Gephyrin phosphorylation facilitates sexually dimorphic development and function of parvalbumin interneurons in the mouse hippocampus.

The precise function of specialized GABAergic interneuron subtypes is required to provide appropriate synaptic inhibition for regulating principal neuron excitability and synchronization within brain circuits. Of these, parvalbumin-type (PV neuron) dysfunction is a feature of several sex-biased psychiatric and brain disorders, although, the underlying developmental mechanisms are unclear. While the transcriptional action of sex hormones generates sexual dimorphism during brain development, whether kinase signaling contributes to sex differences in PV neuron function remains unexplored. In the hippocampus, we report that gephyrin, the main inhibitory post-synaptic scaffolding protein, is phosphorylated at serine S268 and S270 in a developmentally-dependent manner in both males and females. When examining GphnS268A/S270A mice in which site-specific phosphorylation is constitutively blocked, we found that sex differences in PV neuron density in the hippocampal CA1 present in WT mice were abolished, coincident with a female-specific increase in PV neuron-derived terminals and increased inhibitory input onto principal cells. Electrophysiological analysis of CA1 PV neurons indicated that gephyrin phosphorylation is required for sexually dimorphic function. Moreover, while male and female WT mice showed no difference in hippocampus-dependent memory tasks, GphnS268A/S270A mice exhibited sex- and task-specific deficits, indicating that gephyrin phosphorylation is differentially required by males and females for convergent cognitive function. In fate mapping experiments, we uncovered that gephyrin phosphorylation at S268 and S270 establishes sex differences in putative PV neuron density during early postnatal development. Furthermore, patch-sequencing of putative PV neurons at postnatal day 4 revealed that gephyrin phosphorylation contributes to sex differences in the transcriptomic profile of developing interneurons. Therefore, these early shifts in male-female interneuron development may drive adult sex differences in PV neuron function and connectivity. Our results identify gephyrin phosphorylation as a new substrate organizing PV neuron development at the anatomical, functional, and transcriptional levels in a sex-dependent manner, thus implicating kinase signaling disruption as a new mechanism contributing to the sex-dependent etiology of brain disorders.

Modulation of sleep/wake patterns by gephyrin phosphorylation status.

Sleep/wake cycles intricately shape physiological activities including cognitive brain functions, yet the precise molecular orchestrators of sleep remain elusive. Notably, the clinical impact of benzodiazepine drugs underscores the pivotal role of GABAergic neurotransmission in sleep regulation. However, the specific contributions of distinct GABAA receptor subtypes and their principal scaffolding protein, gephyrin, in sleep dynamics remain unclear. The evolving role of synaptic phospho-proteome alterations at excitatory and inhibitory synapses suggests a potential avenue for modulating gephyrin and, consequently, GABAARs for sleep through on-demand kinase recruitment. Our study unveils the distinctive roles of two prevalent GABAA receptor subtypes, α1- and α2-GABAARs, in influencing sleep duration and electrical sleep activity. Notably, the absence of α1-GABAARs emerges as central in sleep regulation, manifesting significant alterations in both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep during dark or active phases, accompanied by altered electroencephalogram (EEG) patterns across various frequencies. Gephyrin proteomics analysis reveals sleep/wake-dependent interactions with a repertoire of known and novel kinases. Crucially, we identify the regulation of gephyrin interaction with ERK1/2, and phosphorylations at serines 268 and 270 are dictated by sleep/wake cycles. Employing AAV-eGFP-gephyrin or its phospho-null variant (S268A/S270A), we disrupt sleep either globally or locally to demonstrate gephyrin phosphorylation as a sleep regulator. In summary, our findings support the local cortical sleep hypothesis and we unveil a molecular mechanism operating at GABAergic synapses, providing critical insights into the intricate regulation of sleep.

Protocol for the culturing of primary hippocampal mouse neurons for functional in vitro studies.

Primary hippocampal cultures grown from genetically modified mice provide a simplified context to study molecular mechanisms underlying neuronal development, synaptogenesis, and synapse plasticity in vitro. Here, we describe a simple protocol for culturing hippocampal neurons from P0 to P2 mice and a strategy for inducing alterations in synaptic strength at inhibitory and excitatory synapses in vitro. We also describe approaches for immunofluorescent labeling, image acquisition, and quantification of synaptic proteins. For complete details on the use and execution of this protocol, please refer to Cramer et al.1.

Protocol for in vivo analysis of pre- and post-synaptic protein function in mice.

Studying synapses in vivo presents challenges due to the complexity of accurately targeting and visualizing specific synaptic proteins within the brain. Here, we present a protocol for in vivo analysis of pre- and post-synaptic protein function in mice. We describe steps for combining adeno-associated virus (AAV)-mediated gene transfer to manipulate specific neuron subtypes. We also describe immunofluorescence on artificial cerebrospinal fluid (ACSF)-perfused brain sections to enhance the visualization of synaptic proteins. For complete details on the use and execution of this protocol, please refer to Cramer et al.1.

The gephyrin scaffold modulates cortical layer 2/3 pyramidal neuron responsiveness to single whisker stimulation.

Gephyrin is the main scaffolding protein at inhibitory postsynaptic sites, and its clusters are the signaling hubs where several molecular pathways converge. Post-translational modifications (PTMs) of gephyrin alter GABAA receptor clustering at the synapse, but it is unclear how this affects neuronal activity at the circuit level. We assessed the contribution of gephyrin PTMs to microcircuit activity in the mouse barrel cortex by slice electrophysiology and in vivo two-photon calcium imaging of layer 2/3 (L2/3) pyramidal cells during single-whisker stimulation. Our results suggest that, depending on the type of gephyrin PTM, the neuronal activities of L2/3 pyramidal neurons can be differentially modulated, leading to changes in the size of the neuronal population responding to the single-whisker stimulation. Furthermore, we show that gephyrin PTMs have their preference for selecting synaptic GABAA receptor subunits. Our results identify an important role of gephyrin and GABAergic postsynaptic sites for cortical microcircuit function during sensory stimulation.