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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 GABA A 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 GABA A receptor subunits. Our results identify an important role of gephyrin and GABAergic postsynaptic sites for cortical microcircuit function during sensory stimulation.

From single-cell RNA-sequencing (scRNA-seq) and spatial transcriptomics (ST), one can extract high-dimensional gene expression patterns that can be described by intercellular communication networks or decoupled gene modules. These two descriptions of information flow are often assumed to occur independently. However, intercellular communication drives directed flows of information that are mediated by intracellular gene modules, in turn triggering outflows of other signals. Methodologies to describe such intercellular flows are lacking. We present FlowSig, a method that infers communication-driven intercellular flows from scRNA-seq or ST data using graphical causal modeling and conditional independence. We benchmark FlowSig using newly generated experimental cortical organoid data and synthetic data generated from mathematical modeling. We demonstrate FlowSig’s utility by applying it to various studies, showing that FlowSig can capture stimulation-induced changes to paracrine signaling in pancreatic islets, demonstrate shifts in intercellular flows due to increasing COVID-19 severity and reconstruct morphogen-driven activator–inhibitor patterns in mouse embryogenesis. Using single-cell and spatial transcriptomics data, FlowSig provides a unified signaling modeling framework by connecting intercellular communication mediated by ligand–receptor interactions and intracellular gene expression modules.

Orexins (also called hypocretins) are hypothalamic neuropeptides that carry out essential functions in the central nervous system; however, little is known about their release and range of action in vivo owing to the limited resolution of current detection technologies. Here we developed a genetically encoded orexin sensor (OxLight1) based on the engineering of circularly permutated green fluorescent protein into the human type-2 orexin receptor. In mice OxLight1 detects optogenetically evoked release of endogenous orexins in vivo with high sensitivity. Photometry recordings of OxLight1 in mice show rapid orexin release associated with spontaneous running behavior, acute stress and sleep-to-wake transitions in different brain areas. Moreover, two-photon imaging of OxLight1 reveals orexin release in layer 2/3 of the mouse somatosensory cortex during emergence from anesthesia. Thus, OxLight1 enables sensitive and direct optical detection of orexin neuropeptides with high spatiotemporal resolution in living animals. OxLight1 is a genetically encoded sensor for the orexin neuropeptides. It has been applied in fiber photometry recordings and two-photon imaging in mice during a variety of behaviors.

In developing brain neuronal migration, dendrite outgrowth and dendritic spine outgrowth are controlled by Cdc42, a small GTPase of the Rho family, and its activators. Cdc42 function in promoting actin polymerization is crucial for glutamatergic synapse regulation. Here, we focus on GABAergic synapse-specific activator of Cdc42, collybistin (CB) and examine functional differences between its splice isoforms CB1 and CB2. We report that CB1 and CB2 differentially regulate GABAergic synapse formation in vitro along proximal-distal axis and adult-born neuron maturation in vivo. The functional specialization between CB1 and CB2 isoforms arises from their differential protein half-life, in turn regulated by ubiquitin conjugation of the unique CB1 C-terminus. We report that CB1 and CB2 negatively regulate Cdc42; however, Cdc42 activation is dependent on CB interaction with gephyrin. During hippocampal adult neurogenesis CB1 regulates neuronal migration, while CB2 is essential for dendrite outgrowth. Finally, using mice lacking Gabra2 subunit, we show that CB1 function is downstream of GABAARs, and we can rescue adult neurogenesis deficit observed in Gabra2 KO. Overall, our results uncover previously unexpected role for CB isoforms downstream of α2-containing GABAARs during neuron maturation in a Cdc42 dependent mechanism.

A diverse set of GABAA receptors (GABAARs) enable synaptic plasticity adaptations at inhibitory postsynaptic sites in collaboration with the scaffolding protein gephyrin. Early studies helped to identify distinctions between GABAAR subtypes allocated within specific functional circuits, but their contribution to the changing dynamics of a microcircuit remains unclear. Here, using the whisker-barrel system in mouse, we assessed the contribution of specific synaptic GABAAR subtypes and gephyrin scaffolding changes to sensory processing in vivo. We monitored spontaneous and evoked Ca2+ transients in layer 2/3 pyramidal cells with the genetically encoded Ca2+ sensor RCaMP1.07. Using Gabra1 or Gabra2 global and conditional knockout mice, we uncovered that alpha1- and alpha2-GABAARs determine the sparseness of L2/3 pyramidal neuron encoding. In a cell-type dependent manner, alpha1-GABAARs and alpha2-GABAARs affected neuronal excitability and the reliability of neuronal responses after whisker stimulation. We also discerned that gephyrin with its diverse post-translational modifications (PTMs) shows preference for specific GABAAR subtype to facilitate microcircuit activity. Our results underscore the relevance of the diversity of GABAARs within a cortical microcircuit. Competing Interest Statement The authors have declared no competing interest. Footnotes * we have added new electrophysiology data using GABARA1 and GABRA2 KO mice. We have reanalysed the 2-P data using additional control GCaMP only cells. We present the loss of GABAAR ubunits upon cre expression in vivo. We provide morphology analysis showing gephyrin PTM determine GABAAR subunit preference at synapses

In developing brain neuronal migration, dendrite outgrowth and dendritic spine outgrowth are controlled by Cdc42, a small GTPase of the Rho family, and its activators. Cdc42 function in promoting actin polymerization is crucial for glutamatergic synapse regulation. Here, we focus on GABAergic synapse-specific activator of Cdc42, collybistin (CB) and examine functional differences between its splice isoforms CB1 and CB2. We report that CB1 and CB2 differentially regulate GABAergic synapse formation in vitro along proximal-distal axis and adult-born neuron maturation in vivo. The functional specialization between CB1 and CB2 isoforms arises from their differential protein half-life, in turn regulated by ubiquitin conjugation of the unique CB1 C-terminus. We report that CB1 and CB2 negatively regulate Cdc42; however, Cdc42 activation is dependent on CB interaction with gephyrin. During hippocampal adult neurogenesis CB1 regulates neuronal migration, while CB2 is essential for dendrite outgrowth. Finally, using mice lacking Gabra2 subunit, we show that CB1 function is downstream of GABA.sub.A Rs, and we can rescue adult neurogenesis deficit observed in Gabra2 KO. Overall, our results uncover previously unexpected role for CB isoforms downstream of [alpha]2-containing GABA.sub.A Rs during neuron maturation in a Cdc42 dependent mechanism.

In developing brain neuronal migration, dendrite outgrowth and dendritic spine outgrowth are controlled by Cdc42, a small GTPase of the Rho family, and its activators. Cdc42 function in promoting actin polymerization is crucial for glutamatergic synapse regulation. Here, we focus on GABAergic synapse-specific activator of Cdc42, collybistin (CB) and examine functional differences between its splice isoforms CB1 and CB2. We report that CB1 and CB2 differentially regulate GABAergic synapse formation in vitro along proximal-distal axis and adult-born neuron maturation in vivo. The functional specialization between CB1 and CB2 isoforms arises from their differential protein half-life, in turn regulated by ubiquitin conjugation of the unique CB1 C-terminus. We report that CB1 and CB2 negatively regulate Cdc42; however, Cdc42 activation is dependent on CB interaction with gephyrin. During hippocampal adult neurogenesis CB1 regulates neuronal migration, while CB2 is essential for dendrite outgrowth. Finally, using mice lacking Gabra2 subunit, we show that CB1 function is downstream of GABAARs, and we can rescue adult neurogenesis deficit observed in Gabra2 KO. Overall, our results uncover previously unexpected role for CB isoforms downstream of [alpha] 2-containing GABAARs during neuron maturation in a Cdc42 dependent mechanism.

The human neocortex exhibits characteristic regional patterning (arealization) critical for higher-order cognitive function. Disrupted arealization is strongly implicated in neurodevelopmental disorders (NDDs), but current neocortical organoid models largely fail to recapitulate this patterning, limiting mechanistic understanding. Here, we establish a straightforward method for generating arealized organoids through short-term early exposure to anterior (FGF8) or posterior (BMP4/CHIR-99021) morphogens. These treatments created distinct anterior and posterior signaling centers, supporting long-lasting polarization, which we validated with single-cell RNA sequencing that revealed area-specific molecular signatures matching prenatal human cortex. To demonstrate the utility of this platform, we modeled Fragile X Syndrome (FXS) in organoids with distinct anterior and posterior regional identities. FXS organoids showed highly disrupted SOX4/SOX11 expression gradients along the anterior-posterior axis, consistent with alterations found in autism spectrum disorder (ASD) and demonstrate how regional patterning defects may contribute to NDD pathology. Together, our study provides a robust platform for generating neocortical organoids with anterior-posterior molecular signatures and highlights the importance of modeling NDDs using experimental platforms with neuroanatomic specificity.