Peripheral effects of vagus nerve stimulation on anxiety and extinction of conditioned fear in rats.

Vagus nerve stimulation (VNS) enhances extinction of conditioned fear in rats. Previous findings support the hypothesis that VNS effects on extinction are due to enhanced consolidation of extinction memories through promotion of plasticity in extinction-related brain pathways however, alternative explanations are plausible. According to one hypothesis, VNS may produce a hedonic effect and enhance extinction through counter-conditioning. According to another hypothesis, VNS reduces anxiety during exposure and this weakens the association of conditioned stimuli with aversive conditioned responses. The present set of experiments (1) used conditioned place preference (CPP) to identify potential rewarding effects associated with VNS and (2) examined the peripheral effects of VNS on anxiety and extinction enhancement. Male Sprague-Dawley rats were surgically implanted with cuff electrodes around the vagus nerve and subjected to a CPP task in which VNS and sham stimulation were each paired with one of two distinct contexts over the course of 5 d. Following this procedure, rats did not show a place preference, suggesting that VNS is not rewarding or aversive. The role of the peripheral parasympathetic system in the anxiolytic effect of VNS on the elevated plus maze was examined by blocking peripheral muscarinic receptors with intraperitoneal administration of methyl scopolamine prior to VNS. Methyl scopolamine blocked the VNS-induced reduction in anxiety but did not interfere with VNS enhancement of extinction of conditioned fear, indicating that the anxiety-reducing effect of VNS is not necessary for the extinction enhancement.

Utility of an "Allosteric Site-Impaired" M2 Muscarinic Acetylcholine Receptor as a Novel Construct for Validating Mechanisms of Action of Synthetic and Putative Endogenous Allosteric Modulators.

Muscarinic acetylcholine receptors (mAChRs) are exemplar models for understanding G protein-coupled receptor (GPCR) allostery, possessing a "common" allosteric site in an extracellular vestibule (ECV) for synthetic modulators including gallamine, strychnine, and brucine. In addition, there is intriguing evidence of endogenous peptides/proteins that may target this region at the M2 mAChR. A common feature of synthetic and endogenous M2 mAChR negative allosteric modulators (NAMs) is their cationic nature. Using a structure-based approach, we previously designed a mutant M2 mAChR (N410K+T423K) to specifically abrogate binding of ECV cationic modulators (Dror et al., 2013). Herein, we used this "allosteric site-impaired" receptor to investigate allosteric interactions of synthetic modulators as well as basic peptides (poly-l-arginine, endogenously produced protamine, and major basic protein). Using [3H]N-methylscopolamine equilibrium and kinetic binding and functional assays of guanosine 5'-O-[γ-thio]triphosphate [35S] binding and extracellular signal-regulated kinases 1 and 2 phosphorylation, we found modest effects of the mutations on potencies of orthosteric antagonists and an increase in the affinity of the cognate agonist, acetylcholine, likely reflecting the effect of the mutations on the access/egress of these ligands into the orthosteric pocket. More importantly, we noted a significant abrogation in affinity for all synthetic or peptidic modulators at the mutant mAChR, validating their allosteric nature. Collectively, these findings provide evidence for a hitherto-unappreciated role of endogenous cationic peptides interacting allosterically at the M2 mAChR and identify the allosteric site-impaired GPCR as a tool for validating NAM activity as well as a potential candidate for future chemogenetic strategies to understand the physiology of endogenous allosteric substances.

Characterization of methanthelinium binding and function at human M1-M5 muscarinic acetylcholine receptors.

Firstly, it was determined whether methanthelinium bromide (MB) binds to human M1-M5 (hM1-hM5) muscarinic acetylcholine receptors in comparison to the classical muscarinic antagonist N-methylscopolamine (NMS). [3H]NMS dissociation binding experiments revealed an allosteric retardation of dissociation at 100 µM of MB ranging from none in hM3 to 4.6-fold in hM2 receptors. Accordingly, global non-linear regression analysis of equilibrium inhibition binding curves between [3H]NMS (0.2 and 2.0 nM) and MB was applied and compared using either an allosteric or a competitive model. The allosteric cooperativity of MB binding within MB/NMS/hM receptor complexes was strongly negative and undistinguishable from a competitive interaction throughout all subtypes. Applying the competitive model to the equilibrium binding data of MB and NMS, suggested competition at all hM subtypes: logKI (± S.E.) hM3 = 8.71 ± 0.15, hM1 = 8.68 ± 0.14, hM5 = 8.58 ± 0.07, hM2 = 8.27 ± 0.07 to hM4 = 8.25 ± 0.11. Secondly, the effects of MB on acetylcholine (ACh) induced hM receptor function showed very strong negative allosteric cooperativity at all subtypes pointing against an allosteric antagonism of MB with ACh. Competition with ACh was characterized by logKB: hM1 = 9.53 ± 0.05, hM4 = 9.33 ± 0.05, hM5 = 8.80 ± 0.05, hM2 = 8,79 ± 0.06, to hM3 = 8.43 ± 0.04. In conclusion, MB, below 1 µM, binds competitively and non-selectively (except for the difference between hM3 vs. hM4) to all five hM receptor subtypes with nanomolar affinity and is able to functionally inhibit ACh responses in a competitive fashion, with a slight subtype preference for hM1 and hM4.

Reduced NPY Y1 receptor hippocampal expression and signs of decreased vagal modulation of heart rate in mice.

Central neuropeptide Y (NPY) signaling participates in the regulation of cardiac autonomic outflow, particularly via activation of NPY-Y1 receptors (Y1Rs). However, the specific brain areas and neural pathways involved have not been completely identified yet. Here, we evaluate the role of hippocampal Y1Rs in the modulation of the autonomic control of cardiac function using a conditional knockout mouse model. Radiotelemetric transmitters were implanted in 4-month-old male mice exhibiting reduced forebrain expression (rfb) of the Y1R (Npy1rrfb, n=10) and their corresponding controls (Npy1r2lox, n=8). ECG signals were recorded (i) during resting conditions, (ii) under selective pharmacological manipulation of cardiac vagal activity, and (iii) during acute and chronic psychosocial stress challenges, and analyzed via time- and frequency-domain analysis of heart rate variability. Npy1rrfb mice showed a lower Npy1r mRNA density in the dentate gyrus and in the CA1 region of the hippocampus. Under resting undisturbed conditions, Npy1rrfb mice exhibited (i) a higher heart rate, (ii) a reduced overall heart rate variability, and (iii) lower values of the indices of vagal modulation compared to Npy1r2lox counterparts. Following pharmacological vagal inhibition, heart rate was higher in control but not in Npy1rrfb mice compared to their respective baseline values, suggesting that tonic vagal influences on heart rate were reduced in Npy1rrfb mice. The magnitude of the heart rate response to acute stressors was smaller in Npy1rrfb mice compared to Npy1r2lox counterparts, likely due to a concurrent lower vagal withdrawal. These findings suggest that reduced Y1R expression leads to a decrease in resting vagal modulation and heart rate variability, which, in turn, may determine a reduced cardiac autonomic responsiveness to acute stress challenges.

Pharmacological evidence is consistent with a prominent role of spatial memory in complex navigation.

The ability to learn about the spatial environment plays an important role in navigation, migration, dispersal, and foraging. However, our understanding of both the role of cognition in the development of navigation strategies and the mechanisms underlying these strategies is limited. We tested the hypothesis that complex navigation is facilitated by spatial memory in a population of Chrysemys picta that navigate with extreme precision (±3.5 m) using specific routes that must be learned prior to age three. We used scopolamine, a muscarinic acetylcholine receptor antagonist, to manipulate the cognitive spatial abilities of free-living turtles during naturally occurring overland movements. Experienced adults treated with scopolamine diverted markedly from their precise navigation routes. Naive juveniles lacking experience (and memory) were not affected by scopolamine, and thereby served as controls for perceptual or non-spatial cognitive processes associated with navigation. Further, neither adult nor juvenile movement was affected by methylscopolamine, a form of scopolamine that does not cross the blood-brain barrier, a control for the peripheral effects of scopolamine. Together, these results are consistent with a role of spatial cognition in complex navigation and highlight a cellular mechanism that might underlie spatial cognition. Overall, our findings expand our understanding of the development of complex cognitive abilities of vertebrates and the neurological mechanisms of navigation.

Apolipoprotein E4 reduces evoked hippocampal acetylcholine release in adult mice.

Apolipoprotein E4 (apoE4) is the most prevalent genetic risk factor for Alzheimer's disease. We utilized apoE4-targeted replacement mice (approved by the Tel Aviv University Animal Care Committee) to investigate whether cholinergic dysfunction, which increases during aging and is a hallmark of Alzheimer's disease, is accentuated by apoE4. This revealed that levels of the pre-synaptic cholinergic marker, vesicular acetylcholine transporter in the hippocampus and the corresponding electrically evoked release of acetylcholine, are similar in 4-month-old apoE4 and apolipoprotein E3 (apoE3) mice. Both parameters decrease with age. This decrease is, however, significantly more pronounced in the apoE4 mice. The levels of cholinacetyltransferase (ChAT), acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) were similar in the hippocampus of young apoE4 and apoE3 mice and decreased during aging. For ChAT, this decrease was similar in the apoE4 and apoE3 mice, whereas it was more pronounced in the apoE4 mice, regarding their corresponding AChE and BuChE levels. The level of muscarinic receptors was higher in the apoE4 than in the apoE3 mice at 4 months and increased to similar levels with age. However, the relative representation of the M1 receptor subtype decreased during aging in apoE4 mice. These results demonstrate impairment of the evoked release of acetylcholine in hippocampus by apoE4 in 12-month-old mice but not in 4-month-old mice. The levels of ChAT and the extent of the M2 receptor-mediated autoregulation of ACh release were similar in the adult mice, suggesting that the apoE4-related inhibition of hippocampal ACh release in these mice is not driven by these parameters. Evoked ACh release from hippocampal and cortical slices is similar in 4-month-old apoE4 and apoE3 mice but is specifically and significantly reduced in hippocampus, but not cortex, of 12-month-old apoE4 mice. This effect is accompanied by decreased VAChT levels. These findings show that the hipocampal cholinergic nerve terminals are specifically affected by apoE4 and that this effect is age dependent.

New insight into active muscarinic receptors with the novel radioagonist [³H]iperoxo.

Activation of G protein-coupled receptors involves major conformational changes of the receptor protein ranging from the extracellular transmitter binding site to the intracellular G protein binding surface. GPCRs such as the muscarinic acetylcholine receptors are commonly probed with radioantagonists rather than radioagonists due to better physicochemical stability, higher affinity, and indifference towards receptor coupling states of the former. Here we introduce tritiated iperoxo, a superagonist at muscarinic M2 receptors with very high affinity. In membrane suspensions of transfected CHO-cells, [³H]iperoxo - unlike the common radioagonists [³H]acetylcholine and [³H]oxotremorine M - allowed labelling of each of the five muscarinic receptor subtypes in radioagonist displacement and saturation binding studies. [³H]iperoxo revealed considerable differences in affinity between the even- and the odd-numbered muscarinic receptor subtypes with affinities for the M2 and M4 receptor in the picomolar range. Probing ternary complex formation on the M2 receptor, [³H]iperoxo dissociation was not influenced by an archetypal allosteric inverse agonist, reflecting activation-related rearrangement of the extracellular loop region. At the inner side of M2, the preferred Gi protein acted as a positive allosteric modulator of [³H]iperoxo binding, whereas Gs and Gq were neutral in spite of their robust coupling to the activated receptor. In intact CHO-hM2 cells, endogenous guanylnucleotides promoted receptor/G protein-dissociation resulting in low-affinity agonist binding which, nevertheless, was still reported by [³H]iperoxo. Taken together, the muscarinic superagonist [³H]iperoxo is the best tool currently available for direct probing activation-related conformational transitions of muscarinic receptors.

Long-term activation upon brief exposure to xanomleline is unique to M1 and M4 subtypes of muscarinic acetylcholine receptors.

Xanomeline is an agonist endowed with functional preference for M1/M4 muscarinic acetylcholine receptors. It also exhibits both reversible and wash-resistant binding to and activation of these receptors. So far the mechanisms of xanomeline selectivity remain unknown. To address this question we employed microfluorometric measurements of intracellular calcium levels and radioligand binding to investigate differences in the short- and long-term effects of xanomeline among muscarinic receptors expressed individually in Chinese hamster ovary cells. 1/One-min exposure of cells to xanomeline markedly increased intracellular calcium at hM1 and hM4, and to a lesser extent at hM2 and hM3 muscarinic receptors for more than 1 hour. 2/Unlike the classic agonists carbachol, oxotremorine, and pilocarpine 10-min exposure to xanomeline did not cause internalization of any receptor subtype. 3/Wash-resistant xanomeline selectively prevented further increase in intracellular calcium by carbachol at hM1 and hM4 receptors. 4/After transient activation xanomeline behaved as a long-term antagonist at hM5 receptors. 5/The antagonist N-methylscopolamine (NMS) reversibly blocked activation of hM1 through hM4 receptors by xanomeline. 6/NMS prevented formation of xanomeline wash-resistant binding and activation at hM2 and hM4 receptors and slowed them at hM1, hM3 and hM5 receptors. Our results show commonalities of xanomeline reversible and wash-resistant binding and short-time activation among the five muscarinic receptor subtypes. However long-term receptor activation takes place in full only at hM1 and hM4 receptors. Moreover xanomeline displays higher efficacy at hM1 and hM4 receptors in primary phasic intracellular calcium release. These findings suggest the existence of particular activation mechanisms specific to these two receptors.

Low vagally-mediated heart rate variability and increased susceptibility to ventricular arrhythmias in rats bred for high anxiety.

In humans, there is a documented association between anxiety disorders and cardiovascular disease. Putative underlying mechanisms may include an impairment of the autonomic nervous system control of cardiac function. The primary objective of the present study was to characterize cardiac autonomic modulation and susceptibility to arrhythmias in genetic lines of rats that differ largely in their anxiety level. To reach this goal, electrocardiographic recordings were performed in high-anxiety behavior (HAB, n=10) and low-anxiety behavior (LAB, n=10) rats at rest, during stressful stimuli and under autonomic pharmacological manipulations, and analyzed by means of time- and frequency-domain indexes of heart rate variability. During resting conditions, HAB rats displayed a reduced heart rate variability, mostly in terms of lower parasympathetic (vagal) modulation compared to LAB rats. In HAB rats, this relatively low cardiac vagal control was associated with smaller heart rate responsiveness to acute stressors compared to LAB counterparts. In addition, beta-adrenergic pharmacological stimulation induced a larger incidence of ventricular tachyarrhythmias in HABs compared to LABs. At sacrifice, a moderate increase in heart-body weight ratio was observed in HAB rats. We conclude that high levels of anxiety-related behavior in rats are associated with signs of i) impaired autonomic modulation of heart rate (low vagally-mediated heart rate variability), ii) poor adaptive heart rate responsiveness to stressful stimuli, iii) increased arrhythmia susceptibility, and iv) cardiac hypertrophy. These results highlight the utility of the HAB/LAB model for investigating the mechanistic basis of the comorbidity between anxiety disorders and cardiovascular disease.

Vagal withdrawal and susceptibility to cardiac arrhythmias in rats with high trait aggressiveness.

Personality characteristics, e.g. aggressiveness, have long been associated with an increased risk of cardiac disease. However, the underlying mechanisms remain unclear. In this study we used a rodent model for characterizing cardiac autonomic modulation in rats that differ widely in their level of aggressive behavior. To reach this goal, high-aggressive (HA, n = 10) and non-aggressive (NA, n = 10) rats were selected from a population (n = 121) of adult male Wild-type Groningen rats on the basis of their latency time to attack (ALT, s) a male intruder in a resident-intruder test lasting 600 s. In order to obtain information on their cardiac autonomic modulation, ECG recordings were subsequently obtained via radiotelemetry at rest, during stressful stimuli and under autonomic pharmacological manipulations, and analyzed by means of time- and frequency-domain indexes of heart rate variability. During resting conditions, HA rats (ALT<90 s) displayed reduced heart rate variability, mostly in terms of lower vagal modulation compared to NA rats (ALT>600 s). Exposure to stressful stimuli (i.e. restraint and psychosocial stress) provoked similar tachycardic responses between the two groups. However, under stress conditions HA rats displayed a reduced vagal antagonism and an increased incidence of tachyarrhythmias compared to NA rats. In addition, beta-adrenergic pharmacological stimulation induced a much larger incidence of ventricular tachyarrhythmias in HA rats compared to NA counterparts. These findings are consistent with the view that high levels of aggressive behavior in rats are associated to signs of cardiac autonomic impairment and increased arrhythmogenic susceptibility that may predict vulnerability to cardiac morbidity and mortality.

Heterotropic cooperativity within and between protomers of an oligomeric M(2) muscarinic receptor.

At least four allosteric sites have been found to mediate the dose-dependent effects of gallamine on the binding of [(3)H]quinuclidinylbenzilate (QNB) and N-[(3)H]methylscopolamine (NMS) to M(2) muscarinic receptors in membranes and solubilized preparations from porcine atria, CHO cells, and Sf9 cells. The rate of dissociation of [(3)H]QNB was affected in a bell-shaped manner with at least one Hill coefficient (n(H)) greater than 1, indicating that at least three allosteric sites are involved. The level of binding of [(3)H]QNB was decreased in a biphasic manner, revealing at least two allosteric sites; binding of [(3)H]NMS was affected in a triphasic, serpentine manner, revealing at least three sites, and values of n(H) >1 pointed to at least four sites. Several lines of evidence indicate that all effects of gallamine were allosteric in nature and could be observed at equilibrium. The rates of equilibration and dissociation suggest that the receptor was predominately oligomeric, and the heterogeneity revealed by gallamine can be attributed to differences in its affinity for the constituent protomers of a tetramer. Those differences appear to arise from inter- and intramolecular cooperativity between gallamine and the radioligand.

Reversal of scopolamine-induced disruption of prepulse inhibition by clozapine in mice.

Prepulse inhibition (PPI) of the acoustic startle reflex refers to the reduction of the startle response to an intense acoustic pulse stimulus when it is shortly preceded by a weak non-startling prepulse stimulus and provides a cross-species measure of sensory-motor gating. PPI is typically impaired in schizophrenia patients, and a similar impairment can be induced in rats by systemic scopolamine, a muscarinic cholinergic receptor antagonist that can evoke a range of cognitive and psychotic symptoms in healthy humans that are commonly referred to as the "anti-muscarinic syndrome" resembling some clinical features of schizophrenia. Scopolamine-induced PPI disruption has therefore been proposed as an anti-muscarinic animal model of schizophrenia, but parallel investigations in the mouse remain scant and the outcomes are mixed and often confounded by an elevation of startle reactivity. Here, we distinguished the PPI-disruptive and the confounding startle-enhancing effects of scopolamine (1 and 10mg/kg, i.p.) in C57BL/6 wild-type mice by showing that the latter partly stemmed from a shift in spontaneous baseline reactivity. With appropriate correction for between-group differences in startle reactivity, we went on to confirm that the PPI-disruptive effect of scopolamine could be nullified by clozapine pre-treatment (1.5mg/kg, i.p.) in a dose-dependent manner. This is the first demonstration that scopolamine-induced PPI disruption is sensitive to atypical antipsychotic drugs. In concert with previous data showing its sensitivity to haloperidol the present finding supports the predictive validity of the anti-muscarinic PPI disruption model for both typical and atypical antipsychotic drug action.

Intracellular distribution of functional M(1) -muscarinic acetylcholine receptors in N1E-115 neuroblastoma cells.

Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M(1) -mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M(1) -mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-(3) H]-benzilate ([(3) H]-QNB) whereas the hydrophilic probe, 1-[N-methyl-(3) H] scopolamine ([(3) H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M(1) -mAChR antibody also detected both cell-surface and intracellular M(1) -mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca(2+) mobilization were completely inhibited by a cell-impermeable M(1) antagonist, muscarinic toxin -7 and the G(q/11) inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of G(q/11) (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M(1) -mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes.

Characterization of muscarinic receptors in the human bladder mucosa: direct quantification of subtypes using 4-DAMP mustard.

OBJECTIVE: To characterize pharmacologically relevant muscarinic receptors in the human bladder mucosa and detrusor muscle using radioligand binding assays with [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) and 4-DAMP mustard. METHODS: Muscarinic receptors in homogenates of bladder mucosa, detrusor muscle, and parotid gland were measured using the radioligand [3H]NMS. 4-DAMP mustard was used to inactivate M3 receptors irreversibly. RESULTS: Specific [3H]NMS binding in the homogenates of the mucosa and detrusor muscle was saturable and of high affinity as shown by dissociation constants (Kd) of 260 ±82 and 237 ±49 pM, respectively. Antimuscarinic agents (oxybutynin, propiverine, tolterodine, and darifenacin) and their active metabolites competed with [3H]NMS for the binding sites in the human mucosa in a concentration-dependent manner. These agents exhibited similar affinity in the detrusor muscle. The Bmax. values of [3H]NMS in the detrusor, bladder mucosa, and parotid gland were significantly decreased by pretreatment with 4-DAMP mustard (36%, 41% and 63%, respectively). CONCLUSION: The density and binding affinity profile of the muscarinic receptor population in the human bladder mucosa was shown to be similar to that of the detrusor muscle. The density of the M3 subtype in the mucosa was similar to that in the detrusor muscle but lower than that in the parotid gland.

Sensorimotor gating and vigilance-dependent choice accuracy: a within-subject correlative analysis in wild-type C57BL/6 mice.

Deterioration in attention and related processes is an early sign in schizophrenia predictive of disease development. Amongst the various translational paradigms for assessing attention in rodents, it is not known if they are equivalent in detecting individual differences. Answers here are pertinent to their use in the general human population for identifying individuals at high risk of developing schizophrenia. The present study employed a within-subject approach to examine in mice two common paradigms for assessing attention that differ markedly in their implementation. An operant-based two-choice visual discrimination task (2-CVDT) that depends on effortful attention to brief visual cues was contrasted with prepulse inhibition (PPI) of the acoustic startle reflex, a well-established test of pre-attentive gating whereby processing of a startle-eliciting stimulus is inhibited by a preceding weak prepulse stimulus. Here, we revealed a correlation showing that individual mice with low PPI tended to perform poorly in the 2-CVDT in terms of choice accuracy but not response speed. This specific positive correlation suggests that the two readouts might be regulated via common attentional mechanisms, which might be critically dependent on normal muscarinic and N-methyl-d-asparate receptor functions. As demonstrated here, blockade of either receptor type by scopolamine or dizocilpine impaired 2-CVDT performance at doses that have been shown to disrupt PPI in mice. Further studies contrasting these two paradigms would be warranted to characterize the possible underlying psychological constructs that give rise to this correlation and to clarify whether the two paradigms may effectively capture schizophrenia-related cognitive deficits belonging to orthogonal domains.

Assessment of novel muscarinic acetylcholine receptors in rat cerebral cortex by a tissue segment binding method.

Muscarinic acetylcholine receptors (mAChRs) of rat cerebral cortex were evaluated using a tissue segment radioligand binding assay. [(3)H]-Quinuclidinyl benzilate (QNB, a hydrophobic ligand) specifically bound to mAChRs in the cortex segments. The total mAChRs level was approximately 2,000 fmol/mg protein, which was estimated after incubation for 120 min at 37 degrees C or for 8 h at 4 degrees C. These mAChRs were a mixture of high- and low-affinity sites for N-methylscopolamine (NMS) in a 70:30 ratio. In contrast, only a single high-affinity site for NMS was detected following incubation for 30 min at 37 degrees C, whose abundance was about 70% of that of the total mAChRs. Atropine showed a single affinity for mAChRs under all conditions. These indicate that mAChRs are constitutively expressed not only on plasma membrane sites but also at intracellular sites in rat cerebral cortex and that the receptors at both sites have different affinities for NMS. Acetylcholine completely inhibited [(3)H]-QNB binding to both mAChRs without any change in the subcellular distribution, suggesting the possibility that acetylcholine can access, and bind to, both mAChRs in intact tissue. Two different affinity states for acetylcholine were detected only in plasma membrane mAChRs at 37 degrees C. The present study demonstrates a unique subcellular distribution, and distinct pharmacological profiles, of mAChRs in rat cerebral cortex.

Pharmacological magnetic resonance imaging of muscarinic acetylcholine receptor activation in rat brain.

The central cholinergic system is involved in several cognitive functions such as attention, consciousness, learning and memory. Functional imaging of this neurotransmitter system may provide novel opportunities in the diagnosis and evaluation of cognitive disorders. The aim of this study was to investigate the spatial and temporal activation patterns of muscarinic acetylcholine receptor (mAChR) stimulation in rat brain with pharmacological magnetic resonance imaging (phMRI). We performed blood oxygenation level-dependent (BOLD) MRI and contrast-enhanced cerebral blood volume (CBV)-weighted MRI combined with injection of pilocarpine, a non-selective mAChR agonist. BOLD and CBV responses were assessed after pretreatment with methyl-scopolamine in order to block peripheral muscarinic effects. Region-of-interest analysis in individual animals and group-level independent component analysis failed to show significant BOLD signal changes following pilocarpine injection. However, with contrast-enhanced CBV-weighted MRI, positive CBV responses were detected in the cerebral cortex, thalamus, and hippocampus whereas a negative CBV response was observed in the striatum. Thus, pilocarpine-induced significant activation responses in brain regions that are known to have a high density of muscarinic receptors. Our study demonstrates that phMRI of mAChR stimulation in rats allows functional assessment of the cholinergic system in vivo.

Detection of novel functional selectivity at M3 muscarinic acetylcholine receptors using a Saccharomyces cerevisiae platform.

"Functional selectivity", although new to many chemists and biologists only a few years ago, has now become a dominant theme in drug discovery. This concept posits that different ligands engender unique receptor conformations such that only a subset of signaling pathways linked to a given receptor are recruited. However, successful exploitation of the phenomenon to achieve pathway-based selectivity requires the ability to routinely detect it when assessing ligand behavior. We have utilized different strains of the yeast S. cerevisiae, each expressing a specific human Galpha/yeast Gpa1 protein chimera coupled to a MAP kinase-linked reporter gene readout, to investigate the signaling of the M(3) muscarinic receptor, a G protein-coupled receptor (GPCR) for which various antagonists are used clinically. Using this novel platform, we found that the "antagonists", atropine, N-methylscopolamine, and pirenzepine, were inverse agonists for Gpa1/Galpha(q) but low efficacy agonists for Gpa1/Galpha(12.) Subsequent studies with atropine performed in mammalian 3T3 cells validated these findings by demonstrating inverse agonism for G(q/11)-mediated calcium mobilization but positive agonism for G(12)-mediated membrane ruffling. This is the first study to utilize a yeast platform to discover pathway-biased functional selectivity in a GPCR. In addition to the likely applicability of this approach for identifying biased signaling by novel chemical entities, our findings also suggest that currently marketed medications may exhibit hitherto unappreciated functional selectivity.

Acute ethanol exposure elevates muscarinic tone in the septohippocampal system.

The septohippocampal system has been implicated in the cognitive deficits associated with ethanol consumption, but the cellular basis of ethanol action awaits full elucidation. In the medial septum/diagonal band of Broca (MS/DB), a muscarinic tone, reflective of firing activity of resident cholinergic neurons, regulates that of their noncholinergic, putatively GABAergic, counterparts. Here we tested the hypothesis that ethanol alters this muscarinic tone. The spontaneous firing activity of cholinergic and noncholinergic MS/DB neurons were monitored in acute MS/DB slices from C57Bl/6 mice. Exposing the entire slice to ethanol increased firing in both cholinergic and noncholinergic neurons. However, applying ethanol focally to individual MS/DB neurons increased firing only in cholinergic neurons. The differential outcome suggested different mechanisms of ethanol action on cholinergic and noncholinergic neurons. Indeed, with bath-perfused ethanol, the muscarinic antagonist methyl scopolamine prevented the increase in firing in noncholinergic, but not cholinergic, MS/DB neurons. Thus, the effect on noncholinergic neuronal firing was secondary to ethanol's direct action of acutely increasing muscarinic tone. We propose that the acute ethanol-induced elevation of muscarinic tone in the MS/DB contributes to the altered net flow of neuronal activity in the septohippocampal system that underlies compromised cognitive function.

Role of acetylcholine in negative patterning in turtles (Chrysemys picta).

Turtles were run on a negative patterning task involving 2 positive elements, a key with white stripes on a black background, and a solid red key, and a compound stimulus combining the 2 elements, white stripes on a red background. Injections of scopolamine, methylscopolamine, or saline were started at the same time that the compound stimulus was introduced, after the animals had been autoshaped to press the key for each of the elements. Scopolamine disrupted the learning of negative patterning, but methylscopolamine had no effect. In contrast, learning of a simple discrimination between the elements was not affected by scopolamine. These results show that muscarinic cholinergic receptors are involved in the learning of negative patterning in turtles.