Oxolane Ammonium Salts (Muscarine-Like)-Synthesis and Microbiological Activity.

Commercially available 2-deoxy-D-ribose was used to synthesize the appropriate oxolane derivative-(2R,3S)-2-(hydroxymethyl)oxolan-3-ol-by reduction and dehydration/cyclization in an acidic aqueous solution. Its monotosyl derivative, as a result of the quaternization reaction, allowed us to obtain eight new muscarine-type derivatives containing a quaternary nitrogen atom and a hydroxyl group linked to the oxolane ring. Their structure was fully confirmed by the results of NMR, MS and IR analyses. The crystal structure of the pyridinium derivative showed a high similarity of the conformation of the oxolane ring to previously published crystal structures of muscarine. Two reference strains of Gram-negative bacteria (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853), two reference strains of Gram-positive staphylococci (Staphylococcus aureus ATCC 25923 and Staphylococcus aureus ATCC 29213) and four reference strains of pathogenic yeasts of the genus Candida spp. (Candida albicans SC5314, Candida glabrata DSM 11226, Candida krusei DSM 6128 and Candida parapsilosis DSM 5784) were selected for the evaluation of the antimicrobial potential of the synthesized compounds. The derivative containing the longest (decyl) chain attached to the quaternary nitrogen atom turned out to be the most active.

The Fatal Mushroom Neurotoxin Muscarine is Released from a Harmless Phosphorylated Precursor upon Cellular Injury.

l-(+)-Muscarine (1)-producing mushrooms pose a severe threat to human health as ingestion can result in circulatory collapse or even death. However, their metabolic profile is surprisingly poorly understood, including knowledge of poison release and potentially toxic congeners. In the mycelium of the 1-producing fool's funnel mushroom Clitocybe rivulosa, we identified 4'-phosphomuscarine (2) as the major natural product. Its structure was elucidated by high-resolution mass spectrometry, nuclear magnetic resonance spectroscopy and by comparison with a synthesized reference. We also detected this previously overlooked phosphorylated compound in the fiber cap mushrooms Pseudosperma spectrale and Inocybe nitidiuscula. Studies on the activation of the muscarinic acetylcholine receptor M3 indicate only weak affinity of 2 to this target. Furthermore, we present biological evidence that muscaridine (3), a quaternary amine congener related to and co-occurring with 1, does not activate the muscarinic acetylcholine receptor M3 on human embryonic kidney cells. Our work provides important insight into the metabolic profile and the pharmacology of some of the most poisonous mushrooms. As the harmless 2 can liberate the potentially fatal 1 by unspecific enzymatic ester cleavage, these results are highly relevant for emergency medicine to estimate the true toxicity of these mushrooms.

M4 muscarinic receptors mediate acetylcholine-induced suppressant effects on the cough reflex in the caudal nucleus tractus solitarii of the rabbit.

It has been shown that muscarinic acetylcholine receptors (mAChRs) located within the caudal nucleus tractus solitarii (cNTS) mediate a cholinergic inhibitory control mechanism of the cough reflex. Thus, identification of the involved mAChR subtypes could be of considerable interest for novel therapeutic strategies. In pentobarbital sodium-anesthetized, spontaneously breathing rabbits we investigated the contribution of different mAChR subtypes in the modulation of mechanically and chemically induced cough reflex. Bilateral microinjections of 1 mM muscarine into the cNTS increased respiratory frequency and decreased expiratory activity even to complete suppression. Interestingly, muscarine induced strong cough-suppressant effects up to the complete abolition of the reflex. Microinjections of specific mAChR subtype antagonists (M1-M5) into the cNTS were performed. Only microinjections of the M4 antagonist tropicamide (1 mM) prevented muscarine-induced changes in both respiratory activity and cough reflex. The results are discussed in light of the notion that cough involves the activation of the nociceptive system. They also suggest that M4 receptor agonists may have an important role in cough downregulation within the cNTS.

Amitriptyline inhibits bronchoconstriction and directly promotes dilatation of the airways.

INTRODUCTION: The standard therapy for bronchial asthma consists of combinations of acute (short-acting ß2-sympathomimetics) and, depending on the severity of disease, additional long-term treatment (including inhaled glucocorticoids, long-acting ß2-sympathomimetics, anticholinergics, anti-IL-4R antibodies). The antidepressant amitriptyline has been identified as a relevant down-regulator of immunological TH2-phenotype in asthma, acting-at least partially-through inhibition of acid sphingomyelinase (ASM), an enzyme involved in sphingolipid metabolism. Here, we investigated the non-immunological role of amitriptyline on acute bronchoconstriction, a main feature of airway hyperresponsiveness in asthmatic disease. METHODS: After stimulation of precision cut lung slices (PCLS) from mice (wildtype and ASM-knockout), rats, guinea pigs and human lungs with mediators of bronchoconstriction (endogenous and exogenous acetylcholine, methacholine, serotonin, endothelin, histamine, thromboxane-receptor agonist U46619 and leukotriene LTD4, airway area was monitored in the absence of or with rising concentrations of amitriptyline. Airway dilatation was also investigated in rat PCLS by prior contraction induced by methacholine. As bronchodilators for maximal relaxation, we used IBMX (PDE inhibitor) and salbutamol (ß2-adrenergic agonist) and compared these effects with the impact of amitriptyline treatment. Isolated perfused lungs (IPL) of wildtype mice were treated with amitriptyline, administered via the vascular system (perfusate) or intratracheally as an inhalation. To this end, amitriptyline was nebulized via pariboy in-vivo and mice were ventilated with the flexiVent setup immediately after inhalation of amitriptyline with monitoring of lung function. RESULTS: Our results show amitriptyline to be a potential inhibitor of bronchoconstriction, induced by exogenous or endogenous (EFS) acetylcholine, serotonin and histamine, in PCLS from various species. The effects of endothelin, thromboxane and leukotrienes could not be blocked. In acute bronchoconstriction, amitriptyline seems to act ASM-independent, because ASM-deficiency (Smdp1-/-) did not change the effect of acetylcholine on airway contraction. Systemic as well as inhaled amitriptyline ameliorated the resistance of IPL after acetylcholine provocation. With the flexiVent setup, we demonstrated that the acetylcholine-induced rise in central and tissue resistance was much more marked in untreated animals than in amitriptyline-treated ones. Additionally, we provide clear evidence that amitriptyline dilatates pre-contracted airways as effectively as a combination of typical bronchodilators such as IBMX and salbutamol. CONCLUSION: Amitriptyline is a drug of high potential, which inhibits acute bronchoconstriction and induces bronchodilatation in pre-contracted airways. It could be one of the first therapeutic agents in asthmatic disease to have powerful effects on the TH2-allergic phenotype and on acute airway hyperresponsiveness with bronchoconstriction, especially when inhaled.

[Application of extracorporeal membrane oxygenation technique in patients with refractory shock caused by dichlorvos poisoning].

Dichlorvos poisoning can cause muscarinic (M) -like symptoms, nicotinoid (N) -like symptoms and central nervous system manifestations. When severe poisoning is combined with refractory shock, the mortality rate exceeds 60%. At present, there are more and more studies on ECMO for poisoning, but there is no report on ECMO for treating refractory hypotension caused by dichlorvos poisoning. We analyzed 3 successful cases of veno-arterial extracorporeal membrane oxygenation (VA-ECMO) in the treatment of refractory shock caused by acute severe dichlorvos poisoning to explore the effectiveness of VA-ECMO in patients with severe poisoning.

Rhythmogenic networks are potently modulated by activation of muscarinic acetylcholine receptors in the rodent spinal cord.

Electrical stimulation of the spinal cord is a potent means for activating mammalian stepping in the absence of the descending control from the brain. Previously, we have shown that stimulation of pain delivering (Aδ) sacrocaudal afferents (SCA) has a powerful capacity to activate the sacral and lumbar rhythmogenic networks in the neonatal rodent spinal cord. Relatively little is known about the neural pathways involved in activation of the locomotor networks by Aδ afferents, on their mechanism of action and on the possibility to modulate their activity. We have shown that elevation of the endogenous level of acetylcholine at the sacral cord by blocking cholinesterase could modulate the SCA-induced locomotor rhythm in a muscarinic receptor-dependent mechanism. Here, we review these and more recent findings and report that controlled stimulation of SCA in the presence of muscarine is a potent activator of the locomotor network. The possible mechanisms involved in the muscarinic modulation of the locomotor rhythm are discussed in terms of the differential projections of sacral relay neurons, activated by SCA stimulation, to the lumbar locomotor rhythm generators, and to their target motoneurons. Altogether, our studies show that manipulations of cholinergic networks offer a simple and powerful means to control the activity of locomotor networks in the absence of supraspinal control. Cover Image for this issue: https://doi.org/10.1111/jnc.15079.

Chemistry and Toxicology of Major Bioactive Substances in Inocybe Mushrooms.

Mushroom poisoning has always been a threat to human health. There are a large number of reports about ingestion of poisonous mushrooms every year around the world. It attracts the attention of researchers, especially in the aspects of toxin composition, toxic mechanism and toxin application in poisonous mushroom. Inocybe is a large genus of mushrooms and contains toxic substances including muscarine, psilocybin, psilocin, aeruginascin, lectins and baeocystin. In order to prevent and remedy mushroom poisoning, it is significant to clarify the toxic effects and mechanisms of these bioactive substances. In this review article, we summarize the chemistry, most known toxic effects and mechanisms of major toxic substances in Inocybe mushrooms, especially muscarine, psilocybin and psilocin. Their available toxicity data (different species, different administration routes) published formerly are also summarized. In addition, the treatment and medical application of these toxic substances in Inocybe mushrooms are also discussed. We hope that this review will help understanding of the chemistry and toxicology of Inocybe mushrooms as well as the potential clinical application of its bioactive substances to benefit human beings.

Subcellular distribution of human tyrosine hydroxylase isoforms 1 and 4 in SH-SY5Y cells.

Tyrosine hydroxylase (TH) is the key enzyme that controls the rate of synthesis of the catecholamines. SH-SY5Y cells with stable transfections of either human tyrosine hydroxylase isoform 1 (hTH1) or human tyrosine hydroxylase isoform 4 (hTH4) were used to determined the subcellular distribution of TH protein and phosphorylated TH, under basal conditions and after muscarine stimulation. Muscarine was previously shown to increase the phosphorylation of only serine 19 and serine 40 in hTH1 cells. Under basal conditions, the hTH1 and hTH4 proteins, their serine 19 phosphorylated forms and hTH1 phosphorylated at serine 40 were all similarly distributed; with ~80% in the cytosolic fraction, ~20% in the membrane fraction, and less than 1%, or not detectable, in the nuclear fraction. However, hTH4 phosphorylated at serine 71 had a significantly different distribution with ~65% cytosolic and ~35% membrane associated. Muscarine stimulation led to hTH1 being redistributed from the cytosol and nuclear fractions to the membrane fraction and hTH4 being redistributed from the cytosol to the nuclear fraction. These muscarine stimulated redistributions were not due to TH phosphorylation at serine 19, serine 40, or serine 71 and were most likely due to TH binding to proteins whose phosphorylation was increased by muscarine. This is the first study to show a difference in subcellular distribution between two human TH isoforms under basal and stimulated conditions.

Cholinergic modulation of striatal nitric oxide-producing interneurons.

Striatal GABAergic interneurons that express nitric oxide synthase-so-called low-threshold spike interneurons (LTSIs)-play several key roles in the striatum. But what drives the activity of these interneurons is less well defined. To fill this gap, a combination of monosynaptic rabies virus mapping (msRVm), electrophysiological and optogenetic approaches were used in transgenic mice in which LTSIs expressed either Cre recombinase or a fluorescent reporter. The rabies virus studies revealed a striking similarity in the afferent connectomes of LTSIs and neighboring cholinergic interneurons, particularly regarding connections arising from the parafascicular nucleus of the thalamus and cingulate cortex. While optogenetic stimulation of cingulate inputs excited both cholinergic interneurons and LTSIs, thalamic stimulation excited cholinergic interneurons, but inhibited LTSIs. This inhibition was dependent on cholinergic interneurons and had two components: a previously described GABAergic element and one that was mediated by M4 muscarinic acetylcholine receptors. In addition to this phasic signal, cholinergic interneurons tonically excited LTSIs through a distinct, M1 muscarinic acetylcholine receptor pathway. This coordinated cholinergic modulation of LTSIs predisposed them to rhythmically burst in response to phasic thalamic activity, potentially reconfiguring striatal circuitry in response to salient environmental stimuli.

Cholinergic activation of enteric glia is a physiological mechanism that contributes to the regulation of gastrointestinal motility.

The reflexive activities of the gastrointestinal tract are regulated, in part, by precise interactions between neurons and glia in the enteric nervous system (ENS). Intraganglionic enteric glia are a unique type of peripheral glia that surround enteric neurons and regulate neuronal function, activity, and survival. Enteric glia express numerous neurotransmitter receptors that allow them to sense neuronal activity, but it is not clear if enteric glia monitor acetylcholine (ACh), the primary excitatory neurotransmitter in the ENS. Here, we tested the hypothesis that enteric glia detect ACh and that glial activation by ACh contributes to the physiological regulation of gut functions. Our results show that myenteric enteric glia express both the M3 and M5 subtypes of muscarinic receptors (MRs) and that muscarine drives intracellular calcium (Ca2+) signaling predominantly through M3R activation. To elucidate the functional effects of activation of glial M3Rs, we used GFAP::hM3Dq mice that express a modified human M3R (hM3Dq) exclusively on glial fibrillary acidic protein (GFAP) positive glia to directly activate glial hM3Dqs using clozapine- N-oxide. Using spatiotemporal mapping analysis, we found that the activation of glial hM3Dq receptors enhances motility reflexes ex vivo. Continuous stimulation of hM3Dq receptors in vivo, drove changes in gastrointestinal motility without affecting neuronal survival in the ENS and glial muscarinic receptor activation did not alter neuron survival in vitro. Our results provide the first evidence that GFAP intraganglionic enteric glia express functional muscarinic receptors and suggest that the activation of glial muscarinic receptors contributes to the physiological regulation of functions. NEW & NOTEWORTHY Enteric glia are emerging as novel regulators of enteric reflex circuits, but little is still known regarding the effects of specific transmitter pathways on glia and the resulting consequences on enteric reflexes. Here, we provide the first evidence that enteric glia monitor acetylcholine in the enteric nervous system and that glial activation by acetylcholine is a physiological mechanism that contributes to the functional regulation of intestinal reflexes.

Antinociception of the spirocyclopiperazinium salt compound LXM-15 via activating α7 nAChR and M4 mAChR and inhibiting CaMKIIα/cAMP/CREB/CGRP signalling pathway in mice.

The aim of this study was to investigate the analgesic effect of the spirocyclopiperazinium salt compound LXM-15 by intragastric administration in thermal and chemical pain models and further to elucidate the possible molecular mechanisms. The results showed that LXM-15 exerted significant antinociception in hot-plate test, formalin test and acetic acid writhing test. Western blot analysis showed that LXM-15 significantly reduced the upregulation of phosphorylation of calcium/calmodulin -dependent protein kinase IIα (CaMKIIα) and cAMP response element-binding protein (CREB), and further decreased the elevation of calcitonin gene related peptide (CGRP) in the dorsal root ganglion (DRG) and spinal cord in mice. ELISA analysis showed the level of cAMP in the spinal cord was decreased by LXM-15. All effects of LXM-15 could be blocked by methyllycaconitine citrate (MLA, a selective α7 nicotinic receptor antagonist) or tropicamide (TRO, a selective M4 muscarinic receptor antagonist). This study first reported that intragastric administration of LXM-15 produced significant analgesic effect, which may be related to the activation of α7 nicotinic acetylcholine receptor and M4 muscarine acetylcholine receptor, and thereby inhibiting CaMKIIα/cAMP/CREB/CGRP signalling pathway.

Faster kinetics of quantal catecholamine release in mouse chromaffin cells stimulated with acetylcholine, compared with other secretagogues.

Adrenal chromaffin cells (CCs) have been used extensively in studies aimed at revealing the intricacies of the Ca2+ -dependent early and late steps of regulated exocytosis. They have also served as invaluable models to study the kinetics of single-vesicle exocytotic events to infer the characteristics of opening and closing of the exocytotic fusion pore. We have here tested the hypothesis that stimulation at room temperature of CCs from mice C57BL/6 with physiological acetylcholine (ACh) and with other secretagogues (dimethylphenylpiperazinium, high K+ , muscarine, histamine, caffeine), alone or in combination, could trigger amperometric spike events with different kinetics. We found that mean secretory spike events in CCs stimulated with ACh had a fast rise rate of 25 pA/ms and a rapid decay time of 6.2 ms, with a small quantal size (0.31 pC). Surprisingly, these parameters considerably differed from those found in CCs stimulated with all other secretagogues that triggered secretory responses with spike events having smaller rise rates, longer decay times and higher quantal sizes. ACh spikes were unaltered by atropine but mitochondrial protonophore carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone markedly slowed down the rate rise and decay time, and augmented the quantal size of mean secretory events. We conclude that the physiological neurotransmitter ACh triggers a fast and efficient exocytotic response that cannot be mimicked by other secretagogues; such response is regulated by the mitochondrial circulation of calcium ions.

Application to several suspected poisoning cases of a validated analytical method for the determination of muscarine in human biological matrices using liquid chromatography with high-resolution mass spectrometry detection.

Despite prevention efforts, many cases of mushroom poisoning are reported around the world every year. Among the different toxins implicated in these poisonings, muscarine may induce parasympathetic neurological damage. Muscarine poisonings are poorly reported in the current literature, implying a lack of available data on muscarine concentrations in human matrices. A validated liquid chromatography with high-resolution mass spectrometry detection (Orbitrap technology) method was developed to determine muscarine concentrations in human urine, plasma, and whole blood samples. Muscarine was determined using 100 µL of biological fluids, and precipitation was used for sample preparation. Liquid chromatography-mass spectrometry was performed using an Accucore Phenyl-X analytical column with the electrospray source in positive ion mode. Muscarine was quantitated in parallel reaction monitoring (PRM) mode with D9-muscarine as the internal standard. The method was validated successfully over the concentration range 0.1-100 µg/L for plasma and whole blood and 1-100 µg/L for urine, with acceptable precision and accuracy (<13.5%), including the lower limit of quantification. Ten real cases of suspected muscarine poisoning were successfully confirmed with this validated method. Muscarine concentrations in these cases ranged from 0.12 to 14 µg/L in whole blood,

Cholinergic Mushroom Poisoning With a Detection of Muscarine Toxin in Urine.

We report an uncommon case of cholinergic poisoning following an ingestion of wild mushrooms. Two middle-aged patients presented to the emergency unit with acute gastrointestinal symptoms including epigastric pain, vomiting and diarrhea, followed by miosis, palpitations and diaphoresis which were compatible with a cholinergic toxidrome. The patients volunteered a history of taking two tablespoons of cooked wild mushrooms collected in a country park. Mildly elevated liver transaminase was noted in one female patient. Mushroom specimens were sent to a mycologist for identification using morphological analysis. Muscarine, a cholinergic toxin found in mushrooms such as Inocybe and Clitocybe species, was subsequently extracted from and identified in the urine specimens of both patients, using a liquid-chromatography tandem mass spectrometry method. In this report, the variable clinical presentation of cholinergic mushroom poisoning is discussed. Key issues in the management of these cases were presented. In addition to conventional mushroom identification methods, this report also highlights the use of toxicology tests on different biological and non-biological specimens for diagnosis, prognosis and surveillance purposes.

Phylogenetic analysis, morphological studies, element profiling, and muscarine detection reveal a new toxic Inosperma (Inocybaceae, Agaricales) species from tropical China.

Tropical Asian collections of Inosperma are usually poisonous mushrooms that have caused many poisoning incidents. However, the species diversity and the toxic mechanisms of these Inosperma species are still unclear. In this study, we describe the discovery of Inosperma wuzhishanense sp. nov. from Wuzhishan City, Hainan Province, tropical China. The new species was identified based on morphological and multi-locus (ITS, nrLSU, and RPB2) phylogenetic analyses. The new species is characterized by its reddish-brown pileus, fibrillose stipes with finely protruding fibrils, rather crowded lamellae, smooth and ellipsoid basidiospores, and mostly clavate, thin-walled cheilocystidia. The new species is phylogenetically nested in the Old World tropical clade 2 and is sister to the tropical Indian taxa I. akirnum. Detailed descriptions, color photos of the new species, and comparisons with its closely related species are provided. Additionally, the muscarine content of the new species was analyzed by ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS). The muscarine contents ranged from 4,359.79 ± 83.87 mg/kg to 7,114.03 ± 76.55 mg/kg, 2,748.37 ± 106.85 mg/kg to 4,491.35 ± 467.21 mg/kg, and 2,301.36 ± 83.52 mg/kg to 2,775.90 ± 205.624 mg/kg in the stipe, pileus, and lamellae, respectively. The elemental composition and concentration were determined using inductively coupled plasma-mass spectrometry (ICP-MS). A total of 24 elements were detected. Among the heavy metals detected, arsenic showed the highest level of toxicity with a concentration of 36.76 ± 0.43 mg/kg.

Five new species of Inosperma from China: Morphological characteristics, phylogenetic analyses, and toxin detection.

Many species of Inosperma cause neurotoxic poisoning in humans after consumption around the world. However, the toxic species of Inosperma and its toxin content remain unclear. In the present study, we proposed five new Inosperma species from China, namely, I. longisporum, I. nivalellum, I. sphaerobulbosum, I. squamulosobrunneum, and I. squamulosohinnuleum. Morphological and molecular phylogenetic analyses based on three genes (ITS, nrLSU, rpb2) revealed that these taxa are independent species. A key to 17 species of Inosperma in China is provided. In addition, targeted screening for the most notorious mushroom neurotoxins, muscarine, psilocybin, ibotenic acid, and muscimol, in these five new species was performed by using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Our results show that the neurotoxin contents in these five species varied: I. sphaerobulbosum contains none of the tested neurotoxins; I. nivalellum is muscarine positive; I. longisporum and I. squamulosohinnuleum contain both ibotenic acid and muscimol, and I. squamulosobrunneum only contains muscimol; psilocybin was not detected in these five new species.

Acute mushroom poisoning of Amanita pseudosychnopyramis: A case report from Fujian, China with exact species identification and descriptive study.

Mushroom poisoning is a deeply concerning food safety problem that affects the public in China every year. Although there are statistics on the number of poisonings and incidents, there is a lack of data on the types of toxic mushrooms, clinical manifestations and toxins. A case of wild mushroom poisoning occurred in Xiamen. Descriptive epidemiological investigation, toxins detection, and morphological and phylogenetic identification were immediately performed. The patients exhibited typical neurotoxic symptoms after consuming wild mushrooms, including chills, vertigo, drowsiness, salivation and coma. The average incubation period was 30 min. Treatments that were adopted included fluid infusion, gastric lavage, catharsis, and liver protection treatment. All patients recovered within 10 days. The species was identified as Amanita pseudosychnopyramis, and its contents of muscarine, muscimol and ibotenic acid were 170.3 ± 5.9 mg/kg, 835.4 ± 43.1 mg/kg and 637.9 ± 54.8 mg/kg in dry weight, respectively, as detected by ultrahigh-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). To our knowledge, this is the first report of Amanita pseudosychnopyramis poisoning worldwide.

Toxin screening of Pseudosperma umbrinellum (Agaricals, Basidiomycota): First report of phalloidin in Inocybaceae mushroom.

Pseudosperma species are widely distributed worldwide. Many of them cause poisoning incidents every year, and the toxin responsible for poisoning is muscarine, which could stimulate the parasympathetic nervous system. This study established a method using multiwalled carbon nanotube purification and liquid chromatography-tandem mass spectrometry for the targeted screening of mushroom toxins (muscarine, isoxazole derivatives, tryptamine alkaloids, three amatoxins and three phallotoxins) from Pseudosperma umbrinellum, a common poisonous mushroom distributed in north and northwestern China. Surprisingly, in addition to muscarine, phalloidin was also detected in P. umbrinellum, and the contents were 3022.2 ± 604.4 to 4002.3 ± 804.6 mg/kg (k = 2; p = 95%) muscarine and 5.9 ± 1.2 to 9.3 ± 1.8 mg/kg (k = 2; p = 95%) phalloidin.

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.