EDITOR SPOTLIGHT: Interview with Cholinergic Mechanisms Special Issue Guest Editor Hermona Soreq

Hermona Soreq holds a Hebrew University Slesinger Chair in Molecular Neuroscience and is among the founding members of the Edmond and Lily Safra Center of Brain Sciences (ELSC). Soreq's research (H-impact: 98) focuses on acetylcholine (ACh)-related pathways and combines RNA-sequencing technologies, transgenic engineering, and molecular biology tests with in-depth analysis approaches. Her work addresses microRNAs (miRs) and transfer RNA fragments (tRFs) which have rapidly acquired wide recognition as global controllers of regulatory processes in healthy and diseased brain and body, including anxiety, inflammation, and cognition. Altogether, Soreq's work leads to molecular neuroscience-driven prevention and/or intervention with diseases involving impaired ACh signaling, including schizophrenia, bipolar disorder, Alzheimer's disease, and stress. Hermona led this Special Issue based on the 17th Symposium on Cholinergic Mechanisms (ISCM2022). We interviewed her on the progress in the field, what she wants to achieve as Senior Editor for the Gene Regulation and Genetics category at the Journal of Neurochemistry, key moments, and future directions.     

Acetylcholine turnover rates in rat brain regions during cocaine self-administration

The involvement of cholinergic neurons in the brain processes underlying reinforcement has been recently demonstrated. This experiment assessed the potential role of cholinergic neurons in cocaine reinforcement by measuring the turnover rates of acetylcholine in brain regions of rats self-administering cocaine and in yoked cocaine and yoked vehicle-infused controls. The activity of cholinergic innervations of and/or interneurons in the olfactory tubercle, caudate putamen, diagonal band-pre-optic region, ventral pallidum, lateral and medial hypothalamus, hippocampus, ventral tegmental area and visual cortices reflected by the turnover rates of acetylcholine were significantly altered in rats self-administering cocaine compared to yoked cocaine infused controls. These changes implicate the involvement of cholinergic neurons with cell bodies in the diagonal band-pre-optic region, the medial septum and several brainstem nuclei and interneurons in the caudate-putamen and ventral pallidum in the processes underlying cocaine self-administration. The identified cholinergic neuronal systems may have a broader role in the brain processes for natural reinforcers (i.e. food, water, etc.) since drugs of abuse are believed to produce reinforcing effects through these systems.     

Functional Regulation of Choline Acetyltransferase by Phosphorylation

Choline acetyltransferase (ChAT) catalyzes synthesis of acetylcholine (ACh) in cholinergic neurons. ACh synthesis is regulated by availability of precursors choline and acetyl coenzyme A or by activity of ChAT; ChAT regulates ACh synthesis under some conditions. Posttranslational phosphorylation is a common mechanism for regulating the function of proteins. Analysis of the primary sequence of 69-kD human ChAT indicates that it has putative phosphorylation consensus sequences for multiple protein kinases. ChAT is phosphorylated on serine-440 and threonine-456 by protein kinase C and CaM kinase II, respectively. These phosphorylation events regulate activity of the enzyme, as well as its binding to plasma membrane and interaction with other cellular proteins. It is relevant to investigate differences in constitutive and inducible patterns of phosphorylation of ChAT under physiological conditions and in response to challenges that cholinergic neurons may be exposed to, and to determine how changes in phosphorylation relate to changes in neurochemical transmission.     

Effects of NGF on acetylcholine, acetyl-CoA metabolism, and viability of differentiated and non-differentiated cholinergic neuroblastoma cells

Nerve growth factor (NGF) is a peptide displaying multiple cholinotropic activities. The aim of this work was to explain mechanisms of the positive and negative effects of NGF on phenotypic properties and viability of cholinergic cells. To discriminate these effects we used two p75NTR receptor-positive lines of cholinergic neuroblastoma cells, SN56 and T17 that are devoid of or express high affinity NGF (TrkA) receptors, respectively. cAMP and retinoic acid caused differentiation of both cell lines. In addition to the morphologic maturation, the increase of choline acetyltransferase activity, acetylcholine, Ca and cytoplasmic acetyl-CoA levels and decrease of mitochondrial acetyl-CoA and cell viability were observed. NGF caused similar effects in non-differentiated T17 cells but had no influence on non-differentiated SN56 cells. On the contrary, in both cAMP/all-trans-retinoic acid (RA) differentiated cell lines, NGF resulted in a similar suppression of cholinergic phenotype along with an increase of mitochondrial acetyl-CoA and cell susceptibility to nitric oxide and amyloid-beta25-35. These effects of NGF were prevented by an antibody against the p75NTR receptor. Data indicate that: (i) positive cholinotrophic effects of NGF required activation of both TrkA and p75NTR receptors; (ii) cAMP/RA-evoked differentiation inhibited NGF effects mediated by TrkA receptors and activated its p75NTR-dependent suppressing influences and (iii) a differentiation-evoked decrease of mitochondrial acetyl-CoA and an elevation of mitochondrial Ca could augment impairment of cholinergic neurons by neurotoxic signals.     

Structural models of the nicotinic acetylcholine receptor and its toxin-binding sites

Models of the protein structure of agonist-, competitive antagonist-, and snake neurotoxin-binding sites were designed using the sequence of the first 54 residues of the acetylcholine receptor (AChR) subunit from Torpedo californica. These models are based on the premise that the N-terminal portions of the subunits form the outermost extracellular surface of the AChR and that agonists bind to this portion. The models were developed by predicting the secondary strucutre of the-subunit N-terminal segment from its sequence, then using these predictions to fold the segment into tertiary structures that should bind snake neurotoxins, agonists, and antagonists. Possible gating mechanisms and quaternary structures are suggested by the proposed tertiary structures of the subunits. Experiments are suggested to test aspects of the models.Supported by Armed Forces Radiobiology Research Institute, Defense Nuclear Agency, under Research Work Unit MJ 00032. The views presented in this paper are those of the author. No endorsement by the Defense Nuclear Agency has been given or should be inferred.     

Effects of Septal Grafts on Acetylcholine Release from Rat Hippocampus After 192 IgG-Saporin Lesion

The cholinergic inputs to the rat hippocampus were lesioned by intraseptal injections of 192 IgG-saporin. After 15 days, fetal septal cells were grafted into the hippocampus. Thirteen months later, hippocampal acetylcholine (ACh) release was studied by microdialysis. Lesioning reduced basal ACh release (100%) to 20% of normal, which was compensated for by the graft (71%). Infusion of the serotonin uptake inhibitor citalopram (100 M) enhanced ACh release to the same extent (% of basal release) in all rat groups. Systemic injection of 8-OH-DPAT (0.5 mg/kg, SC), an agonist of 5-HT_1A receptors, caused a smaller ACh release than citalopram. Acetylcholinesterase (AChE) staining and densitometric quantification revealed that the lesion-induced reduction of the AChE-staining density was compensated for by septal grafting. In conclusion, both histochemical and biochemical methods showed that cholinergic hippocampal parameters were drastically impaired by 192 IgG-saporin lesions, but were almost completely restored by septal grafting. The graft responded to intrinsic serotonergic regulation.     

Oral Administration of Gintonin Attenuates Cholinergic Impairments by Scopolamine,Amyloid-β Protein,and Mouse Model of Alzheimer’s Disease

Gintonin is a novel ginseng-derived lysophosphatidic acid (LPA) receptor ligand. Oral administration of gintonin ameliorates learning and memory dysfunctions in Alzheimer’s disease (AD) animal models. The brain cholinergic system plays a key role in cognitive functions. The brains of AD patients show a reduction in acetylcholine concentration caused by cholinergic system impairments. However, little is known about the role of LPA in the cholinergic system. In this study, we used gintonin to investigate the effect of LPA receptor activation on the cholinergic system in vitro and in vivo using wild-type and AD animal models. Gintonin induced [Ca²⁺]_i transient in cultured mouse hippocampal neural progenitor cells (NPCs). Gintonin-mediated [Ca²⁺]_i transients were linked to stimulation of acetylcholine release through LPA receptor activation. Oral administration of gintonin-enriched fraction (25, 50, or 100 mg/kg, 3 weeks) significantly attenuated scopolamine-induced memory impairment. Oral administration of gintonin (25 or 50 mg/kg, 2 weeks) also significantly attenuated amyloid-β protein (Aβ)-induced cholinergic dysfunctions, such as decreased acetylcholine concentration, decreased choline acetyltransferase (ChAT) activity and immunoreactivity, and increased acetylcholine esterase (AChE) activity. In a transgenic AD mouse model, long-term oral administration of gintonin (25 or 50 mg/kg, 3 months) also attenuated AD-related cholinergic impairments. In this study, we showed that activation of G protein-coupled LPA receptors by gintonin is coupled to the regulation of cholinergic functions. Furthermore, this study showed that gintonin could be a novel agent for the restoration of cholinergic system damages due to Aβ and could be utilized for AD prevention or therapy.     

Non-neuronal nicotinic acetylcholine receptors: Cholinergic regulation of the immune processes

Nicotinic acetylcholine receptors (nAChRs) were initially discovered and studied as mediators of fast synaptic transmission in neuromuscular junctions and autonomic ganglia. Later on, they were found in the brain and in many nonexcitable tissues where they regulate vital cellular functions and the activity of other receptors. Primary immune organs, the bone marrow and thymus, are innervated with cholinergic nerves, which mediate the control of lymphopoiesis provided by the autonomic nervous system. In addition, lymphocytes are able to produce endogenous acetylcholine that can regulate the immune processes in an auto/paracrine way. Correspondingly, both T and B lymphocytes express functional nAChRs involved in the regulation of development and activation of these cells. This review describes the structure and roles of nAChRs in the immune system with regard to its potential regulation by the autonomic nervous system, as well as by self sources of endogenous agonists. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 307–314, July–October, 2007.     

家兔脊髓蛛网膜下腔注射乙酰胆碱对血压和心率的影响

将乙酰胆碱(ACh)注入麻醉家兔脊髓蛛网膜下腔,观察其对心血管活动的影响。结果表明:(1)脊髓蛛网膜下腔注射50～100μg ACh可使血压下降,心率减慢;(2)预先由脊髓蛛网膜下腔注射阿托品,可阻断ACh引起的降压和降心率作用;(3)脊髓蛛网膜下腔注射六甲双铵、酚妥拉明或心得安均不能阻断上述ACh的心血管反应;(4)切断两侧颈部迷走神经,ACh不再使心率减慢,但其降低血压的作用不受到任何影响。 脊髓中ACh水平升高可通过激活胆碱能M-受体引起血压下降和心率减慢。ACh的这种降压作用既没有中枢肾上腺素能受体活动参与,也不是通过迷走神经实现的,可能是由于脊髓交感血管中枢紧张性降低所造成的。     

Synaptic transmission is impaired at neuronal autonomic synapses in agrin-null mice

Neuronal synapse formation is a multistep process regulated by several pre- and postsynaptic adhesion and signaling proteins. Recently, we found that agrin acts as one such synaptogenic factor at neuronal synapses in the PNS by demonstrating that structural synapse formation is impaired in the superior cervical ganglia (SCG) of z+ agrin-deficient mice and in SCG cultures derived from those animals. Here, we tested whether synaptic function is defective in agrin-null (AGD-/-) ganglia and began to define agrin's mechanism of action. Our electrophysiological recordings of compound action potentials showed that presynaptic stimulation evoked action potentials in approximately 40% of AGD-/- ganglionic neurons compared to 90% of wild-type neurons; moreover, transmission could not be potentiated as in wild-type or z+ agrin-deficient ganglia. Intracellular recordings also showed that nerve-evoked excitatory postsynaptic potentials in AGD-/- neurons were only 1/3 the size of those in wild-type neurons and mostly subthreshold. Consistent with these defects in transmission, we found an approximately 40-50% decrease in synapse number in AGD-/- ganglia and cultures, and decreased levels of differentiation at the residual synapses in culture. Furthermore, surface levels of acetylcholine receptors (AChRs) were equivalent in cultured AGD-/- and wild-type neurons, and depolarization reduced the synaptic localization of AChRs in AGD-/- but not wild-type neurons. These findings provide the first direct demonstration that agrin is required for proper structural and functional development of an interneuronal synapse in vivo. Moreover, they suggest a novel role for agrin, in stabilizing the postsynaptic density of nAChR at nascent neuronal synapses.     

Neurotrophins differentially enhance acetylcholine release, acetylcholine content and choline acetyltransferase activity in basal forebrain neurons

Several lines of evidence indicate that nerve growth factor is important for the development and maintenance of the basal forebrain cholinergic phenotype. In the present study, using rat primary embryonic basal forebrain cultures, we demonstrate the differential regulation of functional cholinergic markers by nerve growth factor treatment (24–96 h). Following a 96‐h treatment, nerve growth factor (1–100 ng/mL) increased choline acetyltransferase activity (168–339% of control), acetylcholine content (141–185%), as well as constitutive (148–283%) and K⁺‐stimulated (162–399%) acetylcholine release, but increased release was not accompanied by increased high‐affinity choline uptake. Enhancement of ACh release was attenuated by vesamicol (1 µm ), suggesting a vesicular source, and was abolished under choline‐free conditions, emphasizing the importance of extracellular choline as the primary source for acetylcholine synthesized for release. A greater proportion of acetylcholine released from nerve growth factor‐treated cultures than from nerve growth factor‐naïve cultures was blocked by voltage‐gated Ca²⁺ channel antagonists, suggesting that nerve growth factor modified this parameter of neurotransmitter release. Cotreatment of NGF (20 ng/mL) with K252a (200 nm ) abolished increases in ChAT activity and prevented enhancement of K⁺‐stimulated ACh release beyond the level associated with K252a, suggesting the involvement of TrkA receptor signaling. Also, neurotrophin‐3, neurotrophin‐4 and brain‐derived neurotrophic factor (all at 5–200 ng/mL) increased acetylcholine release, although they were not as potent as nerve growth factor and higher concentrations were required. High brain‐derived neurotrophic factor concentrations (100 and 200 ng/mL) did, however, increase release to a level similar to nerve growth factor. In summary, long‐term exposure (days) of basal forebrain cholinergic neurons to nerve growth factor, and in a less‐potent fashion the other neurotrophins, enhanced the release of acetylcholine, which was dependent upon a vesicular pool and the availability of extracellular choline.     

Changes in cortical acetyl-CoA metabolism after selective basal forebrain cholinergic degeneration by 192IgG-saporin

The aim of the present study was to reveal whether reduced cortical cholinergic input affects the acetyl-CoA metabolism in cholinoceptive cortical target regions which may play a causative role for the deficits in cerebral glucose metabolism observed in Alzheimer's disease. The effect of cortical cholinergic denervation produced by a single intracerebroventricular application of the cholinergic immunotoxin 192IgG-saporin, on activities of pyruvate dehydrogenase and adenosine triphosphate (ATP)-citrate lyase as well as on the level of synaptoplasmic and mitochondrial acetyl-CoA and acetylcholine release in cortical target regions was studied. Cholinergic lesion produced 83%, 72% and 32% decreases in the activities of choline acetyltransferase, acetylcholinesterase and ATP-citrate lyase in nerve terminals isolated from rat brain cortex, respectively, but no change in pyruvate dehydrogenase activity. Spontaneous and Ca2+-evoked acetylcholine release from synaptosomes was inhibited by 76% and 73%, respectively, following immunolesion. The lesion-induced 39% decrease of acetyl-CoA level in synaptosomal mitochondria was accompanied by 74% increase in synaptoplasmic fraction. Levels of acetyl-CoA and CoASH assayed in fraction of whole brain mitochondria from lesioned cortex were 61% and 48%, respectively, higher as compared to controls. The data suggest a preferential localization of ATP-citrate lyase in cholinergic nerve terminals, where it may contribute to the transport of acetyl-CoA from the mitochondrial to the cytoplasmic compartment. They provide evidence on differential distribution of acetyl-CoA in subcellular compartments of cholinergic and non-cholinergic nerve terminals. There are also indications that cholinergic activity affects acetyl-CoA level and its intracellular distribution in glial and other non-cholinergic cortical cells.     

Identification of a region from the human cholinergic gene locus that targets expression of the vesicular acetylcholine transporter to a subset of neurons in the medial habenular nucleus in transgenic mice

We use a transgenic mouse model system to elucidate the regulatory regions within the human cholinergic gene locus responsible for vesicular acetylcholine transporter gene expression in vivo. In this report we characterized two transgenes for their ability to confer cholinergic-specific expression of the encoded vesicular acetylcholine transporter. An 11.2 kb transgene (named hV11.2) that spanned from about 5 kb upstream of the start of vesicular acetylcholine transporter translation down to the first choline acetyltransferase coding exon gave expression in the somatomotor neurons and a subpopulation of cholinergic neurons in the medial habenular nucleus. The second transgene (named hV6.7), a 5-prime truncated version of hV11.2 that was devoid of 4.5 kb of gene-regulatory sequences completely lacked vesicular acetylcholine transporter expression in vivo. Our data indicate that vesicular acetylcholine transporter expression in somatomotor neurons and in the medial habenular nucleus is uniquely specified within the cholinergic gene locus, and separable from cholinergic expression elsewhere. The identification of these two subdivisions of the cholinergic nervous system suggests that other cholinergic neurons in the CNS and PNS are similarly regulated by additional discrete domains within the cholinergic gene locus.     

Unusual Regulation of Splicing of the Cholinergic Locus in Caenorhabditis elegans

The essential neurotransmitter acetylcholine functions throughout the animal kingdom. In Caenorhabditis elegans, the acetylcholine biosynthetic enzyme [choline acetyltransferase (ChAT)] and vesicular transporter [vesicular acetylcholine transporter (VAChT)] are encoded by the cha-1 and unc-17 genes, respectively. These two genes compose a single complex locus in which the unc-17 gene is nested within the first intron of cha-1, and the two gene products arise from a common pre-messenger RNA (pre-mRNA) by alternative splicing. This genomic organization, known as the cholinergic gene locus (CGL), is conserved throughout the animal kingdom, suggesting that the structure is important for the regulation and function of these genes. However, very little is known about CGL regulation in any species. We now report the identification of an unusual type of splicing regulation in the CGL of C. elegans, mediated by two pairs of complementary sequence elements within the locus. We show that both pairs of elements are required for efficient splicing to the distal acceptor, and we also demonstrate that proper distal splicing depends more on sequence complementarity within each pair of elements than on the sequences themselves. We propose that these sequence elements are able to form stem-loop structures in the pre-mRNA; such structures would favor specific splicing alternatives and thus regulate CGL splicing. We have identified complementary elements at comparable locations in the genomes of representative species of other animal phyla; we suggest that this unusual regulatory mechanism may be a general feature of CGLs.     

Nicotinic and muscarinic responses of embryonic chick ventricular myocardium to acetylcholine: chronotropic and electrophysiological effects

Sensitivity of 7-day-old chick embryo ventricular heart fragments to acetylcholine was investigated. Low doses mainly produced a positive chronotropic effect, whereas high doses of acetylcholine provoked a decrease in the heart beat rhythm. The positive chronotropic effect of acetylcholine was related to the presence of nicotinic receptors that were evidenced within ventricular myocardium by autoradiography. Membrane potential recording showed that acetylcholine hyperpolarizes the diastolic membrane potential when the drug had a negative chronotropic effect. This effect of acetylcholine on the membrane potential was not observed when the drug had a positive chronotropic effect. In many cases, the diastolic membrane potential exhibited spontaneous small depolarizing potentials. Their amplitude was low and their frequency was irregular. These potentials were suppressed by treatment with alpha-bungarotoxin, suggesting that they are triggered by nicotinic receptor activation.     

β-Amyloid and Cholinergic Neurons

It is generally accepted that the crucial events in the pathogeny of Alzheimer's disease (AD) are the increased accumulation of amyloidogenic peptides derived from amyloid precursor protein and the harmful actions of these peptides on neurons, which bring about neurodegeneration. The enhanced -amyloid accumulation is known to be caused by mutations of specific genes in patients who suffer from the familial (hereditary) form of AD but who represent just a minor group within the total population of AD patients. The reasons for -amyloid accumulation are not known in the much larger group of patients with the sporadic form of the disease. A biochemical feature common to either form of the disease is the preferential atrophy and degeneration of cholinergic neurons, which is probably responsible for much of the cognitive decline characteristic of the disease. We present an overview of recent investigations on the interactions between -amyloid and cholinergic neurons.     

Alpha7 nicotinic acetylcholine receptors in lung inflammation and carcinogenesis: Friends or foes?

The lung tissue expresses the cholinergic system including nicotinic acetylcholine receptors (nAChRs) which included in many physiologic and pathologic processes. Mounting evidence revealed that these receptors have important roles in lung carcinogenesis via modulating either stimulatory or inhibitory signaling pathways. Among different members of nicotinic receptors family, alpha7-subtype of nAChR (α7nAChR) is a critical mediator involved in both inflammatory responses and cancers. Several studies have shown that this receptor is the most powerful regulator of responses that stimulate lung cancer processes such as proliferation, angiogenesis, metastasis, and inhibition of apoptosis. Moreover, aside from its roles in the regulation of cancer pathways, there is growing evidence indicating that α7nAChR has profound impacts on lung inflammation through the cholinergic anti-inflammatory pathway. Regarding such diverse effects as well as the critical roles of nicotine as an activator of α7nAChR on lung cancer pathogenesis, its modulation has emerged as a promising target for drug developments. In this review, we aim to highlight the detrimental as well as the possible beneficial influences of α7nAChR downstream signaling cascades in the control of lung inflammation and cancer-associated properties. Consequently, by considering the significant global burden of lung cancer, delineating the complex influences of α7 receptors would be of great interest in designing novel anticancer and anti-inflammatory strategies for the patients suffering from lung cancer.     

家兔肺动脉内注射乙酰胆碱对肺动脉血压的影响

在65只氨基甲酸乙酯麻醉的兔,向肺动脉内注射2—4μg乙酰胆碱(ACh),可使颈动脉压出现短暂下降,回升时,肺动脉压可出现升压或降压两种反应,但以升压反应较为多见。发生这两种反应时,心率无明显变化。上述颈动脉降压反应、肺动脉升压和降压反应均能被阿托品阻断。乙酰胆碱引起的肺动脉升压反应可以被胆碱能N-受体阻断剂六甲双铵阻断,亦可被α-受体阻断剂酚妥拉明部分逆转。β-受体阻断剂心得安能部分逆转乙酰胆碱降低颈动脉压的作用,但对乙酰胆碱升高或降低肺动脉压的作用无任何影响。 结果表明,当血液中乙酰胆碱水平升高使肺动脉压下降主要是由于乙酰胆碱直接使肺血管平滑肌舒张。而促使肺动脉压上升的原因,是由于乙酰胆碱一方面直接使肺血管平滑肌收缩;另一方面还可激活交感神经节中N-受体,促进去甲肾上腺素释放,后者与α-受体结合而引起肺血管收缩所致。     

VAChT-Cre. Fast and VAChT-Cre.Slow: postnatal expression of Cre recombinase in somatomotor neurons with different onset

The cholinergic gene locus (CGL) consists of the genes encoding the choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). To establish a cholinergic-specific Cre-expressing mouse, we constructed a transgene expression vector (VAChT-Cre) with 11.3 kb human CGL in which a Cre-IRES-EGFP unit was inserted in the VAChT open reading frame. The activity of Cre, whose expression was driven by the VAChT promoter, was examined by crossing a reporter mouse (CAG-CAT-Z) in which expression of LacZ is activated upon Cre-mediated recombination. Transgenic lines with the VAChT-Cre construct displayed the restricted Cre expression in a subset of cholinergic neurons in the somatomotor nuclei and medial habenular nucleus, but absent in visceromotor and other central and peripheral cholinergic neurons. Cre expression was first observed at postnatal day 7 and later detected in approximately 40-60% of somatomotor neurons. Based on the onset of Cre expression, we generated two mouse lines (two alleles; VAChT-Cre. Fast and VAChT-Cre.Slow) in which Cre expression reaches maximal levels fast and slow, respectively. The use of VAChT-Cre mice should allow us to deliver Cre to a subset of postnatal motor neurons, thereby bypassing lethality and facilitating analysis of gene function in adult motor neurons.     

Muscarinic toxins: tools for the study of the pharmacological and functional properties of muscarinic receptors

Muscarinic receptors mediate metabotropic actions of acetylcholine in the CNS and PNS and autocrine functions of acetylcholine in non-neuronal systems. Because of the lack of highly selective muscarinic ligands, the precise location, functional role, and roles in various diseases of the five muscarinic receptor subtypes remain unclear. Muscarinic toxins isolated from the venom of Dendroaspis snakes have a natural high affinity and selectivity, associated with roles as competitive antagonists, allosteric modulators, and potential agonists. These toxins may therefore be invaluable tools for studying muscarinic receptors. We review data on the structural and pharmacological characterization of the muscarinic toxins, focusing on recent structure–function studies on toxin–receptor interactions. We discuss the potential benefits of using these toxins for investigating muscarinic function in vivo .