Excitatory Connections Research Articles

Orbitofrontal control of the olfactory cortex regulates olfactory discrimination learning.

Serving as an integral node for cognitive processing and value-based decision-making, the orbitofrontal cortex (OFC) plays a multifaceted role in associative learning and reward-driven behaviours through its widespread synaptic integration with both subcortical structures and sensory cortices. Despite the OFC's robust innervation of the olfactory cortex, the functional implications and underlying mechanisms of this top-down influence remain largely unexplored. In this study, we demonstrated that the OFC formed both direct excitatory and indirect inhibitory synaptic connections with pyramidal neurons in the anterior piriform cortex (aPC). OFC projection predominantly regulated spontaneous and odour-evoked excitatory activity in the aPC of awake mice. Importantly, suppression of this OFC-aPC projection disrupted olfactory discrimination learning, potentially due to a consequent decrease in the excitability of aPC principal output neurons following inhibition of this projection. Whole-cell recordings revealed that olfactory learning increased the intrinsic excitability of aPC neurons while concurrently decreasing OFC input to these neurons. These findings underscore the pivotal influence of orbitofrontal modulation over the olfactory cortex in the context of olfactory learning and provide insight into the associated neurophysiological mechanisms. KEY POINTS: The orbitofrontal cortex (OFC) densely innervates the anterior piriform cortex (aPC) through direct excitatory synaptic connections. The OFC regulates both spontaneous and odour-evoked excitatory activities in the aPC of awake mice. Inhibition of OFC projections disrupts olfactory discrimination learning, probably due to reduced excitability of aPC main output neurons. Following olfactory learning, the intrinsic excitability of aPC neurons increases while the OFC-aPC input decreases, highlighting the importance of adaptable OFC input for olfactory learning. These results provide new perspectives on how the OFC's top-down control modulates sensory integration and associative learning.

Specific inhibition and disinhibition in the higher-order structure of a cortical connectome.

Neurons are thought to act as parts of assemblies with strong internal excitatory connectivity. Conversely, inhibition is often reduced to blanket inhibition with no targeting specificity. We analyzed the structure of excitation and inhibition in the MICrONS $mm^{3}$ dataset, an electron microscopic reconstruction of a piece of cortical tissue. We found that excitation was structured around a feed-forward flow in large non-random neuron motifs with a structure of information flow from a small number of sources to a larger number of potential targets. Inhibitory neurons connected with neurons in specific sequential positions of these motifs, implementing targeted and symmetrical competition between them. None of these trends are detectable in only pairwise connectivity, demonstrating that inhibition is structured by these large motifs. While descriptions of inhibition in cortical circuits range from non-specific blanket-inhibition to targeted, our results describe a form of targeting specificity existing in the higher-order structure of the connectome. These findings have important implications for the role of inhibition in learning and synaptic plasticity.

Learning-induced remodelling of inhibitory synapses in the motor cortex.

Robust structural and functional plasticity occurs at excitatory synapses in the motor cortex in response to learning. It is well established that local spinogenesis and the subsequent maintenance of newly formed spines are crucial for motor learning. However, despite local synaptic inhibition being essential for shaping excitatory synaptic input, less is known about the structural rearrangement of inhibitory synapses following learning. In this study, we co-expressed the structural marker tdTomato and a mEmerald-tagged intrabody against gephyrin to visualize inhibitory synapses in layer 2/3 cortical neurons of wild-type CD1 mice. We found that a 1-day accelerated rotarod paradigm induced robust motor learning in male and female adult CD1 mice. Histological analyses revealed a significant increase in the surface area of gephyrin puncta in neurons within the motor cortex but not in the somatosensory cortex upon motor learning. Furthermore, this learning-induced reorganization of inhibitory synapses only occurred in dendritic shafts and not in the spines. These data suggest that learning induces experience-dependent remodelling of existing inhibitory synapses to fine-tune intrinsic plasticity and input-specific modulation of excitatory connections in the motor cortex.

Dendritic spines of layer 5 pyramidal neurons of the aging somatosensory cortex exhibit reduced volumetric remodeling.

Impairments in synaptic dynamics and stability are observed both in neurodegenerative disorders and in the healthy aging cortex, which exhibits elevated dendritic spine turnover and decreased long-term stability of excitatory connections at baseline, as well as an altered response to plasticity induction. In addition to the discrete gain and loss of synapses, spines also change in size and strength both during learning and in the absence of neural activity, and synaptic volume has been associated with stability and incorporation into memory traces. Furthermore, intrinsic dynamics, an apparently stochastic component of spine volume changes, may serve as a homeostatic mechanism to prevent stabilization of superfluous connections. However, the effects of age on modulation of synaptic weights remain unknown. Using two-photon excitation (2PE) microscopy of spines during chemical plasticity induction in vitro and analyzing longitudinal in vivo 2PE images after a plasticity-inducing manipulation, we characterize the effects of age on volumetric changes of spines of the apical tuft of layer 5 pyramidal neurons of mouse primary somatosensory cortex. Aged mice exhibit decreased volumetric volatility and delayed rearrangement of synaptic weights of persistent connections, as well as greater susceptibility to spine shrinkage in response to chemical long-term depression. These results suggest a deficit in the aging brain's ability to fine-tune synaptic weights to properly incorporate and retain novel memories. This research provides the first evidence of alterations in spine volumetric dynamics in healthy aging and may support a model of impaired processing and learning in the aged somatosensory system.Significance Statement Aging is known to impact cognitive functions and sensory processing, yet the underlying mechanisms at the synaptic level remain unclear. This study investigates dendritic spine dynamics of layer 5 pyramidal neurons in the aging somatosensory cortex. By employing two-photon excitation microscopy and analyzing spine volume changes during plasticity induction, we reveal that aged mice exhibit decreased volumetric volatility and delayed synaptic weight rearrangement. These alterations may impair the brain's ability to fine-tune synaptic weights, which is crucial for incorporating and retaining new memories. This research provides the first evidence of altered spine volumetric dynamics in healthy aging, suggesting a potential mechanism for impaired processing and learning in the aged cortex. Understanding these changes could aid in mitigation of age-related cognitive decline.

Synaptic communication within the microcircuits of pyramidal neurons and basket cells in the mouse prefrontal cortex.

Basket cells are inhibitory interneurons in cortical structures with the potential to efficiently control the activity of their postsynaptic partners. Although their contribution to higher order cognitive functions associated with the medial prefrontal cortex (mPFC) relies on the characteristics of their synaptic connections, the way that they are embedded into local circuits is still not fully uncovered. Here, we determined the synaptic properties of excitatory and inhibitory connections between pyramidal neurons (PNs), cholecystokinin-containing basket cells (CCKBCs) and parvalbumin-containing basket cells (PVBCs) in the mouse mPFC. By performing paired recordings, we revealed that PVBCs receive larger unitary excitatory postsynaptic currents from PNs with shorter latency and faster kinetic properties compared to events evoked in CCKBCs. Also, unitary inhibitory postsynaptic currents in PNs were more reliably evoked by PVBCs than by CCKBCs, yet the former connections showed profound short-term depression. Moreover, we demonstrated that CCKBCs and PVBCs in the mPFC are connected with each other. Because alterations in PVBC function have been linked to neurological and psychiatric conditions such as Alzheimer's disease and schizophrenia and CCKBC vulnerability might play a role in mood disorders, a deeper understanding of the general features of basket cell synapses could serve as a reference point for future investigations with therapeutic objectives. KEY POINTS: Cholecystokinin- (CCKBCs) and parvalbumin-expressing basket cells (PVBCs) have distinct passive and active membrane properties. Pyramidal neurons give rise to larger unitary excitatory postsynaptic currents in PVBCs compared to events in CCKBCs. Unitary inhibitory postsynaptic currents in pyramidal neurons are more reliably evoked by PVBCs than by CCKBCs. Basket cells form chemical synapses and gap junctions with their own cell type. The two basket cell types are connected with each other.

Potentiation of cortico-spinal output via targeted electrical stimulation of the motor thalamus

Cerebral white matter lesions prevent cortico-spinal descending inputs from effectively activating spinal motoneurons, leading to loss of motor control. However, in most cases, the damage to cortico-spinal axons is incomplete offering a potential target for therapies aimed at improving volitional muscle activation. Here we hypothesize that, by engaging direct excitatory connections to cortico-spinal motoneurons, stimulation of the motor thalamus could facilitate activation of surviving cortico-spinal fibers thereby immediately potentiating motor output. To test this hypothesis, we identify optimal thalamic targets and stimulation parameters that enhance upper-limb motor-evoked potentials and grip forces in anesthetized monkeys. This potentiation persists after white matter lesions. We replicate these results in humans during intra-operative testing. We then design a stimulation protocol that immediately improves strength and force control in a patient with a chronic white matter lesion. Our results show that electrical stimulation targeting surviving neural pathways can improve motor control after white matter lesions.

New Insights into Freezing of Gait in Parkinson's Disease from Spectral Dynamic Causal Modeling.

Freezing of gait is one of the most disturbing motor symptoms of Parkinson's disease (PD). However, the effective connectivity between key brain hubs that are associated with the pathophysiological mechanism of freezing of gait remains elusive. The aim of this study was to identify effective connectivity underlying freezing of gait. This study applied spectral dynamic causal modeling (DCM) of resting-state functional magnetic resonance imaging in dedicated regions of interest determined using a data-driven approach. Abnormally increased functional connectivity between the bilateral dorsolateral prefrontal cortex (DLPFC) and the bilateral mesencephalic locomotor region (MLR) was identified in freezers compared with nonfreezers. Subsequently, spectral DCM analysis revealed that increased top-down excitatory effective connectivity from the left DLPFC to bilateral MLR and an independent self-inhibitory connectivity within the left DLPFC in freezers versus nonfreezers (>99% posterior probability) were inversely associated with the severity of freezing of gait. The lateralization of these effective connectivity patterns was not attributable to the initial dopaminergic deficit nor to structural changes in these regions. We have identified novel effective connectivity and an independent self-inhibitory connectivity underlying freezing of gait. Our findings imply that modulating the effective connectivity between the left DLPFC and MLR through neurostimulation or other interventions could be a target for reducing freezing of gait in PD. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Hypothalamic-hindbrain circuit for consumption-induced fear regulation

To ensure survival, animals must sometimes suppress fear responses triggered by potential threats during feeding. However, the mechanisms underlying this process remain poorly understood. In the current study, we demonstrated that when fear-conditioned stimuli (CS) were presented during food consumption, a neural projection from lateral hypothalamic (LH) GAD2 neurons to nucleus incertus (NI) relaxin-3 (RLN3)-expressing neurons was activated, leading to a reduction in CS-induced freezing behavior in male mice. LHGAD2 neurons established excitatory connections with the NI. The activity of this neural circuit, including NIRLN3 neurons, attenuated CS-induced freezing responses during food consumption. Additionally, the lateral mammillary nucleus (LM), which received NIRLN3 projections, along with RLN3 signaling in the LM, mediated the decrease in freezing behavior. Collectively, this study identified an LHGAD2-NIRLN3-LM circuit involved in modulating fear responses during feeding, thereby enhancing our understanding of how animals coordinate nutrient intake with threat avoidance.

Activity in the dorsal hippocampus-mPFC circuit modulates stress-coping strategies during inescapable stress

Anatomical connectivity and lesion-deficit studies have shown that the dorsal and ventral hippocampi contribute to cognitive and emotional processes, respectively. However, the role of the dorsal hippocampus (dHP) in emotional or stress-related behaviors remains unclear. Here, we showed that neuronal activity in the dHP affects stress-coping behaviors in mice via excitatory projections to the medial prefrontal cortex (mPFC). The antidepressant ketamine rapidly induced c-Fos expression in both the dorsal and ventral hippocampi. The suppression of GABAergic transmission in the dHP-induced molecular changes similar to those induced by ketamine administration, including eukaryotic elongation factor 2 (eEF2) dephosphorylation, brain-derived neurotrophic factor (BDNF) elevation, and extracellular signal-regulated kinase (ERK) phosphorylation. These synaptic and molecular changes in the dHP induced a reduction in the immobility time of the mice in the tail-suspension and forced swim tests without affecting anxiety-related behavior. Conversely, pharmacological and chemogenetic potentiation of inhibitory neurotransmission in the dHP CA1 region induced passive coping behaviors during the tests. Transneuronal tracing and electrophysiology revealed monosynaptic excitatory connections between dHP CA1 neurons and mPFC neurons. Optogenetic stimulation of dHP CA1 neurons in freely behaving mice produced c-Fos induction and spike firing in the mPFC neurons. Chemogenetic activation of the dHP-recipient mPFC neurons reversed the passive coping behaviors induced by suppression of dHP CA1 neuronal activity. Collectively, these results indicate that neuronal activity in the dHP modulates stress-coping strategies to inescapable stress and contributes to the antidepressant effects of ketamine via the dHP-mPFC circuit.

Inhibitory neurons marked by a connectivity molecule regulate memory precision.

The CA3 region is central to hippocampal function during learning and memory and has a unique connectivity. CA3 pyramidal neurons are the targets of huge, excitatory mossy fiber synapses from DG axons and have a high degree of excitatory recurrent connectivity. Thus, inhibition likely plays an outsized importance in constraining excitation and shaping CA3 ensembles during learning and memory. Here, we investigate the function of a never-before studied set of dendrite-targeting, GABAergic neurons defined by expression of the synaptic adhesion molecule, Kirrel3. We discovered that activating Kirrel3-expressing GABAergic neurons specifically impairs memory discrimination and inhibits CA3 pyramidal neurons in novel contexts. Kirrel3 is required for DG-to-GABA synapse formation and variants in Kirrel3 are strong risk factors for neurodevelopmental disorders. Thus, our work suggests that Kirrel3-GABA neurons are a critical source of feed-forward inhibition from DG to CA3 during the encoding and retrieval of contextual memories, a function which may be specifically disrupted in some brain disorders.

The neocortical infrastructure for language involves region-specific patterns of laminar gene expression

The language network of the human brain has core components in the inferior frontal cortex and superior/middle temporal cortex, with left-hemisphere dominance in most people. Functional specialization and interconnectivity of these neocortical regions is likely to be reflected in their molecular and cellular profiles. Excitatory connections between cortical regions arise and innervate according to layer-specific patterns. Here, we generated a gene expression dataset from human postmortem cortical tissue samples from core language network regions, using spatial transcriptomics to discriminate gene expression across cortical layers. Integration of these data with existing single-cell expression data identified 56 genes that showed differences in laminar expression profiles between the frontal and temporal language cortex together with upregulation in layer II/III and/or layer V/VI excitatory neurons. Based on data from large-scale genome-wide screening in the population, DNA variants within these 56 genes showed set-level associations with interindividual variation in structural connectivity between the left-hemisphere frontal and temporal language cortex, and with the brain-related disorders dyslexia and schizophrenia which often involve affected language. These findings identify region-specific patterns of laminar gene expression as a feature of the brain's language network.

Abnormal dynamics of functional connectivity density and effective connectivity in overactive bladder.

The study aims to analyze alterations in dynamic functional connectivity density (dFCD) and effective connectivity (dEC) patterns using functional magnetic resonance imaging (fMRI), hypothesizing that overactive bladder (OAB) patients will exhibit distinct dFCD and dEC patterns, reflecting altered neural communication underlying the OAB. Forty-three female OAB patients and 40 female healthy controls (HC) underwent resting-state fMRI. Sliding window correlation was used to calculate the variability of the dFCD. The changes in dFCD-based dEC between the two groups were examined using Granger causal analysis. To describe the time-varying Granger causality, a sliding-window approach was utilized to divide time courses into a group of windows. We adopted a k-means clustering method to cluster all matrices into discrete connectivity states. Compared with HC, OAB females consistently had a dFCD (decreased) in the left anterior cingulate cortex (ACC) and left medial prefrontal cortex (mPFC) (p < 0.05, GRF corrected). In state 1, OAB patients had excitatory effective connections from bilateral ACC to left mPFC in comparison to HC. In state 2, there was an increase in dEC from the SMA to the mPFC. Participants with OAB showed significantly more inhibitory dorsolateral prefrontal cortex (dlPFC) connections between the left ACC and the right ACC in state 4, as well as an excitatory dEC connection between the right dlPFC and the left ACC in state 2 (p < 0.05, GRF corrected). OAB patients demonstrate significant alterations in dFCD and dEC patterns, which may be indicative of the neural mechanisms involved in OAB pathophysiology.

A hypothalamic-amygdala circuit underlying sexually dimorphic aggression

Male animals often display higher levels of aggression than females. However, the neural circuitry mechanisms underlying this sexually dimorphic aggression remain elusive. Here, we identify a hypothalamic-amygdala circuit that mediates male-biased aggression in mice. Specifically, the ventrolateral part of the ventromedial hypothalamus (VMHvl), a sexually dimorphic region associated with eliciting male-biased aggression, projects densely to the posterior substantia innominata (pSI), an area that promotes similar levels of attack in both sexes of mice. Although the VMHvl innervates the pSI unidirectionally through both excitatory and inhibitory connections, it is the excitatory VMHvl-pSI projections that are strengthened in males to promote aggression, whereas the inhibitory connections that reduce aggressive behavior are strengthened in females. Consequently, the convergent hypothalamic input onto the pSI leads to heightened pSI activity in males, resulting in male-biased aggression. Our findings reveal a sexually distinct excitation-inhibition balance of a hypothalamic-amygdala circuit that underlies sexually dimorphic aggression.

Excitatory Projections of Wide Field Collicular Neurons to the Nucleus of the Optic Tract in the Rat.

The superficial layers of the mammalian superior colliculus (SC) contain neurons that are generally responsive to visual stimuli but can differ considerably in morphology and response properties. To elucidate the structure and function of these neurons, we combined extracellular recording and juxtacellular labeling, detailed anatomical reconstruction, and ultrastructural analysis of the synaptic contacts of labeled neurons, using transmission electron microscopy. Our labeled neurons project to different brainstem nuclei. Of particular importance are neurons that fit the morphological criteria of the wide field (WF) neurons and whose dendrites are horizontally oriented. They display a rather characteristic axonal projection pattern to the nucleus of optic tract (NOT); thus, we call them superior collicular WF projecting to the NOT (SCWFNOT) neurons. We corroborated the morphological characterization of this neuronal type as a distinct neuronal class with the help of unsupervised hierarchical cluster analysis. Our ultrastructural data demonstrate that SCWFNOT neurons establish excitatory connections with their targets in the NOT. Although, in rodents, the literature about the WF neurons has focused on their extensive projection to the lateral posterior nucleus of the thalamus, as a conduit for information to reach the visual association areas of the cortex, our data suggest that this subclass of WF neurons may participate in the optokinetic nystagmus.

The stabilized supralinear network accounts for the contrast dependence of visual cortical gamma oscillations.

When stimulated, neural populations in the visual cortex exhibit fast rhythmic activity with frequencies in the gamma band (30-80 Hz). The gamma rhythm manifests as a broad resonance peak in the power-spectrum of recorded local field potentials, which exhibits various stimulus dependencies. In particular, in macaque primary visual cortex (V1), the gamma peak frequency increases with increasing stimulus contrast. Moreover, this contrast dependence is local: when contrast varies smoothly over visual space, the gamma peak frequency in each cortical column is controlled by the local contrast in that column's receptive field. No parsimonious mechanistic explanation for these contrast dependencies of V1 gamma oscillations has been proposed. The stabilized supralinear network (SSN) is a mechanistic model of cortical circuits that has accounted for a range of visual cortical response nonlinearities and contextual modulations, as well as their contrast dependence. Here, we begin by showing that a reduced SSN model without retinotopy robustly captures the contrast dependence of gamma peak frequency, and provides a mechanistic explanation for this effect based on the observed non-saturating and supralinear input-output function of V1 neurons. Given this result, the local dependence on contrast can trivially be captured in a retinotopic SSN which however lacks horizontal synaptic connections between its cortical columns. However, long-range horizontal connections in V1 are in fact strong, and underlie contextual modulation effects such as surround suppression. We thus explored whether a retinotopically organized SSN model of V1 with strong excitatory horizontal connections can exhibit both surround suppression and the local contrast dependence of gamma peak frequency. We found that retinotopic SSNs can account for both effects, but only when the horizontal excitatory projections are composed of two components with different patterns of spatial fall-off with distance: a short-range component that only targets the source column, combined with a long-range component that targets columns neighboring the source column. We thus make a specific qualitative prediction for the spatial structure of horizontal connections in macaque V1, consistent with the columnar structure of cortex.

Hypertension increases sympathetic neuron activity by enhancing intraganglionic cholinergic collateral connections.

Autonomic dysregulation, including sympathetic hyperactivity, is a common feature of hypertension (HT) and other cardiovascular diseases. The CNS plays a role in driving chronic sympathetic activation in disease, but several lines of evidence suggest that neuroplasticity in the periphery may also contribute. The potential contribution of postganglionic sympathetic neurons to sustained sympathetic hyperactivity is not well understood. We recently discovered that noradrenergic sympathetic neurons in the stellate ganglion (SG) have excitatory cholinergic collateral connections to other neurons within the ganglion. We hypothesize that remodelling of these neurons and increased cholinergic collateral transmission contributes to sustained sympathetic hyperactivity in cardiovascular diseases, including HT. To test that hypothesis, we examined the activity of sympathetic neurons in isolated SG under control conditions and after 1week of HT induced by peripheral angiotensin II infusion, using whole-cell patch clamp recordings. Despite the absence of central inputs, we observed elevated spontaneous activity and synaptic transmission in sympathetic SG neurons from hypertensive mice that required generation of action potentials. Genetically disrupting cholinergic transmission in noradrenergic neurons decreased basal neuronal activity and prevented angiotensin II-mediated enhancement of activity. Similar changes in activity, driven by increased collateral transmission, were identified in cardiac projecting neurons and neurons projecting to brown adipose tissue. These changes were not driven by altered A-type K+ currents. This suggests that HT stimulates increased activity throughout the intraganglionic network of collateral connections, contributing to the sustained sympathetic hyperactivity characteristic in cardiovascular disease. KEY POINTS: Sympathetic neurons in ganglia isolated from angiotensin II-treated hypertensive mice are more active than neurons from control mice despite the absence of central activation. The enhanced activity is the result of a ganglionic network of cholinergic collaterals, rather than altered intrinsic excitability. Increased neuronal activity was observed in both cardiac neurons and brown adipose tissue-projecting neurons, which are not involved in cardiovascular homeostasis.

The dance of neurons: Exploring nonlinear dynamics in brain networks

The brain is a complex, nonlinear system, exhibiting ever-evolving patterns of activities, whether in the presence or absence of external stimuli or task demands. Nonlinearity can notably obscure the link between structural constraints enforced on the interaction and its dynamical consequences. Suitable nonlinear dynamical models and their analysis serve as essential tools not only for bridging structural and functional understanding of the brain but also for predictably altering the complex dynamical organization of the brain. Here, starting from a large-scale network of threshold Hodgkin–Huxley style neurons, we formulate the average nonlinear dynamics implicitly following from the Wilson–Cowan assumptions. We investigate the influence of biophysical and structural properties on the complexity of neural dynamics at the microscale level and its relationship with the macroscopic Wilson–Cowan model. Incorporating the elements in the model can help identify more realistic regimes of activity and connect the mathematical prediction of increasing nonlinearity to physical manipulations. Our simulations of the temporal profiles reveal dependency on the binary state of interacting subpopulations and the random property of structural network at the transition points, when different synaptic weights are considered. For substantial configurations of stimulus intensity, our model provides further estimates of the neural population’s dynamics, ranging from simple-periodic to aperiodic patterns and phase transition regimes. This reflects the potential contribution of the microscopic nonlinear scheme to the mean-field approximation in studying the collective behaviour of individual neurons with particularly concentrating on the occurrence of critical phenomena. We show that finite-size effects kick the system in a state of irregular modes to evolve differently from predictions of the original Wilson–Cowan reference. Additionally, we report that the complexity and temporal diversity of neural dynamics, especially in terms of limit cycle trajectory, and synchronization can be induced by either small heterogeneity in the degree of various types of local excitatory connectivity or considerable diversity in the external drive to the excitatory pool.

Descending networks transform command signals into population motor control

To convert intentions into actions, movement instructions must pass from the brain to downstream motor circuits through descending neurons (DNs). These include small sets of command-like neurons that are sufficient to drive behaviours1—the circuit mechanisms for which remain unclear. Here we show that command-like DNs in Drosophila directly recruit networks of additional DNs to orchestrate behaviours that require the active control of numerous body parts. Specifically, we found that command-like DNs previously thought to drive behaviours alone2–4 in fact co-activate larger populations of DNs. Connectome analyses and experimental manipulations revealed that this functional recruitment can be explained by direct excitatory connections between command-like DNs and networks of interconnected DNs in the brain. Descending population recruitment is necessary for behavioural control: DNs with many downstream descending partners require network co-activation to drive complete behaviours and drive only simple stereotyped movements in their absence. These DN networks reside within behaviour-specific clusters that inhibit one another. These results support a mechanism for command-like descending control in which behaviours are generated through the recruitment of increasingly large DN networks that compose behaviours by combining multiple motor subroutines.

All-optical voltage imaging-guided postsynaptic single-cell transcriptome profiling with Voltage-Seq.

Neuronal pathways recruit large postsynaptic populations and maintain connections via distinct postsynaptic response types (PRTs). Until recently, PRTs were accessible as a selection criterion for single-cell RNA sequencing only through probing by low-throughput whole-cell electrophysiology. To overcome these limitations and target neurons on the basis of specific PRTs for soma collection and subsequent single-cell RNA sequencing, we developed Voltage-Seq using the genetically encoded voltage indicator Voltron in acute brain slices from mice. We also created an onsite analysis tool, VoltView, to guide soma collection of specific PRTs using a classifier based on a previously acquired database of connectomes from multiple animals. Here we present our procedure for preparing the optical path, the imaging setup and detailing the imaging and analysis steps, as well as a complete procedure for sequencing library preparation. This enables researchers to conduct our high-throughput all-optical synaptic assay and to obtain single-cell transcriptomic data from selected postsynaptic neurons. This also allows researchers to resolve the connectivity ratio of a specific pathway and explore the diversity of PRTs within that connectome. Furthermore, combining high throughput with quick analysis gives unique access to find specific connections within a large postsynaptic connectome. Voltage-Seq also allows the investigation of correlations between connectivity and gene expression changes in a postsynaptic cell-type-specific manner for both excitatory and inhibitory connections. The Voltage-Seq workflow can be completed in ~6 weeks, including 4-5 weeks for viral expression of the Voltron sensor. The technique requires knowledge of basic laboratory techniques, micromanipulator handling skills and experience in molecular biology and bioinformatics.

MiR-7 controls glutamatergic transmission and neuronal connectivity in a Cdr1as-dependent manner

The circular RNA (circRNA) Cdr1as is conserved across mammals and highly expressed in neurons, where it directly interacts with microRNA miR-7. However, the biological function of this interaction is unknown. Here, using primary cortical murine neurons, we demonstrate that stimulating neurons by sustained depolarization rapidly induces two-fold transcriptional upregulation of Cdr1as and strong post-transcriptional stabilization of miR-7. Cdr1as loss causes doubling of glutamate release from stimulated synapses and increased frequency and duration of local neuronal bursts. Moreover, the periodicity of neuronal networks increases, and synchronicity is impaired. Strikingly, these effects are reverted by sustained expression of miR-7, which also clears Cdr1as molecules from neuronal projections. Consistently, without Cdr1as, transcriptomic changes caused by miR-7 overexpression are stronger (including miR-7-targets downregulation) and enriched in secretion/synaptic plasticity pathways. Altogether, our results suggest that in cortical neurons Cdr1as buffers miR-7 activity to control glutamatergic excitatory transmission and neuronal connectivity important for long-lasting synaptic adaptations.