We have studied the effect of clonal overexpression of neuroligin 3 (NL3) or neuroligin 2 (NL2) in the adult rat cerebral cortex following in utero electroporation (IUEP) at embryonic stage E14. Overexpression of NL3 leads to a large increase in vGAT and GAD65 in the GABAergic contacts that the overexpressing neurons receive. Overexpression of NL2 produced a similar effect but to a lesser extent. In contrast, overexpression of NL3 or NL2 after IUEP, does not affect vGlut1 in the glutamatergic contacts that the NL3 or NL2 overexpressing neurons receive. The NL3 or NL2 overexpressing neurons do not show increased innervation by parvalbumin-containing GABAergic terminals or increased parvalbumin in the same terminals that show increased vGAT. These results indicate that the observed increase in vGAT and GAD65 is not due to increased GABAergic innervation but to increased expression of vGAT and GAD65 in the GABAergic contacts that NL3 or NL2 overexpressing neurons receive. The majority of bright vGAT puncta contacting the NL3 overexpressing neurons have no gephyrin juxtaposed to them indicating that many of these contacts are non-synaptic. This contrasts with the majority of the NL2 overexpressing neurons, which show plenty of synaptic gephyrin clusters juxtaposed to vGAT. Besides having an effect on GABAergic contacts, overexpression of NL3 interferes with the neuronal radial migration, in the cerebral cortex, of the neurons overexpressing NL3.

Collybistin (CB) is a guanine nucleotide exchange factor selectively localized to γ-aminobutyric acid (GABA)ergic and glycinergic postsynapses. Active CB interacts with gephyrin, inducing the submembranous clustering and the postsynaptic accumulation of gephyrin, which is a scaffold protein that recruits GABA receptors (GABA Rs) at the postsynapse. CB is expressed with or without a src homology 3 (SH3) domain. We have previously reported the effects on GABAergic synapses of the acute overexpression of CB or CB in cultured hippocampal (HP) neurons. In the present communication, we are studying the effects on GABAergic synapses after chronic in vivo transgenic expression of CB2 or CB2 in neurons of the adult rat cerebral cortex. The embryonic precursors of these cortical neurons were in utero electroporated with CB or CB DNAs, migrated to the appropriate cortical layer, and became integrated in cortical circuits. The results show that: 1) the strength of inhibitory synapses in vivo can be enhanced by increasing the expression of CB in neurons; and 2) there are significant differences in the results between in vivo and in culture studies. J. Comp. Neurol. 525:1291-1311, 2017. © 2016 Wiley Periodicals, Inc.

It has been proposed that the combinatorial expression of γ‐protocadherins (Pcdh‐γs) and other clustered protocadherins (Pcdhs) provides a code of molecular identity and individuality to neurons, which plays a major role in the establishment of specific synaptic connectivity and formation of neuronal circuits. Particular attention has been directed to the Pcdh‐γ family, for which experimental evidence derived from Pcdh‐γ‐deficient mice shows that they are involved in dendrite self‐avoidance, synapse development, dendritic arborization, spine maturation, and prevention of apoptosis of some neurons. Moreover, a triple‐mutant mouse deficient in the three C‐type members of the Pcdh‐γ family (Pcdh‐γC3, Pcdh‐γC4, and Pcdh‐γC5) shows a phenotype similar to the mouse deficient in whole Pcdh‐γ family, indicating that the latter is largely due to the absence of C‐type Pcdh‐γs. The role of each individual C‐type Pcdh‐γ is not known. We have developed a specific antibody to Pcdh‐γC4 to reveal the expression of this protein in the rat brain. The results show that although Pcdh‐γC4 is expressed at higher levels in the embryo and earlier postnatal weeks, it is also expressed in the adult rat brain. Pcdh‐γC4 is expressed in both neurons and astrocytes. In the adult brain, the regional distribution of Pcdh‐γC4 immunoreactivity is similar to that of Pcdh‐γC4 mRNA, being highest in the olfactory bulb, dentate gyrus, and cerebellum. Pcdh‐γC4 forms puncta that are frequently apposed to glutamatergic and GABAergic synapses. They are also frequently associated with neuron‐astrocyte contacts. The results provide new insights into the cell recognition function of Pcdh‐γC4 in neurons and astrocytes.

Collybistin (CB) is a guanine nucleotide exchange factor (GEF) selectively localized at GABAergic and glycinergic postsynapses. Analysis of mRNA shows that several isoforms of collybistin are expressed in the brain. Some of the isoforms have a SH3 domain (CBSH3+) and some have no SH3 domain (CBSH3−). The CBSH3+ mRNAs are predominantly expressed over CBSH3−. However, in an immunoblot study of mouse brain homogenates, only CBSH3+ protein isoforms were detected, proposing that CBSH3− protein might not be expressed in the brain. The expression or lack of expression of CBSH3− protein is an important issue because CBSH3− has a strong effect in promoting the postsynaptic clustering of gephyrin and GABA-A receptors (GABA A Rs). Moreover CBSH3− is constitutively active; therefore lower expression of CBSH3− protein might play a relatively stronger functional role than the more abundant but self-inhibited CBSH3+ isoforms, which need to be activated. We are now showing that: (a) CBSH3− protein is expressed in the brain; (b) parvalbumin positive (PV+) interneurons show higher expression of CBSH3− protein than other neurons; (c) CBSH3− is associated with GABAergic synapses in various regions of the brain and (d) knocking down CBSH3− in hippocampal neurons decreases the synaptic clustering of gephyrin and GABA A Rs. The results show that CBSH3− protein is expressed in the brain and that it plays a significant role in the size regulation of the GABAergic postsynapse.

In the developing and adult CNS, new oligodendrocytes (OLs) are generated from a population of cells known as oligodendrocyte precursor cells (OPCs). As they begin to differentiate, OPCs undergo a series of highly regulated changes to morphology, gene expression, and membrane organization. This stage represents a critical bottleneck in oligodendrogenesis, and the regulatory program that guides it is still not fully understood. Here we show that in vivo toxin-mediated cleavage of the vesicle associated SNARE proteins VAMP2/3 in the OL lineage of both male and female mice impairs the ability of early OLs to mature into functional, myelinating OLs. In the developing mouse spinal cord, many VAMP2/3-deficient OLs appeared to stall in the premyelinating, early OL stage, resulting in an overall loss of both myelin density and OL number. The Src kinase Fyn, a key regulator of oligodendrogenesis and myelination, is highly expressed among premyelinating OLs, but its expression decreases as OLs mature. We found that OLs lacking VAMP2/3 in the spinal cord white matter showed significantly higher expression of Fyn compared to neighboring control cells, potentially due to an extended premyelinating stage. Overall, our results show that functional VAMP2/3 in OL lineage cells is essential for proper myelin formation and plays a major role in controlling the maturation and terminal differentiation of premyelinating OLs.