Autism-associated neuroligin-3 mutations commonly impair striatal circuits to boost repetitive behaviors.

In humans, neuroligin-3 mutations are associated with autism, whereas in mice, the corresponding mutations produce robust synaptic and behavioral changes. However, different neuroligin-3 mutations cause largely distinct phenotypes in mice, and no causal relationship links a specific synaptic dysfunction to a behavioral change. Using rotarod motor learning as a proxy for acquired repetitive behaviors in mice, we found that different neuroligin-3 mutations uniformly enhanced formation of repetitive motor routines. Surprisingly, neuroligin-3 mutations caused this phenotype not via changes in the cerebellum or dorsal striatum but via a selective synaptic impairment in the nucleus accumbens/ventral striatum. Here, neuroligin-3 mutations increased rotarod learning by specifically impeding synaptic inhibition onto D1-dopamine receptor-expressing but not D2-dopamine receptor-expressing medium spiny neurons. Our data thus suggest that different autism-associated neuroligin-3 mutations cause a common increase in acquired repetitive behaviors by impairing a specific striatal synapse and thereby provide a plausible circuit substrate for autism pathophysiology.

Allelic contribution of Nrxn1α to autism-relevant behavioral phenotypes in mice.

Copy number variations (CNVs) in the Neurexin 1 (NRXN1) gene, which encodes a presynaptic protein involved in neurotransmitter release, are some of the most frequently observed single-gene variants associated with autism spectrum disorder (ASD). To address the functional contribution of NRXN1 CNVs to behavioral phenotypes relevant to ASD, we carried out systematic behavioral phenotyping of an allelic series of Nrxn1 mouse models: one carrying promoter and exon 1 deletion abolishing Nrxn1α transcription, one carrying exon 9 deletion disrupting Nrxn1α protein translation, and one carrying an intronic deletion with no observable effect on Nrxn1α expression. We found that homozygous loss of Nrxn1α resulted in enhanced aggression in males, reduced affiliative social behaviors in females, and significantly altered circadian activities in both sexes. Heterozygous or homozygous loss of Nrxn1α affected the preference for social novelty in male mice, and notably, enhanced repetitive motor skills and motor coordination in both sexes. In contrast, mice bearing an intronic deletion of Nrxn1 did not display alterations in any of the behaviors assessed. These findings demonstrate the importance of Nrxn1α gene dosage in regulating social, circadian, and motor functions, and the variables of sex and genomic positioning of CNVs in the expression of autism-related phenotypes. Importantly, mice with heterozygous loss of Nrxn1, as found in numerous autistic individuals, show an elevated propensity to manifest autism-related phenotypes, supporting the use of models with this genomic architecture to study ASD etiology and assess additional genetic variants associated with autism.

Glutamatergic Neurons in the Piriform Cortex Influence the Activity of D1- and D2-Type Receptor-Expressing Olfactory Tubercle Neurons.

Sensory cortices process stimuli in manners essential for perception. Very little is known regarding interactions between olfactory cortices. The piriform "primary" olfactory cortex, especially its anterior division (aPCX), extends dense association fibers into the ventral striatum's olfactory tubercle (OT), yet whether this corticostriatal pathway is capable of shaping OT activity, including odor-evoked activity, is unknown. Further unresolved is the synaptic circuitry and the spatial localization of OT-innervating PCX neurons. Here we build upon standing literature to provide some answers to these questions through studies in mice of both sexes. First, we recorded the activity of OT neurons in awake mice while optically stimulating principal neurons in the aPCX and/or their association fibers in the OT while the mice were delivered odors. This uncovered evidence that PCX input indeed influences OT unit activity. We then used patch-clamp recordings and viral tracing to determine the connectivity of aPCX neurons upon OT neurons expressing dopamine receptor types D1 or D2, two prominent cell populations in the OT. These investigations uncovered that both populations of neurons receive monosynaptic inputs from aPCX glutamatergic neurons. Interestingly, this input originates largely from the ventrocaudal aPCX. These results shed light on some of the basic physiological properties of this pathway and the cell-types involved and provide a foundation for future studies to identify, among other things, whether this pathway has implications for perception.SIGNIFICANCE STATEMENT Sensory cortices interact to process stimuli in manners considered essential for perception. Very little is known regarding interactions between olfactory cortices. The present study sheds light on some of the basic physiological properties of a particular intercortical pathway in the olfactory system and provides a foundation for future studies to identify, among other things, whether this pathway has implications for perception.

Integrated anatomical and physiological mapping of striatal afferent projections.

The dorsomedial striatum, a key site of reward-sensitive motor output, receives extensive afferent input from cortex, thalamus and midbrain. These projections are integrated by striatal microcircuits containing both spiny projection neurons and local circuit interneurons. To explore target cell specificity of these projections, we compared inputs onto D1-dopamine receptor-positive spiny neurons, parvalbumin-positive fast-spiking interneurons and somatostatin-positive low-threshold-spiking interneurons, using cell type-specific rabies virus tracing and optogenetic-mediated projection neuron recruitment in mice. While the relative proportion of retrogradely labelled projection neurons was similar between target cell types, the convergence of inputs was systematically higher for projections onto fast-spiking interneurons. Rabies virus is frequently used to assess cell-specific anatomical connectivity but it is unclear how this correlates to synaptic connectivity and efficacy. To test this, we compared tracing data with target cell-specific measures of synaptic efficacy for anterior cingulate cortex and parafascicular thalamic projections using novel quantitative optogenetic measures. We found that target-specific patterns of convergence were extensively modified according to region of projection neuron origin and postsynaptic cell type. Furthermore, we observed significant divergence between cell type-specific anatomical connectivity and measures of excitatory synaptic strength, particularly for low-threshold-spiking interneurons. Taken together, this suggests a basic uniform connectivity map for striatal afferent inputs upon which presynaptic-postsynaptic interactions impose substantial diversity of physiological connectivity.

Loss of the neurodevelopmental gene Zswim6 alters striatal morphology and motor regulation.

The zinc-finger SWIM domain-containing protein 6 (ZSWIM6) is a protein of unknown function that has been associated with schizophrenia and limited educational attainment by three independent genome-wide association studies. Additionally, a putatively causal point mutation in ZSWIM6 has been identified in several cases of acromelic frontonasal dysostosis with severe intellectual disability. Despite the growing number of studies implicating ZSWIM6 as an important regulator of brain development, its role in this process has never been examined. Here, we report the generation of Zswim6 knockout mice and provide a detailed anatomical and behavioral characterization of the resulting phenotype. We show that Zswim6 is initially expressed widely during embryonic brain development but becomes restricted to the striatum postnatally. Loss of Zswim6 causes a reduction in striatal volume and changes in medium spiny neuron morphology. These changes are associated with alterations in motor control, including hyperactivity, impaired rotarod performance, repetitive movements, and behavioral hyperresponsiveness to amphetamine. Together, our results show that Zswim6 is indispensable to normal brain function and support the notion that Zswim6 might serve as an important contributor to the pathogenesis of schizophrenia and other neurodevelopmental disorders.

The neurexin ligands, neuroligins and leucine-rich repeat transmembrane proteins, perform convergent and divergent synaptic functions in vivo.

Synaptic cell adhesion molecules, including the neurexin ligands, neuroligins (NLs) and leucine-rich repeat transmembrane proteins (LRRTMs), are thought to organize synapse assembly and specify synapse function. To test the synaptic role of these molecules in vivo, we performed lentivirally mediated knockdown of NL3, LRRTM1, and LRRTM2 in CA1 pyramidal cells of WT and NL1 KO mice at postnatal day (P)0 (when synapses are forming) and P21 (when synapses are largely mature). P0 knockdown of NL3 in WT or NL1 KO neurons did not affect excitatory synaptic transmission, whereas P0 knockdown of LRRTM1 and LRRTM2 selectively reduced AMPA receptor-mediated synaptic currents. P0 triple knockdown of NL3 and both LRRTMs in NL1 KO mice yielded greater reductions in AMPA and NMDA receptor-mediated currents, suggesting functional redundancy between NLs and LRRTMs during early synapse development. In contrast, P21 knockdown of LRRTMs did not alter excitatory transmission, whereas NL manipulations supported a role for NL1 in maintaining NMDA receptor-mediated transmission. These results show that neurexin ligands in vivo form a dynamic synaptic cell adhesion network, with compensation between NLs and LRRTMs during early synapse development and functional divergence upon synapse maturation.

Local regulation of striatal dopamine: A diversity of circuit mechanisms for a diversity of behavioral functions?

Striatal dopamine governs a wide range of behavioral functions, yet local dopamine concentrations can be dissociated from somatic activity. Here, we discuss how dopamine's diverse roles in behavior may be driven by local circuit mechanisms shaping dopamine release. We first look at historical and recent work demonstrating that striatal circuits interact with dopaminergic terminals to either initiate the release of dopamine or modulate the release of dopamine initiated by spiking in midbrain dopamine neurons, with particular attention to GABAergic and cholinergic local circuit mechanisms. Then we discuss some of the first in vivo studies of acetylcholine-dopamine interactions in striatum and broadly discuss necessary future work in understanding the roles of midbrain versus striatal dopamine regulation.

Infralimbic activity during REM sleep facilitates fear extinction memory.

Rapid eye movement (REM) sleep is known to facilitate fear extinction and play a protective role against fearful memories.1,2 Consequently, disruption of REM sleep after a traumatic event may increase the risk for developing PTSD.3,4 However, the underlying mechanisms by which REM sleep promotes extinction of aversive memories remain largely unknown. The infralimbic cortex (IL) is a key brain structure for the consolidation of extinction memory.5 Using calcium imaging, we found in mice that most IL pyramidal neurons are intensively activated during REM sleep. Optogenetically suppressing the IL specifically during REM sleep within a 4-h window after auditory-cued fear conditioning impaired extinction memory consolidation. In contrast, REM-specific IL inhibition after extinction learning did not affect the extinction memory. Whole-cell patch-clamp recordings demonstrated that inactivating IL neurons during REM sleep depresses their excitability. Together, our findings suggest that REM sleep after fear conditioning facilitates fear extinction by enhancing IL excitability and highlight the importance of REM sleep in the aftermath of traumatic events for protecting against traumatic memories.

A chromosome region linked to neurodevelopmental disorders acts in distinct neuronal circuits in males and females to control locomotor behavior.

Biological sex shapes the manifestation and progression of neurodevelopmental disorders (NDDs). These disorders often demonstrate male-specific vulnerabilities; however, the identification of underlying mechanisms remains a significant challenge in the field. Hemideletion of the 16p11.2 region (16p11.2 del/+) is associated with NDDs, and mice modeling 16p11.2 del/+ exhibit sex-specific striatum-related phenotypes relevant to NDDs. Striatal circuits, crucial for locomotor control, consist of two distinct pathways: the direct and indirect pathways originating from D1 dopamine receptor (D1R) and D2 dopamine receptor (D2R) expressing spiny projection neurons (SPNs), respectively. In this study, we define the impact of 16p11.2 del/+ on striatal circuits in male and female mice. Using snRNA-seq, we identify sex- and cell type-specific transcriptomic changes in the D1- and D2-SPNs of 16p11.2 del/+ mice, indicating distinct transcriptomic signatures in D1-SPNs and D2-SPNs in males and females, with a ∼5-fold greater impact in males. Further pathway analysis reveals differential gene expression changes in 16p11.2 del/+ male mice linked to synaptic plasticity in D1- and D2-SPNs and GABA signaling pathway changes in D1-SPNs. Consistent with our snRNA-seq study revealing changes in GABA signaling pathways, we observe distinct changes in miniature inhibitory postsynaptic currents (mIPSCs) in D1- and D2-SPNs from 16p11.2 del/+ male mice. Behaviorally, we utilize conditional genetic approaches to introduce the hemideletion selectively in either D1- or D2-SPNs and find that conditional hemideletion of genes in the 16p11.2 region in D2-SPNs causes hyperactivity in male mice, but hemideletion in D1-SPNs does not. Within the striatum, hemideletion of genes in D2-SPNs in the dorsal lateral striatum leads to hyperactivity in males, demonstrating the importance of this striatal region. Interestingly, conditional 16p11.2 del/+ within the cortex drives hyperactivity in both sexes. Our work reveals that a locus linked to NDDs acts in different striatal circuits, selectively impacting behavior in a sex- and cell type-specific manner, providing new insight into male vulnerability for NDDs. Highlights: - 16p11.2 hemideletion (16p11.2 del/+) induces sex- and cell type-specific transcriptomic signatures in spiny projection neurons (SPNs). - Transcriptomic changes in GABA signaling in D1-SPNs align with changes in inhibitory synapse function. - 16p11.2 del/+ in D2-SPNs causes hyperactivity in males but not females. - 16p11.2 del/+ in D2-SPNs in the dorsal lateral striatum drives hyperactivity in males. - 16p11.2 del/+ in cortex drives hyperactivity in both sexes.

Histone variant H2BE enhances chromatin accessibility in neurons to promote synaptic gene expression and long-term memory.

Regulation of histone proteins affects gene expression through multiple mechanisms including exchange with histone variants. However, widely expressed variants of H2B remain elusive. Recent findings link histone variants to neurological disorders, yet few are well studied in the brain. We applied new tools including novel antibodies, biochemical assays, and sequencing approaches to reveal broad expression of the H2B variant H2BE, and defined its role in regulating chromatin structure, neuronal transcription, and mouse behavior. We find that H2BE is enriched at promoters and a single unique amino acid allows it to dramatically enhance chromatin accessibility. Lastly, we show that H2BE is critical for synaptic gene expression and long-term memory. Together, these data reveal a novel mechanism linking histone variants to chromatin regulation, neuronal function, and memory. This work further identifies the first widely expressed H2B variant and uncovers a single histone amino acid with profound effects on genomic structure.

Distributed processing for value-based choice by prelimbic circuits targeting anterior-posterior dorsal striatal subregions in male mice.

Fronto-striatal circuits have been implicated in cognitive control of behavioral output for social and appetitive rewards. The functional diversity of prefrontal cortical populations is strongly dependent on their synaptic targets, with control of motor output mediated by connectivity to dorsal striatum. Despite evidence for functional diversity along the anterior-posterior striatal axis, it is unclear how distinct fronto-striatal sub-circuits support value-based choice. Here we found segregated prefrontal populations defined by anterior/posterior dorsomedial striatal target. During a feedback-based 2-alternative choice task, single-photon imaging revealed circuit-specific representations of task-relevant information with prelimbic neurons targeting anterior DMS (PL::A-DMS) robustly modulated during choices and negative outcomes, while prelimbic neurons targeting posterior DMS (PL::P-DMS) encoded internal representations of value and positive outcomes contingent on prior choice. Consistent with this distributed coding, optogenetic inhibition of PL::A-DMS circuits strongly impacted choice monitoring and responses to negative outcomes while inhibition of PL::P-DMS impaired task engagement and strategies following positive outcomes. Together our data uncover PL populations engaged in distributed processing for value-based choice.

Ventral striatal islands of Calleja neurons bidirectionally mediate depression-like behaviors in mice.

The ventral striatum is a reward center implicated in the pathophysiology of depression. It contains islands of Calleja, clusters of dopamine D3 receptor-expressing granule cells, predominantly in the olfactory tubercle (OT). These OT D3 neurons regulate self-grooming, a repetitive behavior manifested in affective disorders. Here we show that chronic restraint stress (CRS) induces robust depression-like behaviors in mice and decreases excitability of OT D3 neurons. Ablation or inhibition of these neurons leads to depression-like behaviors, whereas their activation ameliorates CRS-induced depression-like behaviors. Moreover, activation of OT D3 neurons has a rewarding effect, which diminishes when grooming is blocked. Finally, we propose a model that explains how OT D3 neurons may influence dopamine release via synaptic connections with OT spiny projection neurons (SPNs) that project to midbrain dopamine neurons. Our study reveals a crucial role of OT D3 neurons in bidirectionally mediating depression-like behaviors, suggesting a potential therapeutic target.

A blueprint for examining striatal control of cognition.

In a recent study, Bolkan, Stone, and colleagues demonstrated that direct and indirect striatal pathways in mice exert opponent control over choice behavior in a task- and state-dependent manner. This work highlights the need for rigorously controlled behavioral experiments and novel behavioral modeling in investigations of the neural mechanisms of decision making.

Developmental trajectories of thalamic progenitors revealed by single-cell transcriptome profiling and Shh perturbation.

The thalamus is the principal information hub of the vertebrate brain, with essential roles in sensory and motor information processing, attention, and memory. The complex array of thalamic nuclei develops from a restricted pool of neural progenitors. We apply longitudinal single-cell RNA sequencing and regional abrogation of Sonic hedgehog (Shh) to map the developmental trajectories of thalamic progenitors, intermediate progenitors, and post-mitotic neurons as they coalesce into distinct thalamic nuclei. These data reveal that the complex architecture of the thalamus is established early during embryonic brain development through the coordinated action of four cell differentiation lineages derived from Shh-dependent and -independent progenitors. We systematically characterize the gene expression programs that define these thalamic lineages across time and demonstrate how their disruption upon Shh depletion causes pronounced locomotor impairment resembling infantile Parkinson's disease. These results reveal key principles of thalamic development and provide mechanistic insights into neurodevelopmental disorders resulting from thalamic dysfunction.

Copy number variants in neurexin genes: phenotypes and mechanisms.

Neurexins are central to trans-synaptic cell adhesion and signaling during synapse specification and maintenance. The past two decades of human genetics research have identified structural variations in the neurexin gene family, in particular NRXN1 copy number variants (CNVs), implicated in multiple neuropsychiatric and developmental disorders. The heterogeneity and reduced penetrance of NRXN1 deletions, in addition to the pleiotropic, circuit-specific functions of NRXN1, present substantial obstacles to understanding how compromised NRXN1 function predisposes individuals to neuropsychiatric disorders. Here, we provide an updated review of NRXN1 genetics in disease, followed by recently published work using both human induced pluripotent stem cell (iPSC) derived systems and animal models to understand the mechanisms of disease pathophysiology. Finally, we suggest our outlook on how the field should progress to improve our understanding of neurexin mediated disease pathogenesis. We believe that understanding how structural genetic variants in NRXN1 contribute to disease pathophysiology requires parallel approaches in iPSC and mouse model systems, each leveraging their unique strengths - analysis of genetic interactions and background effects in iPSCs and neural circuit and behavioral analysis in mice.

Neurexin1⍺ differentially regulates synaptic efficacy within striatal circuits.

Mutations in genes essential for synaptic function, such as the presynaptic adhesion molecule Neurexin1α (Nrxn1α), are strongly implicated in neuropsychiatric pathophysiology. As the input nucleus of the basal ganglia, the striatum integrates diverse excitatory projections governing cognitive and motor control, and its impairment may represent a recurrent pathway to disease. Here, we test the functional relevance of Nrxn1α in striatal circuits by employing optogenetic-mediated afferent recruitment of dorsal prefrontal cortical (dPFC) and parafascicular thalamic connections onto dorsomedial striatal (DMS) spiny projection neurons (SPNs). For dPFC-DMS circuits, we find decreased synaptic strength specifically onto indirect pathway SPNs in both Nrxn1α+/- and Nrxn1α-/- mice, driven by reductions in neurotransmitter release. In contrast, thalamic excitatory inputs to DMS exhibit relatively normal excitatory synaptic strength despite changes in synaptic N-methyl-D-aspartate receptor (NMDAR) content. These findings suggest that dysregulation of Nrxn1α modulates striatal function in an input- and target-specific manner.

Striatal low-threshold spiking interneurons locally gate dopamine.

The dorsomedial striatum (DMS) is a central hub supporting goal-directed learning and motor performance. Recent evidence has revealed unexpected roles for local inhibitory GABAergic networks in modulating striatal output and behavior.1 The sparse low-threshold spiking interneuron subtype (LTSI), which exhibits robust reward-circumscribed population activity, is a bidirectional regulator of initial goal-directed learning.2 Striatal dopamine signaling is a central reward-related neuromodulatory system mediating goal-directed action and performance, serving as a teaching signal,3 facilitating synaptic plasticity,4 and invigorating motor behaviors.5 Given the dynamic modulation of LTSIs during goal-directed behavior, we hypothesized that they could provide a novel GABAergic mechanism of local striatal dopaminergic regulation to shape early learning. We provide anatomical evidence for close proximation of LTSI terminals and dopaminergic processes in striatum, suggesting that LTSIs directly control dopaminergic axon activity. Using in vitro fast scan cyclic voltammetry, we demonstrate that LTSIs directly attenuate optogenetically evoked dopamine via GABAB receptor signaling. In vivo, GRABDA dopamine sensor imaging shows that LTSIs strongly modulate striatal dopamine dynamics during operant learning, while pharmacological stabilization of dopamine via intra-striatal aripiprazole microinjection suppresses the effects of LTSI inhibition on learning. Together, these results uncover an unexpected function for LTSIs in gating striatal dopamine to facilitate goal-directed learning.

Temporal manipulation of Cdkl5 reveals essential postdevelopmental functions and reversible CDKL5 deficiency disorder-related deficits.

CDKL5 deficiency disorder (CDD) is an early onset, neurodevelopmental syndrome associated with pathogenic variants in the X-linked gene encoding cyclin-dependent kinase-like 5 (CDKL5). CDKL5 has been implicated in neuronal synapse maturation, yet its postdevelopmental necessity and the reversibility of CDD-associated impairments remain unknown. We temporally manipulated endogenous Cdkl5 expression in male mice and found that postdevelopmental loss of CDKL5 disrupts numerous behavioral domains, hippocampal circuit communication, and dendritic spine morphology, demonstrating an indispensable role for CDKL5 in the adult brain. Accordingly, restoration of Cdkl5 after the early stages of brain development using a conditional rescue mouse model ameliorated CDD-related behavioral impairments and aberrant NMDA receptor signaling. These findings highlight the requirement of CDKL5 beyond early development, underscore the potential for disease reversal in CDD, and suggest that a broad therapeutic time window exists for potential treatment of CDD-related deficits.

Ventral striatal islands of Calleja neurons control grooming in mice.

The striatum comprises multiple subdivisions and neural circuits that differentially control motor output. The islands of Calleja (IC) contain clusters of densely packed granule cells situated in the ventral striatum, predominantly in the olfactory tubercle (OT). Characterized by expression of the D3 dopamine receptor, the IC are evolutionally conserved, but have undefined functions. Here, we show that optogenetic activation of OT D3 neurons robustly initiates self-grooming in mice while suppressing other ongoing behaviors. Conversely, optogenetic inhibition of these neurons halts ongoing grooming, and genetic ablation reduces spontaneous grooming. Furthermore, OT D3 neurons show increased activity before and during grooming and influence local striatal output via synaptic connections with neighboring OT neurons (primarily spiny projection neurons), whose firing rates display grooming-related modulation. Our study uncovers a new role of the ventral striatum's IC in regulating motor output and has important implications for the neural control of grooming.

GABAergic Restriction of Network Dynamics Regulates Interneuron Survival in the Developing Cortex.

During neonatal development, sensory cortices generate spontaneous activity patterns shaped by both sensory experience and intrinsic influences. How these patterns contribute to the assembly of neuronal circuits is not clearly understood. Using longitudinal in vivo calcium imaging in un-anesthetized mouse pups, we show that spatially segregated functional assemblies composed of interneurons and pyramidal cells are prominent in the somatosensory cortex by postnatal day (P) 7. Both reduction of GABA release and synaptic inputs onto pyramidal cells erode the emergence of functional topography, leading to increased network synchrony. This aberrant pattern effectively blocks interneuron apoptosis, causing increased survival of parvalbumin and somatostatin interneurons. Furthermore, the effect of GABA on apoptosis is mediated by inputs from medial ganglionic eminence (MGE)-derived but not caudal ganglionic eminence (CGE)-derived interneurons. These findings indicate that immature MGE interneurons are fundamental for shaping GABA-driven activity patterns that balance the number of interneurons integrating into maturing cortical networks.