Interhemispheric inhibition of the homotopic motor cortex is believed to be effective for accurate unilateral motor function. However, the cellular mechanisms underlying interhemispheric inhibition during unilateral motor behavior remain unclear. Furthermore, the impact of the neuromodulator acetylcholine on interhemispheric inhibition and the associated cellular mechanisms are not well understood. To address this knowledge gap, we conducted recordings of neuronal activity from the bilateral motor cortex of mice during the paw-reaching task. Subsequently, we analyzed interhemispheric spike correlation at the cell-pair level, classifying putative cell types to explore the underlying cellular circuitry mechanisms of interhemispheric inhibition. We found a cell-type pair-specific enhancement of the interhemispheric spike correlation when the mice were engaged in the reaching task. We also found that the interhemispheric spike correlation was modulated by pharmacological acetylcholine manipulation. The local field responses to contralateral excitation differed along the cortical depths, and muscarinic receptor antagonism enhanced the inhibitory component of the field response in deep layers. The muscarinic subtype M2 receptor is predominantly expressed in deep cortical neurons, including GABAergic interneurons. These results suggest that GABAergic interneurons expressing muscarinic receptors in deep layers mediate the neuromodulation of interhemispheric inhibition in the homotopic motor cortex.

Muscarinic receptors are G protein-coupled receptors (GPCRs) that play a role in various physiological functions. Previous studies have shown that these receptors, along with other GPCRs, are voltage-sensitive; both their affinity toward agonists and their activation are regulated by membrane potential. To our knowledge, whether the effect of antagonists on these receptors is voltage-dependent has not yet been studied. In this study, we used Xenopus oocytes expressing the M2 muscarinic receptor (M2R) to investigate this question. Our results indicate that the potencies of two M2R antagonists, atropine and scopolamine, are voltage-dependent; they are more effective at resting potential than under depolarization. In contrast, the M2R antagonist AF-DX 386 did not exhibit voltage-dependent potency.Furthermore, we discovered that the voltage dependence of M2R activation by acetylcholine remains unchanged in the presence of two allosteric modulators, the negative modulator gallamine and the positive modulator LY2119620. These findings enhance our understanding of GPCRs\' voltage dependence and may have pharmacological implications.

The M2 muscarinic receptor (M2R) is a prototypic class A G protein-coupled receptor (GPCR). Interestingly, Fasciani et al. recently identified an internal translation start site within the M2 receptor mRNA, directing the expression of a C-terminal receptor fragment. Elevated during cellular stress, this polypeptide localizes to mitochondria where it inhibits oxidative phosphorylation.

Acetylcholine is a robust neuromodulator of the limbic system and a critical regulator of arousal and emotions. The anterior cingulate cortex (ACC) and the amygdala (AMY) are key limbic structures that are both densely innervated by cholinergic afferents and interact with each other for emotional regulation. The ACC is composed of functionally distinct dorsal (A24), rostral (A32), and ventral (A25) areas that differ in their connections with the AMY. The structural substrates of cholinergic modulation of distinct ACC microcircuits and outputs to AMY are thought to depend on the laminar and subcellular localization of cholinergic receptors. The present study examines the distribution of muscarinic acetylcholine receptors, m1 and m2, on distinct excitatory and inhibitory neurons and on AMY-targeting projection neurons within ACC areas, via immunohistochemistry and injections of neural tracers into the basolateral AMY in adult rhesus monkeys of both sexes. We found that laminar densities of m1+ and m2+ expressing excitatory and inhibitory neurons depended on area and cell type. Among the ACC areas, ventral subgenual ACC A25 exhibited greater m2+ localization on presynaptic inhibitory axon terminals and greater density of m1+ and m2+ expressing AMY-targeting (tracer+) pyramidal neurons. These patterns suggest robust cholinergic disinhibition and potentiation of amygdalar outputs from the limbic ventral ACC, which may be linked to the hyperexcitability of this subgenual ACC area in depression. These findings reveal the anatomical substrate of diverse cholinergic modulation of specific ACC microcircuits and amygdalar outputs that mediate cognitive-emotional integration and dysfunctions underlying stress and affective disorders.

Muscarinic neurotransmission is fundamentally involved in supporting several brain functions by modulating flow of information in brain neural circuits including the hippocampus which displays a remarkable functional segregation along its longitudinal axis. However, how muscarinic neuromodulation contributes to the functional segregation along the hippocampus remains unclear. In this study we show that the nonselective muscarinic receptor agonist carbachol similarly suppresses basal synaptic transmission in the dorsal and ventral CA1 hippocampal field, in a concentration-depended manner. Furthermore, using a ten-pulse stimulation train of varying frequency we found that carbachol changes the frequency filtering properties more in ventral than dorsal hippocampus by facilitating synaptic inputs at a wide range of input frequencies in the ventral compared with dorsal hippocampus. Using the M2 receptor antagonist gallamine and the M4 receptor antagonist tropicamide, we found that M2 receptors are involved in controlling basal synaptic transmission and short-term synaptic plasticity (STSP) in the ventral but not the dorsal hippocampus, while M4 receptors participate in modulating basal synaptic transmission and STSP in both segments of the hippocampus. These results were corroborated by the higher protein expression levels of M2 receptors in the ventral compared with dorsal hippocampus. We conclude that muscarinic transmission modulates excitatory synaptic transmission and short-term synaptic plasticity along the entire rat hippocampus by acting through M4 receptors and recruiting M2 receptors only in the ventral hippocampus. Furthermore, M4 receptors appear to exert a permissive role on the actions of M2 receptors on STSP in the ventral hippocampus. This dorsoventral differentiation of muscarinic modulation is expected to have important implications in information processing along the endogenous hippocampal circuitry.

Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of 7 transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2 receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single-cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is up-regulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here, it controls oxygen consumption, cell proliferation, and the formation of reactive oxygen species (ROS) by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human-induced pluripotent stem cells (hiPSCs), reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signaling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism that cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration.

G protein-coupled receptors (GPCRs) transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors that are highly modular and could potentially be used to determine GPCR agonist localization across the brain. We previously engineered integrator sensors for the mu- and kappa-opioid receptor agonists called M- and K-Single-chain Protein-based Opioid Transmission Indicator Tool (SPOTIT), respectively. Here, we engineered red versions of the SPOTIT sensors for multiplexed imaging of GPCR agonists. We also modified SPOTIT to create an integrator sensor design platform called SPOTIT for all GPCRs (SPOTall). We used the SPOTall platform to engineer sensors for the beta 2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. Finally, we demonstrated the application of M-SPOTIT and B2AR-SPOTall in detecting exogenously administered morphine, isoproterenol, and epinephrine in the mouse brain via locally injected viruses. The SPOTIT and SPOTall sensor design platform has the potential for unbiased agonist detection of many synthetic and endogenous neuromodulators across the brain.

Acetylcholine is a neurotransmitter that plays a variety of roles in the central nervous system. It was previously shown that blocking muscarinic receptors with a nonselective antagonist prevents a form of experience-dependent plasticity termed \"spatiotemporal sequence learning\" in the mouse primary visual cortex (V1). Muscarinic signaling is a complex process involving the combined activities of five different G protein-coupled receptors, M1-M5, all of which are expressed in the murine brain but differ from each other functionally and in anatomical localization. Here we present electrophysiological evidence that M2, but not M1, receptors are required for spatiotemporal sequence learning in mouse V1. We show in male mice that M2 is highly expressed in the neuropil in V1, especially in thalamorecipient layer 4, and colocalizes with the soma in a subset of somatostatin-expressing neurons in deep layers. We also show that expression of M2 receptors is higher in the monocular region of V1 than it is in the binocular region but that the amount of experience-dependent sequence potentiation is similar in both regions and that blocking muscarinic signaling after visual stimulation does not prevent plasticity. This work establishes a new functional role for M2-type receptors in processing temporal information and demonstrates that monocular circuits are modified by experience in a manner similar to binocular circuits.NEW & NOTEWORTHY Muscarinic acetylcholine receptors are required for multiple forms of plasticity in the brain and support perceptual functions, but the precise role of the five subtypes (M1-M5) are unclear. Here we show that the M2 receptor is specifically required to encode experience-dependent representations of spatiotemporal relationships in both monocular and binocular regions of mouse V1. This work identifies a novel functional role for M2 receptors in coding temporal information into cortical circuits.

Glioblastoma (GB) is a very aggressive human brain tumor. The high growth potential and invasiveness make this tumor surgically and pharmacologically untreatable. Our previous work demonstrated that the activation of the M2 muscarinic acetylcholine receptors (M2 mAChRs) inhibited cell proliferation and survival in GB cell lines and in the cancer stem cells derived from human biopsies. The aim of the present study was to investigate the ability of M2 mAChR to modulate cell migration in two different GB cell lines: U87 and U251. By wound healing assay and single cell migration analysis performed by time-lapse microscopy, we demonstrated the ability of M2 mAChRs to negatively modulate cell migration in U251 but not in the U87 cell line. In order to explain the different effects observed in the two cell lines we have evaluated the possible involvement of the intermediate conductance calcium-activated potassium (IKCa) channel. IKCa channel is present in the GB cells, and it has been demonstrated to modulate cell migration. Using the perforated patch-clamp technique we have found that selective activation of M2 mAChR significantly reduced functional density of the IKCa current in U251 but not in U87 cells. To understand whether the M2 mAChR mediated reduction of ion channel density in the U251 cell line was relevant for the cell migration impairment, we tested the effects of TRAM-34, a selective inhibitor of the IKCa channel, in wound healing assay. We found that it was able to markedly reduce U251 cell migration and significantly decrease the number of invadopodia-like structure formations. These results suggest that only in U251 cells the reduced cell migration M2 mAChR-mediated might involve, at least in part, the IKCa channel.

M4 muscarinic receptors are highly expressed in the striatum and cortex, brain regions that are involved in diseases such as Parkinson\'s disease, schizophrenia, and dystonia. Despite potential therapeutic advantages of specifically targeting the M4 receptor, it has been historically challenging to develop highly selective ligands, resulting in undesired off-target activity at other members of the muscarinic receptor family. Recently, we have reported first-in-class, potent, and selective M4 receptor antagonists. As an extension of that work, we now report the development and characterization of a radiolabeled M4 receptor antagonist, [3H]VU6013720, with high affinity (pKd of 9.5 ± 0.2 at rat M4, 9.7 at mouse M4, and 10 ± 0.1 at human M4 with atropine to define nonspecific binding) and no significant binding at the other muscarinic subtypes. Binding assays using this radioligand in rodent brain tissues demonstrate loss of specific binding in Chrm4 knockout animals. Dissociation kinetics experiments with various muscarinic ligands show differential effects on the dissociation of [3H]VU6013720 from M4 receptors, suggesting a binding site that is overlapping but may be distinct from the orthosteric site. Overall, these results demonstrate that [3H]VU6013720 is the first highly selective antagonist radioligand for the M4 receptor, representing a useful tool for studying the basic biology of M4 as well for the support of M4 receptor-based drug discovery. SIGNIFICANCE STATEMENT: This manuscript describes the development and characterization of a novel muscarinic (M) acetylcholine subtype 4 receptor antagonist radioligand, [3H]VU6013720. This ligand binds to or overlaps with the acetylcholine binding site, providing a highly selective radioligand for the M4 receptor that can be used to quantify M4 protein expression in vivo and probe the selective interactions of acetylcholine with M4 versus the other members of the muscarinic receptor family.