Corticotropin releasing factor alters the functional diversity of accumbal cholinergic interneurons.

Cholinergic interneurons (ChIs) provide the main source of acetylcholine in the striatum and have emerged as a critical modulator of behavioral flexibility, motivation, and associative learning. In the dorsal striatum (DS), ChIs display heterogeneous firing patterns. Here, we investigated the spontaneous firing patterns of ChIs in the nucleus accumbens (NAc) shell, a region of the ventral striatum. We identified four distinct ChI firing signatures: regular single-spiking, irregular single-spiking, rhythmic bursting, and a mixed-mode pattern composed of bursting activity and regular single spiking. ChIs from females had lower firing rates compared with males and had both a higher proportion of mixed-mode firing patterns and a lower proportion of regular single-spiking neurons compared with males. We further observed that across the estrous cycle, the diestrus phase was characterized by higher proportions of irregular ChI firing patterns compared with other phases. Using pooled data from males and females, we examined how the stress-associated neuropeptide corticotropin releasing factor (CRF) impacts these firing patterns. ChI firing patterns showed differential sensitivity to CRF. This translated into differential ChI sensitivity to CRF across the estrous cycle. Furthermore, CRF shifted the proportion of ChI firing patterns toward more regular spiking activity over bursting patterns. Finally, we found that repeated stressor exposure altered ChI firing patterns and sensitivity to CRF in the NAc core, but not the NAc shell. These findings highlight the heterogeneous nature of ChI firing patterns, which may have implications for accumbal-dependent motivated behaviors.NEW & NOTEWORTHY Cholinergic interneurons (ChIs) within the dorsal and ventral striatum can exert a major influence on network output and motivated behaviors. However, the firing patterns and neuromodulation of ChIs within the ventral striatum, specifically the nucleus accumbens (NAc) shell, are understudied. Here, we report that NAc shell ChIs have heterogeneous ChI firing patterns that are labile and can be modulated by the stress-linked neuropeptide corticotropin releasing factor (CRF) and by the estrous cycle.

Cholinergic interneurons in the nucleus accumbens are a site of cellular convergence for corticotropin-releasing factor and estrogen regulation in male and female mice.

Cholinergic interneurons (ChIs) act as master regulators of striatal output, finely tuning neurotransmission to control motivated behaviours. ChIs are a cellular target of many peptide and hormonal neuromodulators, including corticotropin-releasing factor, opioids, insulin and leptin, which can influence an animal's behaviour by signalling stress, pleasure, pain and nutritional status. However, little is known about how sex hormones via estrogen receptors influence the function of these other neuromodulators. Here, we performed in situ hybridisation on mouse striatal tissue to characterise the effect of sex and sex hormones on choline acetyltransferase (Chat), estrogen receptor alpha (Esr1) and corticotropin-releasing factor type 1 receptor (Crhr1) expression. Although we did not detect sex differences in ChAT protein levels in the dorsal striatum or nucleus accumbens, we found that female mice have more Chat mRNA-expressing neurons than males in both the dorsal striatum and nucleus accumbens. At the population level, we observed a sexually dimorphic distribution of Esr1- and Crhr1-expressing ChIs in the ventral striatum that was negatively correlated in intact females, which was abolished by ovariectomy and not present in males. Only in the NAc did we find a significant population of ChIs that co-express Crhr1 and Esr1 in females and to a lesser extent in males. At the cellular level, Crhr1 and Esr1 transcript levels were negatively correlated only during the estrus phase in females, indicating that changes in sex hormone levels can modulate the interaction between Crhr1 and Esr1 mRNA levels.

Coordinated Postnatal Maturation of Striatal Cholinergic Interneurons and Dopamine Release Dynamics in Mice.

Dynamic changes in motor abilities and motivated behaviors occur during the juvenile and adolescent periods. The striatum is a subcortical nucleus critical to action selection, motor learning, and reward processing. Its tonically active cholinergic interneuron (ChI) is an integral regulator of the synaptic activity of other striatal neurons, as well as afferent axonal projections of midbrain dopamine (DA) neurons; however, little is known about its development. Here, we report that ChI spontaneous activity increases during postnatal development of male and female mice, concomitant with a decreased afterhyperpolarization (AHP). We characterized the postnatal development of four currents that contribute to the spontaneous firing rate of ChIs, including ISK, IA, Ih, and INaP We demonstrated that the developmental increase in INaP drives increased ChI firing rates during the postnatal period and can be reversed by the INaP inhibitor, ranolazine. We next addressed whether immature cholinergic signaling may lead to functional differences in DA release during the juvenile period. In the adult striatum, nicotinic acetylcholine receptors (nAChRs) prevent linear summation of DA release in response to trains of high-frequency stimuli. We show that, in contrast, during the second postnatal week, DA release linearly sums with trains of high-frequency stimuli. Consistently, nAChR antagonists exert little effect on dopamine release at postnatal day (P)10, but enhance the summation of evoked DA release in mice older than postnatal day P28. Together, these results reveal that postnatal maturation of ChI activity is due primarily to enhanced INaP and identify an interaction between developing cholinergic signaling and DA neurotransmission in the juvenile striatum.SIGNIFICANCE STATEMENT Motor skills and motivated behavior develop rapidly in juvenile rodents. Recent work has highlighted processes that contribute to the postnatal maturation of striatal principal neurons during development. The functional development of the striatal cholinergic interneuron (ChI), however, has been unexplored. In this study, we tracked the ontogeny of ChI activity and cellular morphology, as well as the developmental trajectory of specific conductances that contribute to the activity of these cells. We further report a link between cholinergic signaling and dopamine (DA) release, revealing a change in the frequency-dependence of DA release during the early postnatal period that is mediated by cholinergic signaling. This study provides evidence that striatal microcircuits are dynamic during the postnatal period and that they undergo coordinated maturation.

Striatal synaptic adaptations in Parkinson's disease.

The striatum is densely innervated by mesencephalic dopaminergic neurons that modulate acquisition and vigor of goal-directed actions and habits. This innervation is progressively lost in Parkinson's disease (PD), contributing to the defining movement deficits of the disease. Although boosting dopaminergic signaling with levodopa early in the course of the disease alleviates these deficits, later this strategy leads to the emergence of debilitating dyskinesia. Here, recent advances in our understanding of how striatal cells and circuits adapt to this progressive de-innervation and to levodopa therapy are discussed. First, we discuss how dopamine (DA) depletion triggers cell type-specific, homeostatic changes in spiny projection neurons (SPNs) that tend to normalize striatal activity but also lead to disruption of the synaptic architecture sculpted by experience. Second, we discuss the roles played by cholinergic and nitric oxide-releasing interneurons in these adaptations. Third, we examine recent work in freely moving mice suggesting that alterations in the spatiotemporal dynamics of striatal ensembles contributes to PD movement deficits. Lastly, we discuss recently published evidence from a progressive model of PD suggesting that contrary to the classical model, striatal pathway imbalance is necessary but not sufficient to produce frank parkinsonism.

M1 muscarinic acetylcholine receptor-mediated inhibition of GABA release from striatal medium spiny neurons onto cholinergic interneurons.

Acetylcholine (ACh) modulates neurotransmitter release in the central nervous system. Although GABAergic transmission onto the striatal cholinergic interneurons (ChIN) is modulated by dopamine receptors, cholinergic modulation of the same synapse is still unknown. In the present study, modulatory roles of ACh in the GABAergic transmission from striatal medium spiny neurons (MSNs) onto ChIN were investigated using optogenetics and whole-cell patch-clamp technique in juvenile and young-adult mice brain slices. GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) were evoked by focal electrical- or blue-light stimulation. Bath application of carbachol, a muscarinic ACh receptor agonist, suppressed the amplitude of IPSCs in a concentration-dependent manner in both age groups. A choline esterase inhibitor, physostigmine, also suppressed the amplitude of IPSCs. In the presence of a membrane permeable M1 muscarine receptor antagonist, pirenzepine, carbachol-induced suppression of IPSCs was antagonized, whereas a M2 muscarine receptor antagonist, a M4 receptor antagonist, or a membrane impermeable M1 receptor antagonist did not antagonize carbachol-induced suppression of IPSCs. Retrograde cannabinoid cascade via cannabinoid receptor 1 was not involved in carbachol-induced inhibition. Furthermore, carbachol did not affect amplitude of inward currents induced by puff application of GABA, whereas coefficient of variation of IPSCs was significantly increased by carbachol. These results suggest that activation of presynaptic M1 muscarine receptors located on the GABAergic terminals including intracellular organelle of MSNs inhibits GABA release onto ChIN.

Diverse Mechanisms Lead to Common Dysfunction of Striatal Cholinergic Interneurons in Distinct Genetic Mouse Models of Dystonia.

Clinical and experimental data indicate striatal cholinergic dysfunction in dystonia, a movement disorder typically resulting in twisted postures via abnormal muscle contraction. Three forms of isolated human dystonia result from mutations in the TOR1A (DYT1), THAP1 (DYT6), and GNAL (DYT25) genes. Experimental models carrying these mutations facilitate identification of possible shared cellular mechanisms. Recently, we reported elevated extracellular striatal acetylcholine by in vivo microdialysis and paradoxical excitation of cholinergic interneurons (ChIs) by dopamine D2 receptor (D2R) agonism using ex vivo slice electrophysiology in Dyt1ΔGAG/+ mice. The paradoxical excitation was caused by overactive muscarinic receptors (mAChRs), leading to a switch in D2R coupling from canonical Gi/o to noncanonical ß-arrestin signaling. We sought to determine whether these mechanisms in Dyt1ΔGAG/+ mice are shared with Thap1C54Y/+ knock-in and Gnal+/- knock-out dystonia models and to determine the impact of sex. We found Thap1C54Y/+ mice of both sexes have elevated extracellular striatal acetylcholine and D2R-induced paradoxical ChI excitation, which was reversed by mAChR inhibition. Elevated extracellular acetylcholine was absent in male and female Gnal+/- mice, but the paradoxical D2R-mediated ChI excitation was retained and only reversed by inhibition of adenosine A2ARs. The Gi/o-preferring D2R agonist failed to increase ChI excitability, suggesting a possible switch in coupling of D2Rs to ß-arrestin, as seen previously in a DYT1 model. These data show that, whereas elevated extracellular acetylcholine levels are not always detected across these genetic models of human dystonia, the D2R-mediated paradoxical excitation of ChIs is shared and is caused by altered function of distinct G-protein-coupled receptors.SIGNIFICANCE STATEMENT Dystonia is a common and often disabling movement disorder. The usual medical treatment of dystonia is pharmacotherapy with nonselective antagonists of muscarinic acetylcholine receptors, which have many undesirable side effects. Development of new therapeutics is a top priority for dystonia research. The current findings, considered in context with our previous investigations, establish a role for cholinergic dysfunction across three mouse models of human genetic dystonia: DYT1, DYT6, and DYT25. The commonality of cholinergic dysfunction in these models arising from diverse molecular etiologies points the way to new approaches for cholinergic modulation that may be broadly applicable in dystonia.

Decreased number of striatal cholinergic interneurons and motor deficits in dopamine receptor 2-expressing-cell-specific Dyt1 conditional knockout mice.

DYT1 early-onset generalized torsion dystonia is a hereditary movement disorder characterized by abnormal postures and repeated movements. It is caused mainly by a heterozygous trinucleotide deletion in DYT1/TOR1A, coding for torsinA. The mutation may lead to a partial loss of torsinA function. Functional alterations of the basal ganglia circuits have been implicated in this disease. Striatal dopamine receptor 2 (D2R) levels are significantly decreased in DYT1 dystonia patients and in the animal models of DYT1 dystonia. D2R-expressing cells, such as the medium spiny neurons in the indirect pathway, striatal cholinergic interneurons, and dopaminergic neurons in the basal ganglia circuits, contribute to motor performance. However, the function of torsinA in these neurons and its contribution to the motor symptoms is not clear. Here, D2R-expressing-cell-specific Dyt1 conditional knockout (d2KO) mice were generated and in vivo effects of torsinA loss in the corresponding cells were examined. The Dyt1 d2KO mice showed significant reductions of striatal torsinA, acetylcholine metabolic enzymes, Tropomyosin receptor kinase A (TrkA), and cholinergic interneurons. The Dyt1 d2KO mice also showed significant reductions of striatal D2R dimers and tyrosine hydroxylase without significant alteration in striatal monoamine contents or the number of dopaminergic neurons in the substantia nigra. The Dyt1 d2KO male mice showed motor deficits in the accelerated rotarod and beam-walking tests without overt dystonic symptoms. Moreover, the Dyt1 d2KO male mice showed significant correlations between striatal monoamines and locomotion. The results suggest that torsinA in the D2R-expressing cells play a critical role in the development or survival of the striatal cholinergic interneurons, expression of striatal D2R mature form, and motor performance. Medical interventions to compensate for the loss of torsinA function in these neurons may affect the onset and symptoms of this disease.

Transneuronal Downregulation of the Premotor Cholinergic System After Corticospinal Tract Loss.

Injury to the supraspinal motor systems, especially the corticospinal tract, leads to movement impairments. In addition to direct disruption of descending motor pathways, spinal motor circuits that are distant to and not directly damaged by the lesion undergo remodeling that contributes significantly to the impairments. Knowing which spinal circuits are remodeled and the underlying mechanisms are critical for understanding the functional changes in the motor pathway and for developing repair strategies. Here, we target spinal premotor cholinergic interneurons (IN) that directly modulate motoneuron excitability via their cholinergic C-bouton terminals. Using a model of unilateral medullary corticospinal tract lesion in male rats, we found transneuronal downregulation of the premotor cholinergic pathway. Phagocytic microglial cells were upregulated in parallel with cholinergic pathway downregulation and both were blocked by minocycline, a microglia activation inhibitor. Additionally, we found a transient increase in interneuronal complement protein C1q expression that preceded cell loss. 3D reconstructions showed ongoing phagocytosis of C1q-expressing cholinergic INs by microglia 3 d after injury, which was complete by 10 d after injury. Unilateral motor cortex inactivation using the GABAA receptor agonist muscimol replicated the changes detected at 3 d after lesion, indicating activity dependence. The neuronal loss after the lesion was rescued by increasing spinal activity using cathodal trans-spinal direct current stimulation. Our finding of activity-dependent modulation of cholinergic premotor INs after CST injury provides the mechanistic insight that maintaining activity, possibly during a critical period, helps to protect distant motor circuits from further damage and, as a result, may improve motor functional recovery and rehabilitation.SIGNIFICANCE STATEMENT Supraspinal injury to the motor system disrupts descending motor pathways, leading to movement impairments. Whether and how intrinsic spinal circuits are remodeled after a brain injury is unclear. Using a rat model of unilateral corticospinal tract lesion in the medulla, we show activity-dependent, transneuronal downregulation of the spinal premotor cholinergic system, which is mediated by microglial phagocytosis, possibly involving a rapid and transient increase in neuronal C1q before neuronal loss. Spinal cord neuromodulation after injury to augment spinal activity rescued the premotor cholinergic system. Our findings provide the mechanistic insight that maintaining activity, possibly during an early critical period, could protect distant motor circuits from further damage mediated by microglia and interneuronal complement protein and improve motor functional outcomes.

Neostriatal GABAergic Interneurons Mediate Cholinergic Inhibition of Spiny Projection Neurons.

UNLABELLED: Synchronous optogenetic activation of striatal cholinergic interneurons ex vivo produces a disynaptic inhibition of spiny projection neurons composed of biophysically distinct GABAAfast and GABAAslow components. This has been shown to be due, at least in part, to activation of nicotinic receptors on GABAergic NPY-neurogliaform interneurons that monosynaptically inhibit striatal spiny projection neurons. Recently, it has been proposed that a significant proportion of this inhibition is actually mediated by activation of presynaptic nicotinic receptors on nigrostriatal terminals that evoke GABA release from the terminals of the dopaminergic nigrostriatal pathway. To disambiguate these the two mechanisms, we crossed mice in which channelrhodopsin is endogenously expressed in cholinergic neurons with Htr3a-Cre mice, in which Cre is selectively targeted to several populations of striatal GABAergic interneurons, including the striatal NPY-neurogliaform interneuron. Htr3a-Cre mice were then virally transduced to express halorhodopsin to allow activation of channelrhodopsin and halorhodopsin, individually or simultaneously. Thus we were able to optogenetically disconnect the interneuron-spiny projection neuron (SPN) cell circuit on a trial-by-trial basis. As expected, optogenetic activation of cholinergic interneurons produced inhibitory currents in SPNs. During simultaneous inhibition of GABAergic interneurons with halorhodopsin, we observed a large, sometimes near complete reduction in both fast and slow components of the cholinergic-evoked inhibition, and a delay in IPSC latency. This demonstrates that the majority of cholinergic-evoked striatal GABAergic inhibition is derived from GABAergic interneurons. These results also reinforce the notion that a semiautonomous circuit of striatal GABAergic interneurons is responsible for transmitting behaviorally relevant cholinergic signals to spiny projection neurons. SIGNIFICANCE STATEMENT: The circuitry between neurons of the striatum has been recently described to be far more complex than originally imagined. One example of this phenomenon is that striatal cholinergic interneurons have been shown to provide intrinsic nicotinic excitation of local GABAergic interneurons, which then inhibit the projection neurons of the striatum. As deficits of cholinergic interneurons are reported in patients with Tourette syndrome, the normal functions of these interneurons are of great interest. Whether this novel route of nicotinic input constitutes a major output of cholinergic interneurons remains unknown. The study addressed this question using excitatory and inhibitory optogenetic technology, so that cholinergic interneurons could be selectively activated and GABAergic interneurons selectively inhibited to determine the causal relationship in this circuit.

Role of Striatal Cholinergic Interneurons in Set-Shifting in the Rat.

The ability to change strategies in different contexts is a form of behavioral flexibility that is crucial for adaptive behavior. The striatum has been shown to contribute to certain forms of behavioral flexibility such as reversal learning. Here we report on the contribution of striatal cholinergic interneurons-a key element in the striatal neuronal circuit-to strategy set-shifting in which an attentional shift from one stimulus dimension to another is required. We made lesions of rat cholinergic interneurons in dorsomedial or ventral striatum using a specific immunotoxin and investigated the effects on set-shifting paradigms and on reversal learning. In shifting to a set that required attention to a previously irrelevant cue, lesions of dorsomedial striatum significantly increased the number of perseverative errors. In this condition, the number of never-reinforced errors was significantly decreased in both types of lesions. When shifting to a set that required attention to a novel cue, rats with ventral striatum lesions made more perseverative errors. Neither lesion impaired learning of the initial response strategy nor a subsequent switch to a new strategy when response choice was indicated by a previously relevant cue. Reversal learning was not affected. These results suggest that in set-shifting the striatal cholinergic interneurons play a fundamental role, which is dissociable between dorsomedial and ventral striatum depending on behavioral context. We propose a common mechanism in which cholinergic interneurons inhibit neurons representing the old strategy and enhance plasticity underlying exploration of a new rule.

Enhanced histamine H2 excitation of striatal cholinergic interneurons in L-DOPA-induced dyskinesia.

Levodopa is the most effective therapy for the motor deficits of Parkinson's disease (PD), but long term treatment leads to the development of L-DOPA-induced dyskinesia (LID). Our previous studies indicate enhanced excitability of striatal cholinergic interneurons (ChIs) in mice expressing LID and reduction of LID when ChIs are selectively ablated. Recent gene expression analysis indicates that stimulatory H2 histamine receptors are preferentially expressed on ChIs at high levels in the striatum, and we tested whether a change in H2 receptor function might contribute to the elevated excitability in LID. Using two different mouse models of PD (6-hydroxydopamine lesion and Pitx3(ak/ak) mutation), we chronically treated the animals with either vehicle or l-DOPA to induce dyskinesia. Electrophysiological recordings indicate that histamine H2 receptor-mediated excitation of striatal ChIs is enhanced in mice expressing LID. Additionally, H2 receptor blockade by systemic administration of famotidine decreases behavioral LID expression in dyskinetic animals. These findings suggest that ChIs undergo a pathological change in LID with respect to histaminergic neurotransmission. The hypercholinergic striatum associated with LID may be dampened by inhibition of H2 histaminergic neurotransmission. This study also provides a proof of principle of utilizing selective gene expression data for cell-type-specific modulation of neuronal activity.

The Orbitofrontal Cortex to Striatal Cholinergic Interneuron Circuit Controls Cognitive Flexibility Shaping Alcohol-Seeking Behavior.

BACKGROUND: A top-down neuronal circuit from the orbitofrontal cortex (OFC) to the dorsomedial striatum (DMS) appears to be critical for cognitive flexibility. However, how OFC projections to different types of neurons in the DMS control cognitive flexibility and contribute to substance seeking and use, which are relatively inflexible behaviors, remains unclear. METHODS: Mice were trained on two-bottle choice and operant alcohol self-administration procedures. The cognitive flexibility of the mice was tested through a place discrimination task. Electrophysiology and in vivo optogenetics were used to test the function of neural circuits in alcohol-seeking behavior. RESULTS: We depicted a connection from the OFC to striatal neurons and found that OFC afferents could elicit functional flexibility in striatal cholinergic interneurons (CINs). A mouse model of chronic alcohol consumption showed impaired cognitive flexibility and reduced burst-pause firing. The impairment of the OFC-DMS circuit resulted in a reduction in glutamatergic transmission in OFC-medium spiny neurons (MSNs) through a CIN-mediated pre-inhibition mechanism. Importantly, remodeling the OFC-DMS circuit by inducing LTP restored cognitive flexibility. Furthermore, CINs were responsible for the impact of remodeling of the OFC-DMS circuit on cognitive flexibility. This regulatory role of CINs preferentially facilitated the potentiation of glutamatergic transmission in D2 receptor-expressing medium spiny neurons (D2-MSNs) but not in D1-MSNs. Finally, activation of the OFC-CIN-D2-MSN circuit decreased alcohol-seeking behavior. CONCLUSIONS: Improving OFC-CIN circuit-mediated cognitive flexibility may provide a novel strategy for treating uncontrolled alcohol-seeking behavior.

Functional GnRH receptor signaling regulates striatal cholinergic neurons in neonatal but not in adult mice.

Reproduction in mammals is controlled by hypothalamic gonadotropin-releasing hormone (GnRH) neurons. Recent studies from our laboratory established that the basal ganglia of the human brain contain additional large populations of GnRH synthesizing neurons which are absent in adult mice. Such extrahypothalamic GnRH neurons mostly occur in the putamen where they correspond to subsets of the striatal cholinergic interneurons (ChINs) and express GnRHR autoreceptors. In an effort to establish a mouse model for functional studies of striatal GnRH/GnRHR signaling, we carried out electrophysiological experiments on acute brain slices from male transgenic mice. Using PN4-7 neonatal mice, half of striatal ChINs responded with transient hyperpolarization and decreased firing rate to 1.2 µM GnRH, whereas medium spiny projection neurons remained unaffected. GnRH acted on its specific receptor because no response was observed in the presence of the GnRHR antagonist Antide. Addition of the membrane-impermeable G protein-coupled receptor inhibitor GDP-ß-S to the internal electrode solution eliminated the effect of GnRH. Further, GnRH was able to inhibit ChINs in presence of tetrodotoxin which blocked action potential mediated events. Collectively, these data indicated that the receptor underlying the effects of GnRH in neonatal mice is localized within ChINs. GnRH responsiveness of ChINs was transient and entirely disappeared in adult mice. These results raise the possibility to use neonatal transgenic mice as a functional model to investigate the role of GnRH/GnRHR signaling discovered earlier in adult human ChINs.

Striatal parvalbumin interneurons are activated in a mouse model of cerebellar dystonia.

Dystonia is thought to arise from abnormalities in the motor loop of the basal ganglia; however, there is an ongoing debate regarding cerebellar involvement. We adopted an established cerebellar dystonia mouse model by injecting ouabain to examine the contribution of the cerebellum. Initially, we examined whether the entopeduncular nucleus (EPN), substantia nigra pars reticulata (SNr), globus pallidus externus (GPe) and striatal neurons were activated in the model. Next, we examined whether administration of a dopamine D1 receptor agonist and dopamine D2 receptor antagonist or selective ablation of striatal parvalbumin (PV, encoded by Pvalb)-expressing interneurons could modulate the involuntary movements of the mice. The cerebellar dystonia mice had a higher number of cells positive for c-fos (encoded by Fos) in the EPN, SNr and GPe, as well as a higher positive ratio of c-fos in striatal PV interneurons, than those in control mice. Furthermore, systemic administration of combined D1 receptor agonist and D2 receptor antagonist and selective ablation of striatal PV interneurons relieved the involuntary movements of the mice. Abnormalities in the motor loop of the basal ganglia could be crucially involved in cerebellar dystonia, and modulating PV interneurons might provide a novel treatment strategy.

Dopaminergic D2 receptor modulation of striatal cholinergic interneurons contributes to sequence learning.

Learning action sequences is necessary for normal daily activities. Medium spiny neurons (MSNs) in the dorsal striatum (dStr) encode action sequences through changes in firing at the start and/or stop of action sequences or sustained changes in firing throughout the sequence. Acetylcholine (ACh), released from cholinergic interneurons (ChIs), regulates striatal function by modulating MSN and interneuron excitability, dopamine and glutamate release, and synaptic plasticity. Cholinergic neurons in dStr pause their tonic firing during the performance of learned action sequences. Activation of dopamine type-2 receptors (D2Rs) on ChIs is one mechanism of ChI pausing. In this study we show that deleting D2Rs from ChIs by crossing D2-floxed with ChAT-Cre mice (D2Flox-ChATCre), which inhibits dopamine-mediated ChI pausing and leads to deficits in an operant action sequence task and lower breakpoints in a progressive ratio task. These data suggest that D2Flox-ChATCre mice have reduced motivation to work for sucrose reward, but show no generalized motor skill deficits. D2Flox-ChATCre mice perform similarly to controls in a simple reversal learning task, indicating normal behavioral flexibility, a cognitive function associated with ChIs. In vivo electrophysiological recordings show that D2Flox-ChatCre mice have deficits in sequence encoding, with fewer dStr MSNs encoding entire action sequences compared to controls. Thus, ChI D2R deletion appears to impair a neural substrate of action chunking. Virally replacing D2Rs in dStr ChIs in adult mice improves action sequence learning, but not the lower breakpoints, further suggesting that D2Rs on ChIs in the dStr are critical for sequence learning, but not for driving the motivational aspects of the task.

Nucleus accumbens local circuit for cue-dependent aversive learning.

Response to threatening environmental stimuli requires detection and encoding of important environmental features that dictate threat. Aversive events are highly salient, which promotes associative learning about stimuli that signal this threat. The nucleus accumbens is uniquely positioned to process this salient, aversive information and promote motivated output, through plasticity on the major projection neurons in the brain area. We describe a nucleus accumbens core local circuit whereby excitatory plasticity facilitates learning and recall of discrete aversive cues. We demonstrate that putative nucleus accumbens substance P release and long-term excitatory plasticity on dopamine 2 receptor-expressing projection neurons are required for cue-dependent fear learning. Additionally, we find that fear learning and recall is dependent on distinct projection neuron subtypes. Our work demonstrates a critical role for nucleus accumbens substance P in cue-dependent aversive learning.

Pathway-specific alterations in striatal excitability and cholinergic modulation in a SAPAP3 mouse model of compulsive motor behavior.

Deletion of the obsessive-compulsive disorder (OCD)-associated gene SAP90/PSD-95-associated protein 3 (Sapap3), which encodes a postsynaptic anchoring protein at corticostriatal synapses, causes OCD-like motor behaviors in mice. While corticostriatal synaptic dysfunction is central to this phenotype, the striatum efficiently adapts to pathological changes, often in ways that expand upon the original circuit impairment. Here, we show that SAPAP3 deletion causes non-synaptic and pathway-specific alterations in dorsolateral striatum circuit function. While somatic excitability was elevated in striatal projection neurons (SPNs), dendritic excitability was exclusively enhanced in direct pathway SPNs. Layered on top of this, cholinergic modulation was altered in opposing ways: striatal cholinergic interneuron density and evoked acetylcholine release were elevated, while basal muscarinic modulation of SPNs was reduced. These data describe how SAPAP3 deletion alters the striatal landscape upon which impaired corticostriatal inputs will act, offering a basis for how pathological synaptic integration and unbalanced striatal output underlying OCD-like behaviors may be shaped.

Binge alcohol drinking alters the differential control of cholinergic interneurons over nucleus accumbens D1 and D2 medium spiny neurons.

Animals studies support the notion that striatal cholinergic interneurons (ChIs) play a central role in basal ganglia function by regulating associative learning, reward processing, and motor control. In the nucleus accumbens (NAc), a brain region that mediates rewarding properties of substance abuse, acetylcholine regulates glutamatergic, dopaminergic, and GABAergic neurotransmission in naïve mice. However, it is unclear how ChIs orchestrate the control of these neurotransmitters/modulators to determine the synaptic excitability of medium spiny neurons (MSNs), the only projecting neurons that translate accumbens electrical activity into behavior. Also unknown is the impact of binge alcohol drinking on the regulation of dopamine D1- and D2 receptor-expressing MSNs (D1- and D2-MSNs, respectively) by ChIs. To investigate this question, we optogenetically stimulated ChIs while recording evoked and spontaneous excitatory postsynaptic currents (sEPSCs) in nucleus accumbens core D1- and D2-MSN of ChAT.ChR2.eYFPxDrd1.tdtomato mice. In alcohol-naïve mice, we found that stimulating NAc ChIs decreased sEPSCs frequency in both D1- and D2-MSNs, presumably through a presynaptic mechanism. Interestingly, ChI stimulation decreased MSN synaptic excitability through different mechanisms in D1- vs. D2-MSNs. While decrease of ChI-mediated sEPSCs frequency in D1-MSNs was mediated by dopamine, the same effect in D2-MSNs resulted from a direct control of glutamate release by ChIs. Interestingly, after 2 weeks of binge alcohol drinking, optogenetic stimulation of ChIs enhanced glutamate release in D1-MSNs, while its effect on D2-MSNs remained unchanged. Taken together, these data suggest that cholinergic interneurons could be a key target for regulation of NAc circuitry and for alcohol consumption.

Hippocampal-evoked inhibition of cholinergic interneurons in the nucleus accumbens.

Cholinergic interneurons (ChIs) in the nucleus accumbens (NAc) play a central role in motivated behaviors and associated disorders. However, while the activation of ChIs has been well studied in the dorsal striatum, little is known about how they are engaged in the NAc. Here, we find that the ventral hippocampus (vHPC) and the paraventricular nucleus of the thalamus (PVT) are the main excitatory inputs to ChIs in the NAc medial shell. While the PVT activates ChIs, the vHPC evokes a pronounced pause in firing through prominent feedforward inhibition. In contrast to the dorsal striatum, this inhibition reflects strong connections onto ChIs from local parvalbumin interneurons. Our results reveal the mechanisms by which different long-range inputs engage ChIs, highlighting fundamental differences in local connectivity across the striatum.

Electrophysiological characterization of the striatal cholinergic interneurons in Dyt1 ΔGAG knock-in mice.

DYT1 dystonia is an inherited early-onset movement disorder characterized by sustained muscle contractions causing twisting, repetitive movements, and abnormal postures. Most DYT1 patients have a heterozygous trinucleotide GAG deletion mutation (ΔGAG) in DYT1/TOR1A, coding for torsinA. Dyt1 heterozygous ΔGAG knock-in (KI) mice show motor deficits and reduced striatal dopamine receptor 2 (D2R). Striatal cholinergic interneurons (ChIs) are essential in regulating striatal motor circuits. Multiple dystonia rodent models, including KI mice, show altered ChI firing and modulation. However, due to the errors in assigning KI mice, it is essential to replicate these findings in genetically confirmed KI mice. Here, we found irregular and decreased spontaneous firing frequency in the acute brain slices from Dyt1 KI mice. Quinpirole, a D2R agonist, showed less inhibitory effect on the spontaneous ChI firing in Dyt1 KI mice, suggesting decreased D2R function on the striatal ChIs. On the other hand, a muscarinic receptor agonist, muscarine, inhibited the ChI firing in both wild-type (WT) and Dyt1 KI mice. Trihexyphenidyl, a muscarinic acetylcholine receptor M1 antagonist, had no significant effect on the firing. Moreover, the resting membrane property and functions of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, µ-opioid receptors, and large-conductance calcium-activated potassium (BK) channels were unaffected in Dyt1 KI mice. The results suggest that the irregular and low-frequency firing and decreased D2R function are the main alterations of striatal ChIs in Dyt1 KI mice. These results appear consistent with the reduced dopamine release and high striatal acetylcholine tone in the previous reports.