Correction: GABAergic regulation of striatal spiny projection neurons depends upon their activity state.

[This corrects the article DOI: 10.1371/journal.pbio.3002483.].

GABAergic regulation of striatal spiny projection neurons depends upon their activity state.

Synaptic transmission mediated by GABAA receptors (GABAARs) in adult, principal striatal spiny projection neurons (SPNs) can suppress ongoing spiking, but its effect on synaptic integration at subthreshold membrane potentials is less well characterized, particularly those near the resting down-state. To fill this gap, a combination of molecular, optogenetic, optical, and electrophysiological approaches were used to study SPNs in mouse ex vivo brain slices, and computational tools were used to model somatodendritic synaptic integration. In perforated patch recordings, activation of GABAARs, either by uncaging of GABA or by optogenetic stimulation of GABAergic synapses, evoked currents with a reversal potential near -60 mV in both juvenile and adult SPNs. Transcriptomic analysis and pharmacological work suggested that this relatively positive GABAAR reversal potential was not attributable to NKCC1 expression, but rather to HCO3- permeability. Regardless, from down-state potentials, optogenetic activation of dendritic GABAergic synapses depolarized SPNs. This GABAAR-mediated depolarization summed with trailing ionotropic glutamate receptor (iGluR) stimulation, promoting dendritic spikes and increasing somatic depolarization. Simulations revealed that a diffuse dendritic GABAergic input to SPNs effectively enhanced the response to dendritic iGluR signaling and promoted dendritic spikes. Taken together, our results demonstrate that GABAARs can work in concert with iGluRs to excite adult SPNs when they are in the resting down-state, suggesting that their inhibitory role is limited to brief periods near spike threshold. This state-dependence calls for a reformulation for the role of intrastriatal GABAergic circuits.

Disruption of mitochondrial complex I induces progressive parkinsonism.

Loss of functional mitochondrial complex I (MCI) in the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson's disease1. Yet, whether this change contributes to Parkinson's disease pathogenesis is unclear2. Here we used intersectional genetics to disrupt the function of MCI in mouse dopaminergic neurons. Disruption of MCI induced a Warburg-like shift in metabolism that enabled neuronal survival, but triggered a progressive loss of the dopaminergic phenotype that was first evident in nigrostriatal axons. This axonal deficit was accompanied by motor learning and fine motor deficits, but not by clear levodopa-responsive parkinsonism-which emerged only after the later loss of dopamine release in the substantia nigra. Thus, MCI dysfunction alone is sufficient to cause progressive, human-like parkinsonism in which the loss of nigral dopamine release makes a critical contribution to motor dysfunction, contrary to the current Parkinson's disease paradigm3,4.

Dysregulation of the Basal Ganglia Indirect Pathway in Early Symptomatic Q175 Huntington's Disease Mice.

The debilitating psychomotor symptoms of Huntington's disease (HD) are linked partly to degeneration of the basal ganglia indirect pathway. At early symptomatic stages, before major cell loss, indirect pathway neurons exhibit numerous cellular and synaptic changes in HD and its models. However, the impact of these alterations on circuit activity remains poorly understood. To address this gap, optogenetic- and reporter-guided electrophysiological interrogation was used in early symptomatic male and female Q175 HD mice. D2 dopamine receptor-expressing striatal projection neurons (D2-SPNs) were hypoactive during synchronous cortical slow-wave activity, consistent with known reductions in dendritic excitability and cortical input strength. Downstream prototypic parvalbumin-expressing external globus pallidus (PV+ GPe) neurons discharged at 2-3 times their normal rate, even during periods of D2-SPN inactivity, arguing that defective striatopallidal inhibition was not the only cause of their hyperactivity. Indeed, PV+ GPe neurons also exhibited abnormally elevated autonomous firing ex vivo Optogenetic inhibition of PV+ GPe neurons in vivo partially and fully ameliorated the abnormal hypoactivity of postsynaptic subthalamic nucleus (STN) and putative PV- GPe neurons, respectively. In contrast to STN neurons whose autonomous firing is impaired in HD mice, putative PV- GPe neuron activity was unaffected ex vivo, implying that excessive inhibition was responsible for their hypoactivity in vivo Together with previous studies, these data demonstrate that (1) indirect pathway nuclei are dysregulated in Q175 mice through changes in presynaptic activity and/or intrinsic cellular and synaptic properties; and (2) prototypic PV+ GPe neuron hyperactivity and excessive target inhibition are prominent features of early HD pathophysiology.SIGNIFICANCE STATEMENT The early symptoms of Huntington's disease (HD) are linked to degenerative changes in the action-suppressing indirect pathway of the basal ganglia. Consistent with this linkage, the intrinsic properties of cells in this pathway exhibit complex alterations in HD and its models. However, the impact of these changes on activity is poorly understood. Using electrophysiological and optogenetic approaches, we demonstrate that the indirect pathway is highly dysregulated in early symptomatic HD mice through changes in upstream activity and/or intrinsic properties. Furthermore, we reveal that hyperactivity of external globus pallidus neurons and excessive inhibition of their targets are key features of early HD pathophysiology. Together, these findings could help to inform the development and targeting of viral-based, gene therapeutic approaches for HD.

Pathway-Specific Remodeling of Thalamostriatal Synapses in a Mouse Model of Parkinson's Disease.

BACKGROUND: The network pathophysiology underlying the motor symptoms of Parkinson's disease (PD) is poorly understood. In models of late-stage PD, there is significant cell-specific remodeling of corticostriatal, axospinous glutamatergic synapses on principal spiny projection neurons (SPNs). Neurons in the centrolateral nucleus (CLN) of the thalamus that relay cerebellar activity to the striatum also make axospinous synapses on SPNs, but the extent to which they are affected in PD has not been definitively characterized. OBJECTIVE: To fill this gap, transgenic mice in which CLN neurons express Cre recombinase were used in conjunction with optogenetic and circuit mapping approaches to determine changes in the CLN projection to SPNs in a unilateral 6-hydroxydopamine (6-OHDA) model of late-stage PD. METHODS: Adeno-associated virus vectors carrying Cre-dependent opsin expression constructs were stereotaxically injected into the CLN of Grp-KH288 mice in which CLN, but not parafascicular nucleus neurons, expressed Cre recombinase. The properties of this projection to identify direct pathway spiny projection neurons (dSPNs) and indirect pathway spiny projection neurons (iSPNs) were then studied in ex vivo brain slices of the dorsolateral striatum from control and 6-OHDA lesioned mice using anatomic, optogenetic, and electrophysiological approaches. RESULTS: Optogenetically evoked excitatory synaptic currents in both iSPNs and dSPNs were reduced in lesioned mice; however, the reduction was significantly greater in dSPNs. In iSPNs, the reduction in evoked responses was attributable to synaptic pruning, because synaptic channelrhodopsin assisted circuit mapping (sCRACm) revealed fewer synapses per cell after lesioning. In contrast, sCRACm mapping of CLN inputs to dSPNs failed to detect any change in synapse abundance in lesioned mice. However, the ratio of currents through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors to those through N-methyl-D-aspartate receptors was significantly reduced in dSPNs. Moreover, the distribution of currents evoked by optical stimulation of individual synapses shifted toward smaller amplitudes by lesioning, suggesting that they had undergone long-term depression. CONCLUSIONS: Taken together, our results demonstrate that the CLN projection to the striatum undergoes a pathway-specific remodeling that could contribute to the circuit imbalance thought to drive the hypokinetic features of PD. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Photoactivatable drugs for nicotinic optopharmacology.

Photoactivatable pharmacological agents have revolutionized neuroscience, but the palette of available compounds is limited. We describe a general method for caging tertiary amines by using a stable quaternary ammonium linkage that elicits a red shift in the activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncageable via one- or two-photon excitation, that is useful to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.

Dysregulation of external globus pallidus-subthalamic nucleus network dynamics in parkinsonian mice during cortical slow-wave activity and activation.

KEY POINTS: Reciprocally connected GABAergic external globus pallidus (GPe) and glutamatergic subthalamic nucleus (STN) neurons form a key network within the basal ganglia. In Parkinson's disease and its models, abnormal rates and patterns of GPe-STN network activity are linked to motor dysfunction. Using cell class-specific optogenetic identification and inhibition during cortical slow-wave activity and activation, we report that, in dopamine-depleted mice, (1) D2 dopamine receptor expressing striatal projection neurons (D2-SPNs) discharge at higher rates, especially during cortical activation, (2) prototypic parvalbumin-expressing GPe neurons are excessively patterned by D2-SPNs even though their autonomous activity is upregulated, (3) despite being disinhibited, STN neurons are not hyperactive, and (4) STN activity opposes striatopallidal patterning. These data argue that in parkinsonian mice abnormal, temporally offset prototypic GPe and STN neuron firing results in part from increased striatopallidal transmission and that compensatory plasticity limits STN hyperactivity and cortical entrainment. ABSTRACT: Reciprocally connected GABAergic external globus pallidus (GPe) and glutamatergic subthalamic nucleus (STN) neurons form a key, centrally positioned network within the basal ganglia. In Parkinson's disease and its models, abnormal rates and patterns of GPe-STN network activity are linked to motor dysfunction. Following the loss of dopamine, the activities of GPe and STN neurons become more temporally offset and strongly correlated with cortical oscillations below 40 Hz. Previous studies utilized cortical slow-wave activity and/or cortical activation (ACT) under anaesthesia to probe the mechanisms underlying the normal and pathological patterning of basal ganglia activity. Here, we combined this approach with in vivo optogenetic inhibition to identify and interrupt the activity of D2 dopamine receptor-expressing striatal projection neurons (D2-SPNs), parvalbumin-expressing prototypic GPe (PV GPe) neurons, and STN neurons. We found that, in dopamine-depleted mice, (1) the firing rate of D2-SPNs was elevated, especially during cortical ACT, (2) abnormal phasic suppression of PV GPe neuron activity was ameliorated by optogenetic inhibition of coincident D2-SPN activity, (3) autonomous PV GPe neuron firing ex vivo was upregulated, presumably through homeostatic mechanisms, (4) STN neurons were not hyperactive, despite being disinhibited, (5) optogenetic inhibition of the STN exacerbated abnormal GPe activity, and (6) exaggerated beta band activity was not present in the cortex or GPe-STN network. Together with recent studies, these data suggest that in dopamine-depleted mice abnormally correlated and temporally offset PV GPe and STN neuron activity is generated in part by elevated striatopallidal transmission, while compensatory plasticity prevents STN hyperactivity and limits cortical entrainment.

Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1.

Parkinson's disease is a pervasive, ageing-related neurodegenerative disease the cardinal motor symptoms of which reflect the loss of a small group of neurons, the dopaminergic neurons in the substantia nigra pars compacta (SNc). Mitochondrial oxidant stress is widely viewed as being responsible for this loss, but why these particular neurons should be stressed is a mystery. Here we show, using transgenic mice that expressed a redox-sensitive variant of green fluorescent protein targeted to the mitochondrial matrix, that the engagement of plasma membrane L-type calcium channels during normal autonomous pacemaking created an oxidant stress that was specific to vulnerable SNc dopaminergic neurons. The oxidant stress engaged defences that induced transient, mild mitochondrial depolarization or uncoupling. The mild uncoupling was not affected by deletion of cyclophilin D, which is a component of the permeability transition pore, but was attenuated by genipin and purine nucleotides, which are antagonists of cloned uncoupling proteins. Knocking out DJ-1 (also known as PARK7 in humans and Park7 in mice), which is a gene associated with an early-onset form of Parkinson's disease, downregulated the expression of two uncoupling proteins (UCP4 (SLC25A27) and UCP5 (SLC25A14)), compromised calcium-induced uncoupling and increased oxidation of matrix proteins specifically in SNc dopaminergic neurons. Because drugs approved for human use can antagonize calcium entry through L-type channels, these results point to a novel neuroprotective strategy for both idiopathic and familial forms of Parkinson's disease.

An autism-associated variant of Epac2 reveals a role for Ras/Epac2 signaling in controlling basal dendrite maintenance in mice.

The architecture of dendritic arbors determines circuit connectivity, receptive fields, and computational properties of neurons, and dendritic structure is impaired in several psychiatric disorders. While apical and basal dendritic compartments of pyramidal neurons are functionally specialized and differentially regulated, little is known about mechanisms that selectively maintain basal dendrites. Here we identified a role for the Ras/Epac2 pathway in maintaining basal dendrite complexity of cortical neurons. Epac2 is a guanine nucleotide exchange factor (GEF) for the Ras-like small GTPase Rap, and it is highly enriched in the adult mouse brain. We found that in vivo Epac2 knockdown in layer 2/3 cortical neurons via in utero electroporation reduced basal dendritic architecture, and that Epac2 knockdown in mature cortical neurons in vitro mimicked this effect. Overexpression of an Epac2 rare coding variant, found in human subjects diagnosed with autism, also impaired basal dendritic morphology. This mutation disrupted Epac2's interaction with Ras, and inhibition of Ras selectively interfered with basal dendrite maintenance. Finally, we observed that components of the Ras/Epac2/Rap pathway exhibited differential abundance in the basal versus apical dendritic compartments. These findings define a role for Epac2 in enabling crosstalk between Ras and Rap signaling in maintaining basal dendrite complexity, and exemplify how rare coding variants, in addition to their disease relevance, can provide insight into cellular mechanisms relevant for brain connectivity.

Ca 2+ -dependent phosphodiesterase 1 regulates the plasticity of striatal spiny projection neuron glutamatergic synapses.

Long-term synaptic plasticity at glutamatergic synapses on striatal spiny projection neurons (SPNs) is central to learning goal-directed behaviors and habits. Although considerable attention has been paid to the mechanisms underlying synaptic strengthening and new learning, little scrutiny has been given to those involved in the attenuation of synaptic strength that attends suppression of a previously learned association. Our studies revealed a novel, non-Hebbian, long-term, postsynaptic depression of glutamatergic SPN synapses induced by interneuronal nitric oxide (NO) signaling (NO-LTD) that was preferentially engaged at quiescent synapses. This form of plasticity was gated by local Ca 2+ influx through CaV1.3 Ca 2+ channels and stimulation of phosphodiesterase 1 (PDE1), which degraded cyclic guanosine monophosphate (cGMP) and blunted NO signaling. Consistent with this model, mice harboring a gain-of-function mutation in the gene coding for the pore-forming subunit of CaV1.3 channels had elevated depolarization-induced dendritic Ca 2+ entry and impaired NO-LTD. Extracellular uncaging of glutamate and intracellular uncaging of cGMP suggested that this Ca 2+ -dependent regulation of PDE1 activity allowed for local regulation of dendritic NO signaling. This inference was supported by simulation of SPN dendritic integration, which revealed that dendritic spikes engaged PDE1 in a branch-specific manner. In a mouse model of Parkinson's disease (PD), NO-LTD was absent not because of a postsynaptic deficit in NO signaling machinery, but rather due to impaired interneuronal NO release. Re-balancing intrastriatal neuromodulatory signaling in the PD model restored NO release and NO-LTD. Taken together, these studies provide novel insights into the mechanisms governing NO-LTD in SPN and its role in psychomotor disorders, like PD.

Ca2+-dependent phosphodiesterase 1 regulates the plasticity of striatal spiny projection neuron glutamatergic synapses.

Long-term synaptic plasticity at glutamatergic synapses on striatal spiny projection neurons (SPNs) is central to learning goal-directed behaviors and habits. Our studies reveal that SPNs manifest a heterosynaptic, nitric oxide (NO)-dependent form of long-term postsynaptic depression of glutamatergic SPN synapses (NO-LTD) that is preferentially engaged at quiescent synapses. Plasticity is gated by Ca2+ entry through CaV1.3 Ca2+ channels and phosphodiesterase 1 (PDE1) activation, which blunts intracellular cyclic guanosine monophosphate (cGMP) and NO signaling. Both experimental and simulation studies suggest that this Ca2+-dependent regulation of PDE1 activity allows for local regulation of dendritic cGMP signaling. In a mouse model of Parkinson disease (PD), NO-LTD is absent because of impaired interneuronal NO release; re-balancing intrastriatal neuromodulatory signaling restores NO release and NO-LTD. Taken together, these studies provide important insights into the mechanisms governing NO-LTD in SPNs and its role in psychomotor disorders such as PD.

Social, communication, and cortical structural impairments in Epac2-deficient mice.

Deficits in social and communication behaviors are common features of a number of neurodevelopmental disorders. However, the molecular and cellular substrates of these higher order brain functions are not well understood. Here we report that specific alterations in social and communication behaviors in mice occur as a result of loss of the EPAC2 gene, which encodes a protein kinase A-independent cAMP target. Epac2-deficient mice exhibited robust deficits in social interactions and ultrasonic vocalizations, but displayed normal olfaction, working and reference memory, motor abilities, anxiety, and repetitive behaviors. Epac2-deficient mice displayed abnormal columnar organization in the anterior cingulate cortex, a region implicated in social behavior in humans, but not in somatosensory cortex. In vivo two-photon imaging revealed reduced dendritic spine motility and density on cortical neurons in Epac2-deficient mice, indicating deficits at the synaptic level. Together, these findings provide novel insight into the molecular and cellular substrates of social and communication behavior.

Cholinergic deficits selectively boost cortical intratelencephalic control of striatum in male Huntington's disease model mice.

Huntington's disease (HD) is a progressive, neurodegenerative disease caused by a CAG triplet expansion in huntingtin. Although corticostriatal dysfunction has long been implicated in HD, the determinants and pathway specificity of this pathophysiology are not fully understood. Here, using a male zQ175+/- knock-in mouse model of HD we carry out optogenetic interrogation of intratelencephalic and pyramidal tract synapses with principal striatal spiny projection neurons (SPNs). These studies reveal that the connectivity of intratelencephalic, but not pyramidal tract, neurons with direct and indirect pathway SPNs increased in early symptomatic zQ175+/- HD mice. This enhancement was attributable to reduced pre-synaptic inhibitory control of intratelencephalic terminals by striatal cholinergic interneurons. Lowering mutant huntingtin selectively in striatal cholinergic interneurons with a virally-delivered zinc finger repressor protein normalized striatal acetylcholine release and intratelencephalic functional connectivity, revealing a node in the network underlying corticostriatal pathophysiology in a HD mouse model.

State-dependent GABAergic regulation of striatal spiny projection neuron excitability.

Synaptic transmission mediated by GABA A receptors (GABA A Rs) in adult, principal striatal spiny projection neurons (SPNs) can suppress ongoing spiking, but its effect on synaptic integration at sub-threshold membrane potentials is less well characterized, particularly those near the resting down-state. To fill this gap, a combination of molecular, optogenetic, optical and electrophysiological approaches were used to study SPNs in mouse ex vivo brain slices, and computational tools were used to model somatodendritic synaptic integration. Activation of GABA A Rs, either by uncaging of GABA or by optogenetic stimulation of GABAergic synapses, evoked currents with a reversal potential near -60 mV in perforated patch recordings from both juvenile and adult SPNs. Molecular profiling of SPNs suggested that this relatively positive reversal potential was not attributable to NKCC1 expression, but rather to a dynamic equilibrium between KCC2 and Cl-/HCO3-cotransporters. Regardless, from down-state potentials, optogenetic activation of dendritic GABAergic synapses depolarized SPNs. This GABAAR-mediated depolarization summed with trailing ionotropic glutamate receptor (iGluR) stimulation, promoting dendritic spikes and increasing somatic depolarization. Simulations revealed that a diffuse dendritic GABAergic input to SPNs effectively enhanced the response to coincident glutamatergic input. Taken together, our results demonstrate that GABA A Rs can work in concert with iGluRs to excite adult SPNs when they are in the resting down-state, suggesting that their inhibitory role is limited to brief periods near spike threshold. This state-dependence calls for a reformulation of the role intrastriatal GABAergic circuits.

α-Synuclein pathology disrupts mitochondrial function in dopaminergic and cholinergic neurons at-risk in Parkinson's disease.

Background: Pathological accumulation of aggregated α-synuclein (aSYN) is a common feature of Parkinson's disease (PD). However, the mechanisms by which intracellular aSYN pathology contributes to dysfunction and degeneration of neurons in the brain are still unclear. A potentially relevant target of aSYN is the mitochondrion. To test this hypothesis, genetic and physiological methods were used to monitor mitochondrial function in substantia nigra pars compacta (SNc) dopaminergic and pedunculopontine nucleus (PPN) cholinergic neurons after stereotaxic injection of aSYN pre-formed fibrils (PFFs) into the mouse brain. Methods: aSYN PPFs were stereotaxically injected into the SNc or PPN of mice. Twelve weeks later, mice were studied using a combination of approaches, including immunocytochemical analysis, cell- type specific transcriptomic profiling, electron microscopy, electrophysiology and two-photon-laser- scanning microscopy of genetically encoded sensors for bioenergetic and redox status. Results: In addition to inducing a significant neuronal loss, SNc injection of PFFs induced the formation of intracellular, phosphorylated aSYN aggregates selectively in dopaminergic neurons. In these neurons, PFF-exposure decreased mitochondrial gene expression, reduced the number of mitochondria, increased oxidant stress, and profoundly disrupted mitochondrial adenosine triphosphate production. Consistent with an aSYN-induced bioenergetic deficit, the autonomous spiking of dopaminergic neurons slowed or stopped. PFFs also up-regulated lysosomal gene expression and increased lysosomal abundance, leading to the formation of Lewy-like inclusions. Similar changes were observed in PPN cholinergic neurons following aSYN PFF exposure. Conclusions: Taken together, our findings suggest that disruption of mitochondrial function, and the subsequent bioenergetic deficit, is a proximal step in the cascade of events induced by aSYN pathology leading to dysfunction and degeneration of neurons at-risk in PD.

Acute and protracted abstinence from methamphetamine bidirectionally changes intrinsic excitability of indirect pathway spiny projection neurons in the dorsomedial striatum.

Methamphetamine (meth) is an addictive psychostimulant and illicit use presents significant personal and socioeconomic harm. Behavioral studies support the involvement of the dorsal striatum in drug-seeking but stimulant induced dysfunction in this region is understudied. The dorsal striatum can be subdivided into the dorsomedial (DMS) and dorsolateral (DLS) striatum with the DMS implicated in goal-directed and DLS in habitual behaviors; both regions are primarily composed of GABAergic direct (dSPNs) and indirect pathway (iSPNs) spiny projection neurons. To examine the effect of repeated meth on SPNs, mice were administered meth (2 mg/kg) for ten consecutive days and intrinsic excitability, dendritic excitability, and spine density were examined. DMS iSPN intrinsic excitability was increased at 1 day but decreased at 21 days of abstinence. In contrast, DMS dSPN intrinsic excitability was unchanged at either timepoint. Dendritic excitability and spine densities were unaltered in DMS iSPNs and dSPNs at 1 and 21 days of abstinence. The effect of repeated meth on iSPN excitability was specific to the DMS; DLS iSPN intrinsic excitability, dendritic excitability, and spine density were unchanged at 1 and 21 days of abstinence. These findings point toward DMS iSPN dysfunction in meth use disorders with differential dysfunction dependent on abstinence duration.

Ca2+ channels couple spiking to mitochondrial metabolism in substantia nigra dopaminergic neurons.

How do neurons match generation of adenosine triphosphate by mitochondria to the bioenergetic demands of regenerative activity? Although the subject of speculation, this coupling is still poorly understood, particularly in neurons that are tonically active. To help fill this gap, pacemaking substantia nigra dopaminergic neurons were studied using a combination of optical, electrophysiological, and molecular approaches. In these neurons, spike-activated calcium (Ca2+) entry through Cav1 channels triggered Ca2+ release from the endoplasmic reticulum, which stimulated mitochondrial oxidative phosphorylation through two complementary Ca2+-dependent mechanisms: one mediated by the mitochondrial uniporter and another by the malate-aspartate shuttle. Disrupting either mechanism impaired the ability of dopaminergic neurons to sustain spike activity. While this feedforward control helps dopaminergic neurons meet the bioenergetic demands associated with sustained spiking, it is also responsible for their elevated oxidant stress and possibly to their decline with aging and disease.

Selective participation of somatodendritic HCN channels in inhibitory but not excitatory synaptic integration in neurons of the subthalamic nucleus.

The activity patterns of subthalamic nucleus (STN) neurons are intimately linked to motor function and dysfunction and arise through the complex interaction of intrinsic properties and inhibitory and excitatory synaptic inputs. In many neurons, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play key roles in intrinsic excitability and synaptic integration both under normal conditions and in disease states. However, in STN neurons, which strongly express HCN channels, their roles remain relatively obscure. To address this deficit, complementary molecular and cellular electrophysiological, imaging, and computational approaches were applied to the rat STN. Molecular profiling demonstrated that individual STN neurons express mRNA encoding several HCN subunits, with HCN2 and 3 being the most abundant. Light and electron microscopic analysis showed that HCN2 subunits are strongly expressed and distributed throughout the somatodendritic plasma membrane. Voltage-, current-, and dynamic-clamp analysis, two-photon Ca(2+) imaging, and computational modeling revealed that HCN channels are activated by GABA(A) receptor-mediated inputs and thus limit synaptic hyperpolarization and deinactivation of low-voltage-activated Ca(2+) channels. Although HCN channels also limited the temporal summation of EPSPs, generated through two-photon uncaging of glutamate, this action was largely shunted by GABAergic inhibition that was necessary for HCN channel activation. Together the data demonstrate that HCN channels in STN neurons selectively counteract GABA(A) receptor-mediated inhibition arising from the globus pallidus and thus promote single-spike activity rather than rebound burst firing.