Astrocytes Assemble Thalamocortical Synapses by Bridging NRX1α and NL1 via Hevin.

Proper establishment of synapses is critical for constructing functional circuits. Interactions between presynaptic neurexins and postsynaptic neuroligins coordinate the formation of synaptic adhesions. An isoform code determines the direct interactions of neurexins and neuroligins across the synapse. However, whether extracellular linker proteins can expand such a code is unknown. Using a combination of in vitro and in vivo approaches, we found that hevin, an astrocyte-secreted synaptogenic protein, assembles glutamatergic synapses by bridging neurexin-1alpha and neuroligin-1B, two isoforms that do not interact with each other. Bridging of neurexin-1alpha and neuroligin-1B via hevin is critical for the formation and plasticity of thalamocortical connections in the developing visual cortex. These results show that astrocytes promote the formation of synapses by modulating neurexin/neuroligin adhesions through hevin secretion. Our findings also provide an important mechanistic insight into how mutations in these genes may lead to circuit dysfunction in diseases such as autism.

Striatal Direct Pathway Targets Npas1+ Pallidal Neurons.

The classic basal ganglia circuit model asserts a complete segregation of the two striatal output pathways. Empirical data argue that, in addition to indirect-pathway striatal projection neurons (iSPNs), direct-pathway striatal projection neurons (dSPNs) innervate the external globus pallidus (GPe). However, the functions of the latter were not known. In this study, we interrogated the organization principles of striatopallidal projections and their roles in full-body movement in mice (both males and females). In contrast to the canonical motor-promoting response of dSPNs in the dorsomedial striatum (DMSdSPNs), optogenetic stimulation of dSPNs in the dorsolateral striatum (DLSdSPNs) suppressed locomotion. Circuit analyses revealed that dSPNs selectively target Npas1+ neurons in the GPe. In a chronic 6-hydroxydopamine lesion model of Parkinson's disease, the dSPN-Npas1+ projection was dramatically strengthened. As DLSdSPN-Npas1+ projection suppresses movement, the enhancement of this projection represents a circuit mechanism for the hypokinetic symptoms of Parkinson's disease that has not been previously considered. In sum, our results suggest that dSPN input to the GPe is a critical circuit component that is involved in the regulation of movement in both healthy and parkinsonian states.SIGNIFICANCE STATEMENT In the classic basal ganglia model, the striatum is described as a divergent structure: it controls motor and adaptive functions through two segregated, opposing output streams. However, the experimental results that show the projection from direct-pathway neurons to the external pallidum have been largely ignored. Here, we showed that this striatopallidal subpathway targets a select subset of neurons in the external pallidum and is motor-suppressing. We found that this subpathway undergoes changes in a Parkinson's disease model. In particular, our results suggest that the increase in strength of this subpathway contributes to the slowness or reduced movements observed in Parkinson's disease.

Dissociable Roles of Pallidal Neuron Subtypes in Regulating Motor Patterns.

We have previously established that PV+ neurons and Npas1+ neurons are distinct neuron classes in the external globus pallidus (GPe): they have different topographical, electrophysiological, circuit, and functional properties. Aside from Foxp2+ neurons, which are a unique subclass within the Npas1+ class, we lack driver lines that effectively capture other GPe neuron subclasses. In this study, we examined the utility of Kcng4-Cre, Npr3-Cre, and Npy2r-Cre mouse lines (both males and females) for the delineation of GPe neuron subtypes. By using these novel driver lines, we have provided the most exhaustive investigation of electrophysiological studies of GPe neuron subtypes to date. Corroborating our prior studies, GPe neurons can be divided into two statistically distinct clusters that map onto PV+ and Npas1+ classes. By combining optogenetics and machine learning-based tracking, we showed that optogenetic perturbation of GPe neuron subtypes generated unique behavioral structures. Our findings further highlighted the dissociable roles of GPe neurons in regulating movement and anxiety-like behavior. We concluded that Npr3+ neurons and Kcng4+ neurons are distinct subclasses of Npas1+ neurons and PV+ neurons, respectively. Finally, by examining local collateral connectivity, we inferred the circuit mechanisms involved in the motor patterns observed with optogenetic perturbations. In summary, by identifying mouse lines that allow for manipulations of GPe neuron subtypes, we created new opportunities for interrogations of cellular and circuit substrates that can be important for motor function and dysfunction.SIGNIFICANCE STATEMENT Within the basal ganglia, the external globus pallidus (GPe) has long been recognized for its involvement in motor control. However, we lacked an understanding of precisely how movement is controlled at the GPe level as a result of its cellular complexity. In this study, by using transgenic and cell-specific approaches, we showed that genetically-defined GPe neuron subtypes have distinct roles in regulating motor patterns. In addition, the in vivo contributions of these neuron subtypes are in part shaped by the local, inhibitory connections within the GPe. In sum, we have established the foundation for future investigations of motor function and disease pathophysiology.

Parvalbumin+ and Npas1+ Pallidal Neurons Have Distinct Circuit Topology and Function.

The external globus pallidus (GPe) is a critical node within the basal ganglia circuit. Phasic changes in the activity of GPe neurons during movement and their alterations in Parkinson's disease (PD) argue that the GPe is important in motor control. Parvalbumin-positive (PV+) neurons and Npas1+ neurons are the two principal neuron classes in the GPe. The distinct electrophysiological properties and axonal projection patterns argue that these two neuron classes serve different roles in regulating motor output. However, the causal relationship between GPe neuron classes and movement remains to be established. Here, by using optogenetic approaches in mice (both males and females), we showed that PV+ neurons and Npas1+ neurons promoted and suppressed locomotion, respectively. Moreover, PV+ neurons and Npas1+ neurons are under different synaptic influences from the subthalamic nucleus (STN). Additionally, we found a selective weakening of STN inputs to PV+ neurons in the chronic 6-hydroxydopamine lesion model of PD. This finding reinforces the idea that the reciprocally connected GPe-STN network plays a key role in disease symptomatology and thus provides the basis for future circuit-based therapies.SIGNIFICANCE STATEMENT The external pallidum is a key, yet an understudied component of the basal ganglia. Neural activity in the pallidum goes awry in neurologic diseases, such as Parkinson's disease. While this strongly argues that the pallidum plays a critical role in motor control, it has been difficult to establish the causal relationship between pallidal activity and motor function/dysfunction. This was in part because of the cellular complexity of the pallidum. Here, we showed that the two principal neuron types in the pallidum have opposing roles in motor control. In addition, we described the differences in their synaptic influence. Importantly, our research provides new insights into the cellular and circuit mechanisms that explain the hypokinetic features of Parkinson's disease.

Npas1+-Nkx2.1+ Neurons Are an Integral Part of the Cortico-pallido-cortical Loop.

Within the basal ganglia circuit, the external globus pallidus (GPe) is critically involved in motor control. Aside from Foxp2+ neurons and ChAT+ neurons that have been established as unique neuron types, there is little consensus on the classification of GPe neurons. Properties of the remaining neuron types are poorly defined. In this study, we leverage new mouse lines, viral tools, and molecular markers to better define GPe neuron subtypes. We found that Sox6 represents a novel, defining marker for GPe neuron subtypes. Lhx6+ neurons that lack the expression of Sox6 were devoid of both parvalbumin and Npas1. This result confirms previous assertions of the existence of a unique Lhx6+ population. Neurons that arise from the Dbx1+ lineage were similarly abundant in the GPe and displayed a heterogeneous makeup. Importantly, tracing experiments revealed that Npas1+-Nkx2.1+ neurons represent the principal noncholinergic, cortically-projecting neurons. In other words, they form the pallido-cortical arm of the cortico-pallido-cortical loop. Our data further show that pyramidal-tract neurons in the cortex collateralized within the GPe, forming a closed-loop system between the two brain structures. Overall, our findings reconcile some of the discrepancies that arose from differences in techniques or the reliance on preexisting tools. Although spatial distribution and electrophysiological properties of GPe neurons reaffirm the diversification of GPe subtypes, statistical analyses strongly support the notion that these neuron subtypes can be categorized under the two principal neuron classes: PV+ neurons and Npas1+ neurons.SIGNIFICANCE STATEMENT The poor understanding of the neuronal composition in the external globus pallidus (GPe) undermines our ability to interrogate its precise behavioral and disease involvements. In this study, 12 different genetic crosses were used, hundreds of neurons were electrophysiologically characterized, and >100,000 neurons were histologically- and/or anatomically-profiled. Our current study further establishes the segregation of GPe neuron classes and illustrates the complexity of GPe neurons in adult mice. Our results support the idea that Npas1+-Nkx2.1+ neurons are a distinct GPe neuron subclass. By providing a detailed analysis of the organization of the cortico-pallidal-cortical projection, our findings establish the cellular and circuit substrates that can be important for motor function and dysfunction.

Cell and circuit complexity of the external globus pallidus.

The external globus pallidus (GPe) of the basal ganglia has been underappreciated owing to poor understanding of its cells and circuits. It was assumed that the GPe consisted of a homogeneous neuron population primarily serving as a 'relay station' for information flowing through the indirect basal ganglia pathway. However, the advent of advanced tools in rodent models has sparked a resurgence in interest in the GPe. Here, we review recent data that have unveiled the cell and circuit complexity of the GPe. These discoveries have revealed that the GPe does not conform to traditional views of the basal ganglia. In particular, recent evidence confirms that the afferent and efferent connections of the GPe span both the direct and the indirect pathways. Furthermore, the GPe displays broad interconnectivity beyond the basal ganglia, consistent with its emerging multifaceted roles in both motor and non-motor functions. In summary, recent data prompt new proposals for computational rules of the basal ganglia.

Antipsychotic drug efficacy correlates with the modulation of D1 rather than D2 receptor-expressing striatal projection neurons.

Elevated dopamine transmission in psychosis is assumed to unbalance striatal output through D1- and D2-receptor-expressing spiny-projection neurons (SPNs). Antipsychotic drugs are thought to re-balance this output by blocking D2 receptors (D2Rs). In this study, we found that amphetamine-driven dopamine release unbalanced D1-SPN and D2-SPN Ca2+ activity in mice, but that antipsychotic efficacy was associated with the reversal of abnormal D1-SPN, rather than D2-SPN, dynamics, even for drugs that are D2R selective or lacking any dopamine receptor affinity. By contrast, a clinically ineffective drug normalized D2-SPN dynamics but exacerbated D1-SPN dynamics under hyperdopaminergic conditions. Consistent with antipsychotic effect, selective D1-SPN inhibition attenuated amphetamine-driven changes in locomotion, sensorimotor gating and hallucination-like perception. Notably, antipsychotic efficacy correlated with the selective inhibition of D1-SPNs only under hyperdopaminergic conditions-a dopamine-state-dependence exhibited by D1R partial agonism but not non-antipsychotic D1R antagonists. Our findings provide new insights into antipsychotic drug mechanism and reveal an important role for D1-SPN modulation.

Blunted mGluR Activation Disinhibits Striatopallidal Transmission in Parkinsonian Mice.

The prevailing circuit model predicts that hyperactivity of the striatopallidal pathway and subsequently increased inhibition of external globus pallidus (GPe) neurons lead to the hypokinetic symptoms of Parkinson's disease (PD). It is believed that hyperactivity of the striatopallidal pathway is due to inactivity of dopamine receptors on the somatodendritic membrane of striatopallidal neurons, but the exact cellular underpinnings remain unclear. In this study, we show that mouse GPe astrocytes critically control ambient glutamate level, which in turn gates striatopallidal transmission via the activation of presynaptic metabotropic glutamate receptors. This presynaptic inhibition of striatopallidal transmission is diminished after the chronic loss of dopamine. Elevation of intracellular glutamate content in astrocytes restores the proper regulation of the striatopallidal input in PD models. These findings argue that astrocytes are key regulators of the striatopallidal synapse. Targeting this cell class may serve as an alternative therapeutic strategy for PD.