Edited by Thomas SöllnerThe formation of neuronal synapses and the dynamic regulation of their efficacy depend on the proper assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic postsynapses requires the scaffold protein gephyrin and the guanine nucleotide exchange factor collybistin (Cb). In vitro, the pleckstrin homology domain of Cb binds phosphoinositides, specifically phosphatidylinositol 3-phosphate (PI3P). However, whether PI3P is required for inhibitory postsynapse formation is currently unknown. Here, we investigated the role of PI3P at developing GABAergic postsynapses by using a membrane-permeant PI3P derivative, timelapse confocal imaging, electrophysiology, as well as knockdown and overexpression of PI3P-metabolizing enzymes. Our results provide the first in cellula evidence that PI3P located at early/ sorting endosomes regulates the postsynaptic clustering of gephyrin and GABA A receptors and the strength of inhibitory, but not excitatory, postsynapses in cultured hippocampal neurons. In human embryonic kidney 293 cells, stimulation of gephyrin cluster formation by PI3P depends on Cb. We therefore conclude that the endosomal pool of PI3P, generated by the class III phosphatidylinositol 3-kinase, is important for the Cbmediated recruitment of gephyrin and GABA A receptors to developing inhibitory postsynapses and thus the formation of postsynaptic membrane specializations.Phosphoinositides, phosphorylated derivatives of phosphatidylinositol, are critical regulators of intracellular signaling, membrane traffic, and cell compartmentalization (1-3). The functional roles of phosphoinositide metabolism have been studied in great detail at the presynaptic terminal, where phosphoinositide turnover is of critical importance for synaptic vesicle (SV) 3 recycling and synapse function (4). Little, however, is known about specific roles of phosphoinositides at postsynapses.Core components of most inhibitory GABAergic postsynapses are GABA A receptors (GABA A Rs), the cell adhesion protein neuroligin 2 (NL2), the scaffolding protein gephyrin, and the guanine nucleotide exchange factor collybistin (Cb) (5, 6). During the formation of many GABAergic synapses, most notably those at neuronal somata, NL2 is thought to activate Cb, which promotes Cb membrane association and the subsequent recruitment of gephyrin and GABA A Rs (7-9). In vitro binding studies indicate that the pleckstrin homology (PH) domain of Cb specifically binds phosphatidylinositol 3-phosphate (PI3P) (9 -13), and this interaction is thought to be essential for the anchoring of NL2-gephyrin-Cb complexes at the postsynaptic plasma membrane of synapses that depend on Cb (7, 9). The notion that PI3P binding by the PH domain is required for proper Cb function is supported by the observation that deletion of the PH domain (14), or the substitution therein of two arginine residues that are essential for PI3P binding (9, 11), causes a marked reduction in the density of postsynaptic gephyrin cluste...

Summary Mammalian central synapses exhibit vast heterogeneity in signaling strength. To understand the extent of this diversity, how it is achieved, and its functional implications, characterization of a large number of individual synapses is required. Using glutamate imaging, we characterized the evoked release probability and spontaneous release frequency of over 24,000 individual synapses. We found striking variability and no correlation between action potential-evoked and spontaneous synaptic release strength, suggesting distinct regulatory mechanisms. Subpixel localization of individual evoked and spontaneous release events reveals tight spatial regulation of evoked release and enhanced spontaneous release outside of evoked release region. Using on-stage post hoc immune-labeling of vesicle-associated proteins, Ca 2+ -sensing proteins, and soluble presynaptic proteins we were able to show that distinct molecular ensembles are associated with evoked and spontaneous modes of synaptic release.

Striatal output pathways are known to play a crucial role in the control of movement. One possible component for shaping the synaptic output of striatal neuron is the glutamatergic input that originates from cortex and thalamus. Although reports focusing on quantifying glutamatergic-induced morphological changes in striatum exist, the role of glutamatergic input in regulating striatal function remains poorly understood. Using primary neurons from newborn mice of either sex in a reduced two-neuron microcircuit culture system, we examined whether glutamatergic input modulates the output of striatal neurons. We found that glutamatergic input enhanced striatal inhibition in vitro. With a glutamatergic partner from either cortex or thalamus, we attributed this potentiation to an increase in the size of quantal IPSC, suggesting a strengthening of the postsynaptic response to GABAergic signaling. Additionally, a differential effect of cortical and thalamic innervation onto striatal GABAergic neurons output was revealed. We observed that cortical, but not thalamic input, enhanced the number of releasable GABAergic synaptic vesicles and morphological synapses. Importantly, these alterations were reverted by blockade of neuronal activity and glutamate receptors, as well as disruption of BDNF-TrkB signaling. Together, our data indicate, for first time, that GABAergic synapse formation in corticostriatal pairs depends on two parallel, but potentially intersecting, signaling pathways that involve glutamate receptor activation in striatal neurons, as well as BDNF signaling. Understanding how cortical and thalamic inputs refine striatal output will pave the way toward dissecting basal ganglia activity in both physiological and pathological conditions. Significance StatementStriatal GABAergic microcircuits are critical for motor function. However, the mechanisms controlling striatal output, particularly at the level of synaptic strength, are unclear. Using two-neuron culture system, we quantified the synaptic output of individual striatal GABAergic neurons paired with a glutamatergic partner and studied the influence of the excitatory connections that are known to be interregionally formed in vivo. We found that glutamatergic input potentiated striatal inhibitory output, potentially involving an increased feedback and/or feedforward inhibition. Moreover, distinct components of glutamatergic innervation, such as firing activity or release of neurotrophic factors were shown to be required for the glutamatergic-induced phenotype. Investigation, therefore, of two-neuron in vitro microcircuits could be a powerful tool to explore synaptic mechanisms or disease pathophysiology.

Transcriptional dysregulation in Huntington’s disease (HD) causes functional deficits in striatal neurons. Here, we performed Patch-sequencing (Patch-seq) in an in vitro HD model to investigate the effects of mutant Huntingtin (Htt) on synaptic transmission and gene transcription in single striatal neurons. We found that expression of mutant Htt decreased the synaptic output of striatal neurons in a cell autonomous fashion and identified a number of genes whose dysregulation was correlated with physiological deficiencies in mutant Htt neurons. In support of a pivotal role for epigenetic mechanisms in HD pathophysiology, we found that inhibiting histone deacetylase 1/3 activities rectified several functional and morphological deficits and alleviated the aberrant transcriptional profiles in mutant Htt neurons. With this study, we demonstrate that Patch-seq technology can be applied both to better understand molecular mechanisms underlying a complex neurological disease at the single-cell level and to provide a platform for screening for therapeutics for the disease.

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