Rett syndrome is a neurodevelopmental disorder that primarily affects females and is caused by mutations in the methyl-CpG-binding-protein 2 (MECP2) gene. Initially, MeCP2 had been shown to be a repressor of gene transcription. In their 2008 paper, Chahrour and colleagues (DOI: 10.1126/science.1153252) reported that MeCP2 could also function as a transcriptional activator.

Synaptic transmission is initiated via spontaneous or action-potential evoked fusion of synaptic vesicles. At excitatory synapses, glutamatergic receptors activated by spontaneous and evoked neurotransmission are segregated. Although inhibitory synapses also transmit signals spontaneously or in response to action potentials, they differ from excitatory synapses in both structure and function. Therefore, we hypothesized that inhibitory synapses may have different organizing principles. We report picrotoxin, a GABAAR antagonist, blocks neurotransmission in a use-dependent manner at rat hippocampal synapses and therefore can be used to interrogate synaptic properties. Using this tool, we uncovered partial segregation of inhibitory spontaneous and evoked neurotransmission. We found up to 40% of the evoked response is mediated through GABAARs which are only activated by evoked neurotransmission. These data indicate GABAergic spontaneous and evoked neurotransmission processes are partially non-overlapping, suggesting they may serve divergent roles in neuronal signaling.

Spontaneous neurotransmitter release is a fundamental property of synapses in which neurotransmitter filled vesicles release their content independent of presynaptic action potentials (APs). Despite their seemingly random nature, these spontaneous fusion events can be regulated by Ca 21 signaling pathways. Here, we probed the mechanisms that maintain Ca 21 sensitivity of spontaneous release events in synapses formed between hippocampal neurons cultured from rats of both sexes. In this setting, we examined the potential role of vesicle-associated membrane protein 4 (VAMP4), a vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein in spontaneous neurotransmission. Our results show that VAMP4 is required for Ca 21 -dependent spontaneous excitatory neurotransmission, with a limited role in spontaneous inhibitory neurotransmission. Key residues in VAMP4 that regulate its retrieval as well as functional clathrin-mediated vesicle trafficking were essential for the maintenance of VAMP4-mediated spontaneous release. Moreover, high-frequency stimulation (HFS) that typically triggers asynchronous release and retrieval of VAMP4 from the plasma membrane also augments Ca 21 -sensitive spontaneous release for up to 30 min in a VAMP4-dependent manner. This VAMP4-mediated link between asynchronous and spontaneous excitatory neurotransmission might serve as a presynaptic substrate for synaptic plasticity coupling distinct forms of release.

Highlights d Transcription can be directly driven by inhibitory signaling d At rest, neurons sense inhibition, not summation of excitatory/inhibitory current d mIPSCs regulate calcium signaling at excitatory synapses and somas d Blocking mIPSCs downscales excitatory synapses via Bdnf transcription and signaling

Background The use of the CRISPR/Cas9 system is becoming widespread, however current studies have predominantly focused on dividing cells. It is currently unknown if CRISPR/Cas9 can be used in a postmitotic setting to examine non-cell autonomous/presynaptic phenotypes in the resulting genetically heterogeneous cell population. New method A single CRISPR/Cas9 lentivirus was used to transfect a high percentage of primary cultured neurons and target synaptobrevin 2 (Syb2, also called VAMP2). Results Primary hippocampal cultures infected with the Syb2 targeting virus displayed dramatic reductions in Syb2 protein and immunocytochemical staining. In many boutons Syb2 was completely undetected. These cultures recapitulated the known functional phenotypes of Syb2 knockout neurons, which are non-cell autonomous and presynaptic in origin, indicating that Syb2 was knocked out in a large fraction of neurons. Comparison with existing method(s) Previous methods used multiple viruses or sparse transfection methods and only examined cell autonomous or postsynaptic phenotypes. The current method demonstrates that the CRISPR/Cas9 system can be used to alter network dynamics by removing or lowering the target gene from a majority of cells in the culture. Conclusions A combination of CRISPR/Cas9 system and single high efficiency lentivirus infection can be used to examine non-cell autonomous and presynaptic phenotypes in postmitotic neurons.

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Rett syndrome is a leading cause of intellectual disability in females primarily caused by loss of function mutations in the transcriptional regulator MeCP2. Loss of MeCP2 leads to a host of synaptic phenotypes that are believed to underlie Rett syndrome pathophysiology. Synaptic deficits vary by brain region upon MeCP2 loss, suggesting distinct molecular alterations leading to disparate synaptic outcomes. In this study, we examined the contribution of MeCP2's newly described role in miRNA regulation to regional molecular and synaptic impairments. Two miRNAs, miR‐101a and miR‐203, were identified and confirmed as upregulated in MeCP2 KO mice in the hippocampus and cortex, respectively. miR‐101a overexpression in hippocampal cultures led to opposing effects at excitatory and inhibitory synapses and in spontaneous and evoked neurotransmission, revealing the potential for a single miRNA to broadly regulate synapse function in the hippocampus. These results highlight the importance of regional alterations in miRNA expression and the specific impact on synaptic function with potential implications for Rett syndrome.

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