Striatal Cholinergic Interneurons Control Physical Nicotine Withdrawal via Muscarinic Receptor Signaling.

Striatal cholinergic interneurons (ChIs) provide acetylcholine tone to the striatum and govern motor functions. Nicotine withdrawal elicits physical symptoms that dysregulate motor behavior. Here, the role of striatal ChIs in physical nicotine withdrawal is investigated. Mice under RNAi-dependent genetic inhibition of striatal ChIs (ChIGI) by suppressing the sodium channel subunit NaV1.1, lessening action potential generation and activity-dependent acetylcholine release is first generated. ChIGI markedly reduced the somatic signs of nicotine withdrawal without affecting other nicotine-dependent or striatum-associated behaviors. Multielectrode array (MEA) recording revealed that ChIGI reversed ex vivo nicotine-induced alterations in the number of neural population spikes in the dorsal striatum. Notably, the drug repurposing strategy revealed that a clinically-approved antimuscarinic drug, procyclidine, fully mimicked the therapeutic electrophysiological effects of ChIGI. Furthermore, both ChIGI and procyclidine prevented the nicotine withdrawal-induced reduction in striatal dopamine release in vivo. Lastly, therapeutic intervention with procyclidine dose-dependently diminished the physical signs of nicotine withdrawal. The data demonstrated that the striatal ChIs are a critical substrate of physical nicotine withdrawal and that muscarinic antagonism holds therapeutic potential against nicotine withdrawal.

A mismatch between striatal cholinergic pauses and dopaminergic reward prediction errors.

Striatal acetylcholine and dopamine critically regulate movement, motivation, and reward-related learning. Pauses in cholinergic interneuron (CIN) firing are thought to coincide with dopamine pulses encoding reward prediction errors (RPE) to jointly enable synaptic plasticity. Here, we examine the firing of identified CINs during reward-guided decision-making in freely moving rats and compare this firing to dopamine release. Relationships between CINs, dopamine, and behavior varied strongly by subregion. In the dorsal-lateral striatum, a Go! cue evoked burst-pause CIN spiking, followed by a brief dopamine pulse that was unrelated to RPE. In the dorsal-medial striatum, this cue evoked only a CIN pause, that was curtailed by a movement-selective rebound in firing. Finally, in the ventral striatum, a reward cue evoked RPE-coding increases in both dopamine and CIN firing, without a consistent pause. Our results demonstrate a spatial and temporal dissociation between CIN pauses and dopamine RPE signals and will inform future models of striatal information processing under both normal and pathological conditions.

Striatal cholinergic transmission in an inducible transgenic mouse model of paroxysmal non-kinesiogenic dyskinesia.

Altered interaction between striatonigral dopaminergic (DA) inputs and local acetylcholine (ACh) in striatum has long been hypothesized to play a central role in the pathophysiology of dystonia and dyskinesia. Indeed, previous research using several genetic mouse models of human isolated dystonia identified a shared endophenotype with paradoxical excitation of striatal cholinergic interneuron (ChIs) activity in response to activation of dopamine D2 receptors (D2R). These mouse models lack a dystonic motor phenotype, which leaves a critical gap in comprehending the role of DA and ACh transmission in the manifestations of dystonia. To tackle this question, we used a combination of ex vivo slice physiology and in vivo monitoring of striatal ACh dynamics in the inducible, phenotypically penetrant, transgenic mouse model of paroxysmal non-kinesiogenic dyskinesia (PNKD), an animal with both dystonic and dyskinetic features. We found that, similarly to genetic models of isolated dystonia, the PNKD mouse displays D2R-induced paradoxical excitation of ChI firing in ex vivo striatal brain slices. In vivo, caffeine triggers dystonic symptoms while reversing the D2R-mediated excitation of ChIs and desynchronizing ACh release in PNKD mice. In WT littermate controls, caffeine stimulates spontaneous locomotion through a similar but reversed mechanism involving an excitatory switch of the D2R control of ChI activity, associated with enhanced synchronization of ACh release. These observations suggest that the "paradoxical excitation" of cholinergic interneurons described in isolated dystonia models could represent a compensatory or protective mechanism that prevents manifestation of movement abnormalities and that phenotypic dystonia is possible only when this is absent. These findings also suggest that D2Rs may play an important role in synchronizing the ChI network leading to rhythmic ACh release during heightened movement states. Dysfunction of this interaction and corresponding desynchrony of ACh release may contribute to aberrant movements.

Regional heterogeneity in the membrane properties of mouse striatal neurons.

The cytoarchitecture of the striatum is remarkably homogeneous, in contrast to the regional variation in striatal functions. Whether differences in the intrinsic membrane properties of striatal neurons contribute to regional heterogeneity has not been addressed systematically. We made recordings throughout the young adult mouse striatum under identical conditions, with synaptic input blocked, from four major striatal neuron types, namely, the two subtypes of spiny projection neurons (SPNs), cholinergic interneurons (ChIs), and fast-spiking GABAergic interneurons (FSIs), sampling at least 100 cells per cell type. Regional variation manifested across all cell types. All cell types in the nucleus accumbens (NAc) shell had higher input impedance and increased excitability. Cells in the NAc core were differentiated from the caudate-putamen (CPu) for both SPN subtypes by smaller action potentials and increased excitability. Similarity between the two SPN subtypes showed regional variation, differing more in the NAc than in the CPu. So, in the Str, both the intrinsic properties of interneurons and projection neurons are regionally heterogeneous, with the greatest difference between the NAc and CPu; greater excitability of NAc shell neurons may make the region more susceptible to activity-dependent plasticity.

Elevated antibody binding to striatal cholinergic interneurons in patients with pediatric acute-onset neuropsychiatric syndrome.

Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) is characterized by the abrupt onset of significant obsessive-compulsive symptoms (OCS) and/or severe food restriction, together with other neuropsychiatric manifestations. An autoimmune pathogenesis triggered by infection has been proposed for at least a subset of PANS. The older diagnosis of Pediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcus (PANDAS) describes rapid onset of OCD and/or tics associated with infection with Group A Streptococcus. The pathophysiology of PANS and PANDAS remains incompletely understood. We recently found serum antibodies from children with rigorously defined PANDAS to selectively bind to cholinergic interneurons (CINs) in the striatum. Here we examine this binding in children with relapsing and remitting PANS, a more heterogeneous condition, collected in a distinct clinical context from those examined in our previous work, from children with a clinical history of Streptococcus infection. IgG from PANS cases showed elevated binding to striatal CINs in both mouse and human brain. Patient plasma collected during symptom flare decreased a molecular marker of CIN activity, phospho-riboprotein S6, in ex vivo brain slices; control plasma did not. Neither elevated antibody binding to CINs nor diminished CIN activity was seen with plasma collected from the same children during remission. These findings replicate what we have seen previously in PANDAS and support the hypothesis that at least a subset of PANS cases have a neuroimmune pathogenesis. Given the critical role of CINs in modulating basal ganglia function, these findings confirm striatal CINs as a locus of interest in the pathophysiology of both PANS and PANDAS.

Cholinergic Interneurons in the Accumbal Shell Region Regulate Binge Alcohol Self-Administration in Mice: An In Vivo Calcium Imaging Study.

Recently, we and others have shown that manipulating the activity of cholinergic interneurons (CIN) present in the NAc can modulate binge alcohol consumption. The present study is designed to examine the relationship between binge alcohol consumption and the activity of the CIN in real time by using an in vivo microendoscopic technique. We hypothesized that mice exposed to Drinking in the Dark (DID)-a recognized mouse model for binge drinking-would exhibit increased activity in the accumbal shell region (NAcSh). To test this hypothesis, male mice expressing Cre-recombinase in the cholinergic neurons were exposed to binge alcohol consumption (alcohol group), employing the DID method, and utilized in vivo calcium imaging to observe CIN activity in real time during alcohol consumption. The control (sucrose) group was exposed to 10% (w/v) sucrose. As compared to sucrose, mice in the alcohol group displayed a significant increase in the frequency and amplitude of discharge activity, which was measured using calcium transients in the CIN present in the NAcSh. In summary, our findings suggest that the activity of CIN in the NAcSh plays a crucial role in alcohol self-administration. These results emphasize the potential significance of targeting CIN activity as a therapeutic approach for addressing AUD.

Cholinergic interneurons in the dorsal striatum play an important role in the acquisition of duration memory.

The present study aimed to examine the effect of cholinergic interneuron lesions in the dorsal striatum on duration-memory formation. Cholinergic interneurons in the dorsal striatum may be involved in the formation of duration memory since they are among the main inputs to the dorsal striatal muscarinic acetylcholine-1 receptors, which play a role in the consolidation of duration memory. Rats were sufficiently trained using a peak-interval 20 s procedure and then infused with anti-choline acetyltransferase-saporin into the dorsal striatum to cause selective ablation of cholinergic interneurons. To make the rats acquire new duration-memories, we trained them with a peak interval 40 s after lesion. Before lesion, the peak times (an index of duration memory) for sham-lesioned and lesioned groups were similar at approximately 20 s. In the peak interval 40 s session, the peak times for the sham-lesioned and lesioned groups were approximately 30 and 20 s, respectively. After additional peak interval 40 s sessions, the peak times of both groups were shifted to approximately 40 s. Those results suggest that the cholinergic interneuron lesion delayed new duration-memory acquisition. Subsequent experiments showed that cholinergic interneuron lesions did not retard the shift of peak time to the original target time (20 s). Following experiment without changing the target time after lesion showed that cholinergic interneuron lesions did not change their peak times. Our findings suggest that cholinergic interneurons in the dorsal striatum are involved in new duration-memory acquisition but not in the utilization of already acquired duration memory and interval timing.

Cholinergic interneurons in the shell region of the nucleus accumbens regulate binge alcohol consumption: A chemogenetic and genetic lesion study.

BACKGROUND: Binge drinking, characterized by heavy episodic alcohol consumption, poses significant health hazards and increases the likelihood of developing an alcohol use disorder (AUD). Given the growing prevalence of this behavior and its negative consequences, there is a need to explore novel therapeutic targets. Accumulating evidence suggests that cholinergic interneurons (CIN) within the shell region of the nucleus accumbens (NAcSh) play a critical role in reward and addiction. However, their specific involvement in binge alcohol administration remains unclear. We hypothesized that CIN in the NAcSh regulates binge alcohol consumption. METHODS: To test this hypothesis, we used male ChAT-cre mice expressing Cre-recombinase in cholinergic neurons. We performed chemogenetic manipulation using Designer Receptor Exclusively Activated by Designer Drugs (DREADD) to examine the activity, and genetic ablation of CIN in the NAcSh to examine the amount of alcohol consumed in mice exposed to binge alcohol consumption using the 4-Days Drinking-in-Dark (DID) paradigm. The impact of CIN manipulations in the NAcSh on sucrose self-administration was used to control for taste and caloric effects. Additionally, in a separate group of mice, c-Fos immunofluorescence was employed to verify chemogenetic activation or inhibition. Histological and immunohistochemical techniques were used to verify microinfusion sites, DREADD expression in CINs, and genetic ablation. RESULTS: We found that, while chemogenetic activation of CIN in the NAcSh caused a significant increase in alcohol consumption, chemogenetic inhibition or genetic ablation of CIN significantly reduced the amount of alcohol consumed without affecting sucrose self-administration. The chemogenetic inhibition caused a significant reduction, whereas activation caused a significant increase, in the number of c-Fos-labeled CIN in the NAcSh. CONCLUSIONS: Our findings highlight the crucial involvement of CIN in the NAcSh in modulating binge alcohol consumption, suggesting that targeting these neurons could serve to modify alcohol-related behaviors.

The Integration of Top-down and Bottom-up Inputs to the Striatal Cholinergic Interneurons.

BACKGROUND: Cholinergic interneurons (ChIs) are important for learning and memory. They exhibit a multiphasic excitation-pause-rebound response to reward or sensory cues indicating a reward, believed to gate dopamine-dependent learning. Although ChIs receive extensive top-down inputs from the cortex and bottom-up inputs from the thalamus and midbrain, it is unclear which inputs are involved in the development of ChI multiphasic activity. METHODS: We used a single-unit recording of putative ChIs (pChIs) in response to cortical and visual stimulation to investigate how top-down and bottom-up inputs regulate the firing pattern of ChIs. RESULTS: We demonstrated that cortical stimulation strongly regulates pChIs, with the maximum firing rate occurring at the peak of the inverted local field potential (iLFP), reflecting maximum cortical stimulation. Pauses in pChIs occurred during the descending phase of iLFP, indicating withdrawal of excitatory cortical input. Visual stimulation induced long pauses in pChIs, but it is unlikely that bottom- up inputs alone induce pauses in behaving animals. Also, the firing pattern of ChIs triggered by visual stimulation did not correlate with the iLFP as it did after cortical stimulation. Top-down and bottom-up inputs independently regulate the firing pattern of ChIs with similar efficacy but notably produce a well-defined pause in ChI firing. CONCLUSION: This study provides in vivo evidence that the multiphasic ChI response may require both top-down and bottom-up inputs. The findings suggest that the firing pattern of ChIs correlated to the iLFP might be a useful tool for estimating the degree of contribution of top-down and bottom-up inputs in regulating the firing activity of ChIs.

Loss-of-function of GNAL dystonia gene impairs striatal dopamine receptors-mediated adenylyl cyclase/ cyclic AMP signaling pathway.

Loss-of-function mutations in the GNAL gene are responsible for DYT-GNAL dystonia. However, how GNAL mutations contribute to synaptic dysfunction is still unclear. The GNAL gene encodes the Gαolf protein, an isoform of stimulatory Gαs enriched in the striatum, with a key role in the regulation of cAMP signaling. Here, we used a combined biochemical and electrophysiological approach to study GPCR-mediated AC-cAMP cascade in the striatum of the heterozygous GNAL (GNAL+/-) rat model. We first analyzed adenosine type 2 (A2AR), and dopamine type 1 (D1R) receptors, which are directly coupled to Gαolf, and observed that the total levels of A2AR were increased, whereas D1R level was unaltered in GNAL+/- rats. In addition, the striatal isoform of adenylyl cyclase (AC5) was reduced, despite unaltered basal cAMP levels. Notably, the protein expression level of dopamine type 2 receptor (D2R), that inhibits the AC5-cAMP signaling pathway, was also reduced, similar to what observed in different DYT-TOR1A dystonia models. Accordingly, in the GNAL+/- rat striatum we found altered levels of the D2R regulatory proteins, RGS9-2, spinophilin, Gß5 and ß-arrestin2, suggesting a downregulation of D2R signaling cascade. Additionally, by analyzing the responses of striatal cholinergic interneurons to D2R activation, we found that the receptor-mediated inhibitory effect is significantly attenuated in GNAL+/- interneurons. Altogether, our findings demonstrate a profound alteration in the A2AR/D2R-AC-cAMP cascade in the striatum of the rat DYT-GNAL dystonia model, and provide a plausible explanation for our previous findings on the loss of dopamine D2R-dependent corticostriatal long-term depression.

Regulation of ethanol-mediated dopamine elevation by glycine receptors located on cholinergic interneurons in the nucleus accumbens.

Alcohol use disorder is one of the major psychiatric disorders worldwide, and there are many factors and effects contributing to the disorder, for example, the experience of ethanol reward. The rewarding and reinforcing properties of ethanol have been linked to activation of the mesolimbic dopamine system, an effect that appears to involve glycine receptors (GlyRs) in the nucleus accumbens. On which neuronal subtypes these receptors are located is, however, not known. The aim of this study was to explore the role of GlyRs on cholinergic interneurons (CIN) in sustaining extracellular dopamine levels and in ethanol-induced dopamine release. To this end, CIN were ablated by anti-choline acetyltransferase-saporin administered locally in the nucleus accumbens of male Wistar rats. Changes in dopamine levels induced by ablation, ethanol and/or a GlyR antagonist were monitored using in vivo microdialysis. The GlyRs antagonist strychnine depressed extracellular dopamine in a similar manner independent on local ablation, suggesting that GlyRs on CIN are not important for sustaining the extracellular dopamine tone. However, a low concentration of strychnine hampered ethanol-induced dopamine release in sham-treated animals, whilst no reduction was seen in ablated animals, suggesting that GlyRs located on CIN are involved in ethanol-induced dopamine release. Further, in ablated rats, ethanol-induced increases of the extracellular levels of the GlyR agonists glycine and taurine were attenuated. In conclusion, this study suggests that CIN are not important for GlyR-mediated regulation of basal dopamine output, but that CIN ablation blunts the ethanol-induced dopamine release, putatively by reducing the release of GlyR agonists.

Dopaminergic Input Regulates the Sensitivity of Indirect Pathway Striatal Spiny Neurons to Brain-Derived Neurotrophic Factor.

Motor dysfunction in Parkinson's disease (PD) is closely linked to the dopaminergic depletion of striatal neurons and altered synaptic plasticity at corticostriatal synapses. Dopamine receptor D1 (DRD1) stimulation is a crucial step in the formation of long-term potentiation (LTP), whereas dopamine receptor D2 (DRD2) stimulation is needed for the formation of long-term depression (LTD) in striatal spiny projection neurons (SPNs). Tropomyosin receptor kinase B (TrkB) and its ligand brain-derived neurotrophic factor (BDNF) are centrally involved in plasticity regulation at the corticostriatal synapses. DRD1 activation enhances TrkB's sensitivity for BDNF in direct pathway spiny projection neurons (dSPNs). In this study, we showed that the activation of DRD2 in cultured striatal indirect pathway spiny projection neurons (iSPNs) and cholinergic interneurons causes the retraction of TrkB from the plasma membrane. This provides an explanation for the opposing synaptic plasticity changes observed upon DRD1 or DRD2 stimulation. In addition, TrkB was found within intracellular structures in dSPNs and iSPNs from Pitx3-/- mice, a genetic model of PD with early onset dopaminergic depletion in the dorsolateral striatum (DLS). This dysregulated BDNF/TrkB signaling might contribute to the pathophysiology of direct and indirect pathway striatal projection neurons in PD.

Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS): Myth or Reality? The State of the Art on a Controversial Disease.

Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS) syndrome is one of the most controversial diseases in pediatric rheumatology. Despite first being described more than 25 years ago as the sudden and rapid onset of obsessive-compulsive disorder (OCD) and/or tic disorder symptoms as complications of a Group A beta-hemolytic Streptococcus (GAS) infection, precise epidemiological data are still lacking, and there are no strong recommendations for its treatment. Recent advances in the comprehension of PANDAS pathophysiology are largely attributable to animal model studies and the understanding of the roles of Ca++/calmodulin-dependent protein kinase (CaM kinase) II, disrupted dopamine release in the basal ganglia, and striatal cholinergic interneurons. The diagnosis of PANDAS should be made after an exclusion process and should include prepubescent children with a sudden onset of OCD and/or a tic disorder, with a relapsing/remitting disease course, a clear temporal association between GAS infection and onset or exacerbation of symptoms, and the association with other neurological abnormalities such as motoric hyperactivity and choreiform movements. Antibiotic medications are the primary therapeutic modality. Nonetheless, there is a paucity of randomized studies and validated data, resulting in a scarcity of solid recommendations.

Acetylcholine release from striatal cholinergic interneurons is controlled differently depending on the firing pattern.

How is the quantal size in neurotransmitter release adjusted for various firing levels? We explored the possible mechanisms that regulate acetylcholine (ACh) release from cholinergic interneurons using an ultra-mini superfusion system. After preloading [3 H]ACh in rat striatal cholinergic interneurons, the release was elicited by electrical stimulation under a condition in which presynaptic cholinergic and dopaminergic feedback was inhibited. [3 H]ACh release was reproducible at intervals of more than 10 min; shorter intervals resulted in reduced levels of ACh release. Upon persistent stimulation for 10 min, ACh release transiently increased, before gradually decreasing. Vesamicol, an inhibitor of the vesicular ACh transporter (VAChT), had no effect on the release induced by the first single pulse, but it reduced the release caused by subsequent pulses. Vesamicol also reduced the [3 H]ACh release evoked by multiple pulses, and the inhibition was enhanced by repetitive stimulation. The decreasing phase of [3 H]ACh release during persistent stimulation was accelerated by vesamicol treatment. Thus, it is likely that releasable ACh was slowly compensated for via VAChT during and after stimulation, changing the vesicular ACh content. In addition, ACh release per pulse decreased under high-frequency stimulation. The present results suggest that ACh release from striatal cholinergic interneurons may be adjusted by changes in the quantal size due to slow replenishment via VAChT, and by a reduction in release probability upon high-frequency stimulation. These two distinct processes likely enable the fine tuning of neurotransmission and neuroprotection/limitation against excessive output and have important physiological roles in the brain.

The dopamine neuron synaptic map in the striatum.

Dopamine neurons project to the striatum to control movement, cognition, and motivation via slower volume transmission as well as faster dopamine, glutamate, and GABA synaptic actions capable of conveying the temporal information in dopamine neuron firing. To define the scope of these synaptic actions, recordings of dopamine-neuron-evoked synaptic currents were made in four major striatal neuron types, spanning the entire striatum. This revealed that inhibitory postsynaptic currents are widespread, while excitatory postsynaptic currents are localized to the medial nucleus accumbens and the anterolateral-dorsal striatum, and that all synaptic actions are weak in the posterior striatum. Synaptic actions in cholinergic interneurons are the strongest, variably mediating inhibition throughout the striatum and excitation in the medial accumbens, capable of controlling their activity. This mapping shows that dopamine neuron synaptic actions extend throughout the striatum, preferentially target cholinergic interneurons, and define distinct striatal subregions.

Role of α6-Nicotinic Receptors in Alcohol-Induced GABAergic Synaptic Transmission and Plasticity to Cholinergic Interneurons in the Nucleus Accumbens.

The prevailing view is that enhancement of dopamine (DA) transmission in the mesolimbic system, consisting of DA neurons in the ventral tegmental area (VTA) that project to the nucleus accumbens (NAc), underlies the reward properties of ethanol (EtOH) and nicotine (NIC). We have shown previously that EtOH and NIC modulation of DA release in the NAc is mediated by α6-containing nicotinic acetylcholine receptors (α6*-nAChRs), that α6*-nAChRs mediate low-dose EtOH effects on VTA GABA neurons and EtOH preference, and that α6*-nAChRs may be a molecular target for low-dose EtOH. However, the most sensitive target for reward-relevant EtOH modulation of mesolimbic DA transmission and the involvement of α6*-nAChRs in the mesolimbic DA reward system remains to be elucidated. The aim of this study was to evaluate EtOH effects on GABAergic modulation of VTA GABA neurons and VTA GABAergic input to cholinergic interneurons (CINs) in the NAc. Low-dose EtOH enhanced GABAergic input to VTA GABA neurons that was blocked by knockdown of α6*-nAChRs. Knockdown was achieved either by α6-miRNA injected into the VTA of VGAT-Cre/GAD67-GFP mice or by superfusion of the α-conotoxin MII[H9A;L15A] (MII). Superfusion of MII blocked EtOH inhibition of mIPSCs in NAc CINs. Concomitantly, EtOH enhanced CIN firing rate, which was blocked by knockdown of α6*-nAChRs with α6-miRNA injected into the VTA of VGAT-Cre/GAD67-GFP mice. The firing rate of CINs was not enhanced by EtOH in EtOH-dependent mice, and low-frequency stimulation (LFS; 1 Hz, 240 pulses) caused inhibitory long-term depression at this synapse (VTA-NAc CIN-iLTD) which was blocked by knockdown of α6*-nAChR and MII. Ethanol inhibition of CIN-mediated evoked DA release in the NAc was blocked by MII. Taken together, these findings suggest that α6*-nAChRs in the VTA-NAc pathway are sensitive to low-dose EtOH and play a role in plasticity associated with chronic EtOH.

Accumbal Histamine Signaling Engages Discrete Interneuron Microcircuits.

BACKGROUND: Central histamine (HA) signaling modulates diverse cortical and subcortical circuits throughout the brain, including the nucleus accumbens (NAc). The NAc, a key striatal subregion directing reward-related behavior, expresses diverse HA receptor subtypes that elicit cellular and synaptic plasticity. However, the neuromodulatory capacity of HA within interneuron microcircuits in the NAc remains unknown. METHODS: We combined electrophysiology, pharmacology, voltammetry, and optogenetics in male transgenic reporter mice to determine how HA influences microcircuit motifs controlled by parvalbumin-expressing fast-spiking interneurons (PV-INs) and tonically active cholinergic interneurons (CINs) in the NAc shell. RESULTS: HA enhanced CIN output through an H2 receptor (H2R)-dependent effector pathway requiring Ca2+-activated small-conductance K+ channels, with a small but discernible contribution from H1Rs and synaptic H3Rs. While PV-IN excitability was unaffected by HA, presynaptic H3Rs decreased feedforward drive onto PV-INs via AC-cAMP-PKA (adenylyl cyclase-cyclic adenosine monophosphate-protein kinase A) signaling. H3R-dependent plasticity was differentially expressed at mediodorsal thalamus and prefrontal cortex synapses onto PV-INs, with mediodorsal thalamus synapses undergoing HA-induced long-term depression. These effects triggered downstream shifts in PV-IN- and CIN-controlled microcircuits, including near-complete collapse of mediodorsal thalamus-evoked feedforward inhibition and increased mesoaccumbens dopamine release. CONCLUSIONS: HA targets H1R, H2R, and H3Rs in the NAc shell to engage synapse- and cell type-specific mechanisms that bidirectionally regulate PV-IN and CIN microcircuit activity. These findings extend the current conceptual framework of HA signaling and offer critical insight into the modulatory potential of HA in the brain.

Synaptic determinants of cholinergic interneurons hyperactivity during parkinsonism.

Parkinson's disease is a neurodegenerative ailment generated by the loss of dopamine in the basal ganglia, mainly in the striatum. The disease courses with increased striatal levels of acetylcholine, disrupting the balance among these modulatory transmitters. These modifications disturb the excitatory and inhibitory balance in the striatal circuitry, as reflected in the activity of projection striatal neurons. In addition, changes in the firing pattern of striatal tonically active interneurons during the disease, including cholinergic interneurons (CINs), are being searched. Dopamine-depleted striatal circuits exhibit pathological hyperactivity as compared to controls. One aim of this study was to show how striatal CINs contribute to this hyperactivity. A second aim was to show the contribution of extrinsic synaptic inputs to striatal CINs hyperactivity. Electrophysiological and calcium imaging recordings in Cre-mice allowed us to evaluate the activity of dozens of identified CINs with single-cell resolution in ex vivo brain slices. CINs show hyperactivity with bursts and silences in the dopamine-depleted striatum. We confirmed that the intrinsic differences between the activity of control and dopamine-depleted CINs are one source of their hyperactivity. We also show that a great part of this hyperactivity and firing pattern change is a product of extrinsic synaptic inputs, targeting CINs. Both glutamatergic and GABAergic inputs are essential to sustain hyperactivity. In addition, cholinergic transmission through nicotinic receptors also participates, suggesting that the joint activity of CINs drives the phenomenon; since striatal CINs express nicotinic receptors, not expressed in striatal projection neurons. Therefore, CINs hyperactivity is the result of changes in intrinsic properties and excitatory and inhibitory inputs, in addition to the modification of local circuitry due to cholinergic nicotinic transmission. We conclude that CINs are the main drivers of the pathological hyperactivity present in the striatum that is depleted of dopamine, and this is, in part, a result of extrinsic synaptic inputs. These results show that CINs may be a main therapeutic target to treat Parkinson's disease by intervening in their synaptic inputs.

Nanoscopic distribution of VAChT and VGLUT3 in striatal cholinergic varicosities suggests colocalization and segregation of the two transporters in synaptic vesicles.

Striatal cholinergic interneurons (CINs) use acetylcholine (ACh) and glutamate (Glut) to regulate the striatal network since they express vesicular transporters for ACh (VAChT) and Glut (VGLUT3). However, whether ACh and Glut are released simultaneously and/or independently from cholinergic varicosities is an open question. The answer to that question requires the multichannel detection of vesicular transporters at the level of single synaptic vesicle (SV). Here, we used super-resolution STimulated Emission Depletion microscopy (STED) to characterize and quantify the distribution of VAChT and VGLUT3 in CINs SVs. Nearest-neighbor distances analysis between VAChT and VGLUT3-immunofluorescent spots revealed that 34% of CINs SVs contain both VAChT and VGLUT3. In addition, 40% of SVs expressed only VAChT while 26% of SVs contain only VGLUT3. These results suggest that SVs from CINs have the potential to store simultaneously or independently ACh and/or Glut. Overall, these morphological findings support the notion that CINs varicosities can signal with either ACh or Glut or both with an unexpected level of complexity.