The development of methodology to identify specific cell populations and circuits within the basal ganglia is rapidly transforming our ability to understand the function of this complex circuit. This mini-symposium highlights recent advances in delineating the organization and function of neural circuits in the external segment of the globus pallidus (GPe). Although long considered a homogeneous structure in the motor-suppressing "indirect-pathway," the GPe consists of a number of distinct cell types and anatomical subdomains that contribute differentially to both motor and nonmotor features of behavior. Here, we integrate recent studies using techniques, such as viral tracing, transgenic mice, electrophysiology, and behavioral approaches, to create a revised framework for understanding how the GPe relates to behavior in both health and disease.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and the Ih current they generate contribute to the pathophysiological mechanisms of absence seizures (ASs), but their precise role in neocortical and thalamic neuronal populations, the main components of the network underlying AS generation, remains controversial. In diverse genetic AS models, Ih amplitude is smaller in neocortical neurons and either larger or unchanged in thalamocortical (TC) neurons compared with nonepileptic strains. A lower expression of neocortical HCN subtype 1 channels is present in genetic AS-prone rats, and HCN subtype 2 knock-out mice exhibit ASs. Furthermore, whereas many studies have characterized Ih contribution to "absence-like" paroxysmal activity in vitro, no data are available on the specific role of cortical and thalamic HCN channels in behavioral seizures. Here, we show that the pharmacological block of HCN channels with the antagonist ZD7288 applied via reverse microdialysis in the ventrobasal thalamus (VB) of freely moving male Genetic Absence Epilepsy Rats from Strasbourg decreases TC neuron firing and abolishes spontaneous ASs. A similar effect is observed on γ-hydroxybutyric acid-elicited ASs in normal male Wistar rats. Moreover, thalamic knockdown of HCN channels via virally delivered shRNA into the VB of male Stargazer mice, another genetic AS model, decreases spontaneous ASs and Ih-dependent electrophysiological properties of VB TC neurons. These findings provide the first evidence that block of TC neuron HCN channels prevents ASs and suggest that any potential anti-absence therapy that targets HCN channels should carefully consider the opposite role for cortical and thalamic Ih in the modulation of absence seizures.SIGNIFICANCE STATEMENT Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play critical roles in the fine-tuning of cellular and network excitability and have been suggested to be a key element of the pathophysiological mechanism underlying absence seizures. However, the precise contribution of HCN channels in neocortical and thalamic neuronal populations to these nonconvulsive seizures is still controversial. In the present study, pharmacological block and genetic suppression of HCN channels in thalamocortical neurons in the ventrobasal thalamic nucleus leads to a marked reduction in absence seizures in one pharmacological and two genetic rodent models of absence seizures. These results provide the first evidence that block of TC neuron HCN channels prevents absence seizures.

Midbrain dopamine (DA) neurons have diverse functions that can in part be explained by their heterogeneity. Although molecularly distinct subtypes of DA neurons have been identified by single-cell gene expression profiling, fundamental features such as their projection patterns have not been elucidated. Progress in this regard has been hindered by the lack of genetic tools for studying DA neuron subtypes. Here we develop intersectional genetic labeling strategies, based on combinatorial gene expression, to map the projections of molecularly defined DA neuron subtypes. We reveal distinct genetically defined dopaminergic pathways arising from the substantia nigra pars compacta and from the ventral tegmental area that innervate specific regions of the caudate putamen, nucleus accumbens and amygdala. Together, the genetic toolbox and DA neuron subtype projections presented here constitute a resource that will accelerate the investigation of this clinically significant neurotransmitter system.

LRRK2 mutations are closely associated with Parkinson's disease (PD). Convergent evidence suggests that LRRK2 regulates striatal function. Here, by using knock-in mouse lines expressing the two most common LRRK2 pathogenic mutations-G2019S and R1441C-we investigated how LRRK2 mutations altered striatal physiology. While we found that both R1441C and G2019S mice displayed reduced nigrostriatal dopamine release, hypoexcitability in indirect-pathway striatal projection neurons, and alterations associated with an impaired striatal-dependent motor learning were observed only in the R1441C mice. We also showed that increased synaptic PKA activities in the R1441C and not G2019S mice underlie the specific alterations in motor learning deficits in the R1441C mice. In summary, our data argue that LRRK2 mutations' impact on the striatum cannot be simply generalized. Instead, alterations in electrochemical, electrophysiological, molecular, and behavioral levels were distinct between LRRK2 mutations. Our findings offer mechanistic insights for devising and optimizing treatment strategies for PD patients.

In the classical model of the basal ganglia, direct pathway striatal projection neurons (dSPNs) send projections to the substantia nigra (SNr) and entopeduncular nucleus to regulate motor function. Recent studies have re-established that dSPNs also possess axon collaterals within the globus pallidus (GPe) (bridging collaterals), yet the significance of these collaterals for behavior is unknown. Here we use in vivo optical and chemogenetic tools combined with deep learning approaches in mice to dissect the roles of dSPN GPe collaterals in motor function. We find that dSPNs projecting to the SNr send synchronous motor-related information to the GPe via axon collaterals. Inhibition of native activity in dSPN GPe terminals impairs motor activity and function via regulation of Npas1 neurons. We propose a model by which dSPN GPe axon collaterals (striatopallidal Go pathway) act in concert with the canonical terminals in the SNr to support motor control by inhibiting Npas1 neurons.

Memory formation requires de novo protein synthesis, and memory disorders may result from misregulated synthesis of critical proteins that remain largely unidentified. Plasma membrane ion channels and receptors are likely candidates given their role in regulating neuron excitability, a candidate memory mechanism. Here we conduct targeted molecular monitoring and quantitation of hippocampal plasma membrane proteins from mice with intact or impaired contextual fear memory to identify putative candidates. Here we report contextual fear memory deficits correspond to increased Trpc3 gene and protein expression, and demonstrate TRPC3 regulates hippocampal neuron excitability associated with memory function. These data provide a mechanistic explanation for enhanced contextual fear memory reported herein following knockdown of TRPC3 in hippocampus. Collectively, TRPC3 modulates memory and may be a feasible target to enhance memory and treat memory disorders.

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.

Parkinson's disease is a common neurodegenerative disorder characterized by a profound motor disability that is traceable to the emergence of synchronous, rhythmic spiking in neurons of the external segment of the globus pallidus (GPe). The origins of this pathophysiology are poorly defined for the generation of pacemaking. After the induction of a parkinsonian state in mice, there was a progressive decline in autonomous GPe pacemaking, which normally serves to desynchronize activity. The loss was attributable to the downregulation of an ion channel that is essential in pacemaking, the hyperpolarization and cyclic nucleotide-gated (HCN) channel. Viral delivery of HCN2 subunits restored pacemaking and reduced burst spiking in GPe neurons. However, the motor disability induced by dopamine (DA) depletion was not reversed, suggesting that the loss of pacemaking was a consequence, rather than a cause, of key network pathophysiology, a conclusion that is consistent with the ability of L-type channel antagonists to attenuate silencing after DA depletion.

Although ethanol is one of the most widely used drugs, we still lack a full understanding of which neuronal subtypes are affected by this drug. Pacemaker neurons exert powerful control over brain circuit function, but little is known about ethanol effects on these types of neurons. Neurons in the external globus pallidus (GPe) generate pacemaker activity that controls basal ganglia, circuitry associated with habitual and compulsive drug use. We performed patch-clamp recordings from GPe neurons and found that bath application of ethanol dose-dependently decreased the firing rate of low-frequency GPe neurons, but did not alter the firing of high-frequency neurons. GABA or glutamate receptor antagonists did not block the ethanol effect. The GPe is comprised of a heterogeneous population of neurons. We used Lhx6-EGFP and Npas1-tdTm mice strains to identify low-frequency neurons. Lhx6 and Npas1 neurons exhibited decreased firing with ethanol, but only Npas1 neurons were sensitive to 10 mM ethanol. Large-conductance voltage and Ca2+-activated K+ (BK) channel have a key role in the ethanol effect on GPe neurons, as the application of BK channel inhibitors blocked the ethanol-induced firing decrease. Ethanol also increased BK channel open probability measured in single-channel recordings from Npas1-tdTm neurons. In addition, in vivo electrophysiological recordings from GPe showed that ethanol decreased the firing of a large subset of low-frequency neurons. These findings indicate how selectivity of ethanol effects on pacemaker neurons can occur, and enhance our understanding of the mechanisms contributing to acute ethanol effects on the basal ganglia.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder. The debilitating choreic movements that plague HD patients have been attributed to striatal degeneration induced by the loss of cortically supplied brain-derived neurotrophic factor (BDNF). Here, we show that in mouse models of early symptomatic HD, BDNF delivery to the striatum and its activation of tyrosine-related kinase B (TrkB) receptors were normal. However, in striatal neurons responsible for movement suppression, TrkB receptors failed to properly engage postsynaptic signaling mechanisms controlling the induction of potentiation at corticostriatal synapses. Plasticity was rescued by inhibiting p75 neurotrophin receptor (p75NTR) signaling or its downstream target phosphatase-and-tensin-homolog-deleted-on-chromosome-10 (PTEN). Thus, corticostriatal synaptic dysfunction early in HD is attributable to a correctable defect in the response to BDNF, not its delivery.

Antipsychotic drugs are still widely prescribed to control various severe neuropsychiatric symptoms in the elderly and dementia patients although they are off-label use in the United States. However, clinical practice shows greater side effects and lower efficacy of antipsychotics for this vulnerable population and the mechanisms surrounding this aged-related sensitivity are not well understood. Our previous studies have shown that aging-induced epigenetic alterations may be involved in the increasing severity of typical antipsychotic haloperidol induced side effects in aged mice. Still, it is unknown if similar epigenetic mechanisms extend to atypical antipsychotics, which are most often prescribed to dementia patients combined with severe neuropsychiatric symptoms. In this study, we report that atypical antipsychotic risperidone also causes increased motor side effect behaviors in aged mice and 5xFAD mice. Histone deacetylase (HDAC) inhibitor Valproic Acid and Entinostat can mitigate the risperidone induced motor side effects. We further showed besides D2R, reduced expression of 5-HT2A, one of the primary atypical antipsychotic targets in the striatum of aged mice that are also mitigated by HDAC inhibitors. Finally, we demonstrate that specific histone acetylation mark H3K27 is hypoacetylated at the 5htr2a and Drd2 promoters in aged mice and can be reversed with HDAC inhibitors. Our work here establishes evidence for a mechanism where aging reduces expression of 5-HT2A and D2R, the key atypical antipsychotic drug targets through epigenetic alteration. HDAC inhibitors can restore 5-HT2A and D2R expression in aged mice and decrease the motor side effects in aged and 5xFAD mice.

The striatum is widely viewed as the fulcrum of pathophysiology in Parkinson's disease (PD) and L-DOPA-induced dyskinesia (LID). In these disease states, the balance in activity of striatal direct pathway spiny projection neurons (dSPNs) and indirect pathway spiny projection neurons (iSPNs) is disrupted, leading to aberrant action selection. However, it is unclear whether countervailing mechanisms are engaged in these states. Here we report that iSPN intrinsic excitability and excitatory corticostriatal synaptic connectivity were lower in PD models than normal; L-DOPA treatment restored these properties. Conversely, dSPN intrinsic excitability was elevated in tissue from PD models and suppressed in LID models. Although the synaptic connectivity of dSPNs did not change in PD models, it fell with L-DOPA treatment. In neither case, however, was the strength of corticostriatal connections globally scaled. Thus, SPNs manifested homeostatic adaptations in intrinsic excitability and in the number but not strength of excitatory corticostriatal synapses.

Huntington's disease (HD) is initially characterized by an inability to suppress unwanted movements, a deficit attributable to impaired synaptic activation of striatal indirect pathway spiny projection neurons (iSPNs). To better understand the mechanisms underlying this deficit, striatal neurons in ex vivo brain slices from mouse genetic models of HD were studied using electrophysiological, optical and biochemical approaches. Distal dendrites of iSPNs from symptomatic HD mice were hypoexcitable, a change that was attributable to increased association of dendritic Kv4 potassium channels with auxiliary KChIP subunits. This association was negatively modulated by TrkB receptor signaling. Dendritic excitability of HD iSPNs was rescued by knocking-down expression of Kv4 channels, by disrupting KChIP binding, by restoring TrkB receptor signaling or by lowering mutant-Htt (mHtt) levels with a zinc finger protein. Collectively, these studies demonstrate that mHtt induces reversible alterations in the dendritic excitability of iSPNs that could contribute to the motor symptoms of HD.

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.

Lineage tracing aims to identify the progeny of a defined population of dividing progenitor cells, a daunting task in the developing central nervous system where thousands of cell types are generated. In mice, lineage analysis has been accomplished using Cre recombinase to indelibly label a defined progenitor population and its progeny. However, the interpretation of historical recombination events is hampered by the fact that driver genes are often expressed in both progenitors and postmitotic cells. Genetically inducible approaches provide temporal specificity but are afflicted by mosaicism and toxicity. Here, we present PRISM, a progenitor-restricted intersectional fate mapping approach in which Flp recombinase expression is both dependent on Cre and restricted to neural progenitors, thus circumventing the aforementioned confounds. This tool can be used in conjunction with existing Cre lines making it broadly applicable. We applied PRISM to resolve two developmentally important, but contentious, lineages-Shh and Cux2.