-pulse ratio (PPR). Moreover, the decay rate of eIPSCs was also slowed. In line with eIPSC, spontaneous IPSC (sIPSC) amplitude, frequency and decay tau were altered. Thus, our study suggests that alterations in the ECM mediated by gene-environment interactions might be linked to the enhanced and prolonged GABA action that compensates for the decreased density of PV neurons. This might be one of the causes of the excitatory/inhibitory imbalance in the mPFC of psychiatric patients.

The with-no-lysine (WNK) family of serine-threonine kinases and its downstream kinases of STE20/SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1) may regulate intracellular Cl− homeostasis through phosphorylation of cation-Cl− co-transporters. WNK3 is expressed in fetal and postnatal brains, and its expression level increases during development. Its roles in neurons, however, remain uncertain. Using WNK3 knockout (KO) mice, we investigated the role of WNK3 in the regulation of the intracellular Cl− concentration ([Cl−]i) and the excitability of layer V pyramidal neurons in the medial prefrontal cortex (mPFC). Gramicidin-perforated patch-clamp recordings in neurons from acute slice preparation at the postnatal day 21 indicated a significantly depolarized reversal potential for GABAA receptor-mediated currents by 6 mV, corresponding to the higher [Cl−]i level by ~4 mM in KO mice than in wild-type littermates. However, phosphorylation levels of SPAK and OSR1 and those of neuronal Na+-K+-2Cl− co-transporter NKCC1 and K+-Cl− co-transporter KCC2 did not significantly differ between KO and wild-type mice. Meanwhile, the resting membrane potential of neurons was more hyperpolarized by 7 mV, and the minimum stimulus current necessary for firing induction was increased in KO mice. These were due to an increased inwardly rectifying K+ (IRK) conductance, mediated by classical inwardly rectifying (Kir) channels, in KO neurons. The introduction of an active form of WNK3 into the recording neurons reversed these changes. The potential role of KCC2 function in the observed changes of KO neurons was investigated by applying a selective KCC2 activator, CLP290. This reversed the enhanced IRK conductance in KO neurons, indicating that both WNK3 and KCC2 are intimately linked in the regulation of resting K+ conductance. Evaluation of synaptic properties revealed that the frequency of miniature excitatory postsynaptic currents (mEPSCs) was reduced, whereas that of inhibitory currents (mIPSCs) was slightly increased in KO neurons. Together, the impact of these developmental changes on the membrane and synaptic properties was manifested as behavioral deficits in pre-pulse inhibition, a measure of sensorimotor gating involving multiple brain regions including the mPFC, in KO mice. Thus, the basal function of WNK3 would be the maintenance and/or development of both intrinsic and synaptic excitabilities.

The excitatory action of gamma-aminobutyric-acid (GABA) in the median-eminence (ME) led to the steady-state release of corticotropin-releasing hormone (CRH) from CRH axon terminals, which modulates the hypothalamic-pituitary-adrenal (HPA) axis. However, in ME, the source of excitatory GABAergic input is unknown. We examined agouti-related peptide (AgRP) expressing neurons in the arcuate nucleus as a possible source for excitatory GABAergic input. Here, we show that a subpopulation of activated AgRP neurons directly project to the CRH axon terminals in ME elevates serum corticosterone levels in 60% food-restricted mice. This increase in serum corticosterone is not dependent on activation of CRH neuronal soma in the paraventricular nucleus. Furthermore, conditional deletion of Na+-K+-2Cl– cotransporter-1 (NKCC1), which promotes depolarizing GABA action, from the CRH axon terminals results in significantly lower corticosterone levels in response to food restriction. These findings highlight the important role of a subset of AgRP neurons in HPA axis modulation via NKCC1-dependent GABAergic excitation in ME.

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