mACh Receptor Research Articles

Effects of Free and Nanoencapsulated Benznidazole in Acute Trypanosoma cruzi Infection: Role of Cholinergic Pathway and Redox Status

Background/Objectives: The Trypanosoma cruzi infection promotes an intense inflammatory process that affects several tissues. The cholinergic system may exert a regulatory immune response and control the inflammatory process. This study aimed to evaluate the comparative effect of free and nanoencapsulated benznidazole in acute T. cruzi infection to assess hematological, biochemical, and oxidative status triggered by the cholinergic system. Methods: For this, fifty female Swiss mice were distributed in eight groups, i.e., uninfected and infected animals under four treatment protocols: untreated (control—CT); vehicle treatment (Eudragit L 100—EL-100); benznidazole treatment (BNZ); and nanoencapsulated benznidazole treatment (NBNZ). After eight treatment days, the animals were euthanized for sample collection. Results: The peak of parasitemia was at day 7 p.i., and the BNZ and NBNZ controlled and reduced the parasite rate but showed no efficacy in terms of total elimination of parasites analyzed by RT-PCR in both infected groups. The infection promotes significant anemia, leukopenia, and thrombocytopenia, which the BNZ improves. There was an increase in AChE activity during infection, leading to a pro-inflammatory response and an increase in M1 and M2 mACh receptors in the BNZ group, showing that the treatment interacted with the cholinergic pathway. In addition, a pro-oxidative response was characterized in the infection and mainly in the infected BNZ and NBNZ groups. The histopathological analysis showed significative splenomegaly and inflammatory infiltrate in the heart, liver, and spleen. Conclusions: The administration of the BNZ or NBNZ reverses hematological, hepatic, and renal alterations through cholinergic signaling and stimulates a pro-inflammatory response during acute T. cruzi infection.

Development of simultaneous determination of dopamine 2, histamine 1, and muscarinic acetylcholine receptor occupancies by antipsychotics using liquid chromatography with tandem mass spectrometry

Receptor occupancy is an indicator of antipsychotic efficacy and safety. It is desirable to simultaneously determine the occupancy of multiple brain receptors as an indicator of the efficacy and central side effects of antipsychotics because many of these drugs have binding affinities for various receptors, such as dopamine 2 (D2), histamine 1 (H1), and muscarinic acetylcholine (mACh) receptors. The purpose of this study was to develop a method for the simultaneous measurement of multiple receptor occupancies in the brain by the simultaneous quantification of unlabeled tracer levels using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Rats were pre-administered with a vehicle, displacer, or olanzapine, and mixed solutions of raclopride, doxepin, and 3-quinuclidinyl benzilate (3-QNB) were administered (3, 10, and 30 μg/kg). The brain tissue and plasma tracer concentrations were quantified 45 min later using LC-MS/MS, and the binding potential was calculated. The highest binding potential was observed at 3 μg/kg raclopride, 10 μg/kg doxepin, and 30 μg/kg 3-QNB. Tracer-specific binding at these optimal tracer doses in the cerebral cortex was markedly reduced by pre-administration of displacers. D2, H1, and mACh receptor occupancy by olanzapine increased in a dose-dependent manner, reaching 70–95%, 19–43%, and 12–45%, respectively, at an olanzapine dose range of 3–10 mg/kg. These results suggest that simultaneous determination of in vivo D2, H1, and mACh receptor occupancy is possible using LC-MS/MS.

Regulation of Src family kinases by muscarinic acetylcholine receptors in heterologous cells and neurons.

Five muscarinic acetylcholine (mACh) receptor subtypes are divided into two classes: the M1 class (M1, M3, and M5) and the M2 class (M2 and M4). The former is coupled to Gq proteins, while the latter is coupled to Gi/o proteins. Accumulating evidence indicates that mACh receptors play a significant role in the regulation of the Src family kinase (SFK), a subfamily of non-receptor tyrosine kinases. mACh receptors exert their roles in a subtype-dependent fashion and preferentially target Src and Fyn, two members of SFKs that are expressed in the brain and enriched at synaptic sites. While the M1 receptor positively modulates SFK activity, the M4 receptor inhibits it. By modulating SFKs, mACh receptors are actively involved in the regulation of expression and function of a variety of receptors, structural proteins, and signaling molecules. In particular, the M4 receptor and the dopamine D1 receptor are coexpressed in striatonigral projection neurons of the striatum. Gi/o-coupled M4 and Gq-coupled D1 receptors antagonistically regulate SFK activity, thereby forming a dynamic balance controlling glutamate receptor activity, excitability of neurons, and synaptic plasticity. In summary, mACh receptors play a crucial role in regulating SFK activity in heterologous cells and neurons.

Activation of mACh Receptors, but Not the nACh Receptors, in VTA Ameliorates Anxiety-Like Behaviors in PTSD Model Rats

Activation of mACh Receptors, but Not the nACh Receptors, in VTA Ameliorates Anxiety-Like Behaviors in PTSD Model Rats

Muscarinic and NMDA Receptors in the Substantia Nigra Play a Role in Reward-Related Learning.

Reward-related learning, where animals form associations between rewards and stimuli (i.e., conditioned stimuli [CS]) that predict or accompany those rewards, is an essential adaptive function for survival. In this study, we investigated the mechanisms underlying the acquisition and performance of conditioned approach learning with a focus on the role of muscarinic acetylcholine (mACh) and NMDA glutamate receptors in the substantia nigra (SN), a brain region implicated in reward and motor processes. Using RNAscope in situ hybridization assays, we found that dopamine neurons of the SN express muscarinic (mACh5), NMDA2a, NMDA2b, and NMDA2d receptor mRNA but not mACh4. NMDA, but not mACh5, receptor mRNA was also found on SN GABA neurons. In a conditioned approach paradigm, rats were exposed to 3 or 7 conditioning sessions during which light/tone (CS) presentations were paired with delivery of food pellets, followed by a test session with CS-only presentations. Intra-SN microinjections of scopolamine (a mACh receptor antagonist) or AP-5 (a NMDA receptor antagonist) were made either prior to each conditioning session (to test their effects on acquisition) or prior to the CS-only test (to test their effects on expression of the learned response). Scopolamine and AP-5 produced dose-dependent significant reductions in the acquisition, but not performance, of conditioned approach. These results suggest that SN mACh and NMDA receptors are key players in the acquisition, but not the expression, of reward-related learning. Importantly, these findings redefine the role of the SN, which has traditionally been known for its involvement in motor processes, and suggest that the SN possesses attributes consistent with a function as a hub of integration of primary reward and CS signals.

Localization of muscarinic acetylcholine receptor-dependent rhythm-generating modules in the Drosophila larval locomotor network.

Mechanisms of rhythm generation have been extensively studied in motor systems that control locomotion over terrain in limbed animals; however, much less is known about rhythm generation in soft-bodied terrestrial animals. Here we explored how muscarinic acetylcholine receptor (mAChR)-modulated rhythm-generating networks are distributed in the central nervous system (CNS) of soft-bodied Drosophila larvae. We measured fictive motor patterns in isolated CNS preparations, using a combination of Ca2+ imaging and electrophysiology while manipulating mAChR signaling pharmacologically. Bath application of the mAChR agonist oxotremorine potentiated bilaterally asymmetric activity in anterior thoracic regions and promoted bursting in posterior abdominal regions. Application of the mAChR antagonist scopolamine suppressed rhythm generation in these regions and blocked the effects of oxotremorine. Oxotremorine triggered fictive forward crawling in preparations without brain lobes. Oxotremorine also potentiated rhythmic activity in isolated posterior abdominal CNS segments as well as isolated anterior brain and thoracic regions, but it did not induce rhythmic activity in isolated anterior abdominal segments. Bath application of scopolamine to reduced preparations lowered baseline Ca2+ levels and abolished rhythmic activity. Overall, these results suggest that mAChR signaling plays a role in enabling rhythm generation at multiple sites in the larval CNS. This work furthers our understanding of motor control in soft-bodied locomotion and provides a foundation for study of rhythm-generating networks in an emerging genetically tractable locomotor system.NEW & NOTEWORTHY Using a combination of pharmacology, electrophysiology, and Ca2+ imaging, we find that signaling through mACh receptors plays a critical role in rhythmogenesis in different regions of the Drosophila larval CNS. mAChR-dependent rhythm generators reside in distal regions of the larval CNS and provide functional substrates for central pattern-generating networks (CPGs) underlying headsweep behavior and forward locomotion. This provides new insights into locomotor CPG operation in soft-bodied animals that navigate over terrain.

Oryeongsan (Wulingsan) ameliorates impaired ANP secretion of atria from spontaneously hypertensive rats

Oryeongsan (ORS), a herbal medicine formula, has long been used for the treatment of impaired body water balance in Asian countries. Recently, it was shown that ORS administration modulates the renin-angiotensin system (RAS). Purpose of the present study was to determine characteristics of atrial ANP secretion and effects of ORS on the secretion in the atria from spontaneously hypertensive rats (SHR). Normotensive WKY groups (WKY-V, WKY-ORS, WKY-LOS) and hypertensive SHR groups (SHR-V, SHR-ORS, SHR-LOS) treated with vehicle, ORS, and losartan as a positive control group, respectively, were used. Experiments were performed in perfused beating atria (1.3 Hz) allowing atrial distension, acetylcholine (ACh) stimulation, and serial collection of atrial perfusates. The secreted ANP concentration was measured using radioimmunoassay. Interstitial fluid (ISF) translocation was measured using [3H]inulin clearance. Stepwise increase in atrial distension by 1.1, 2.0, and 2.7 cmH2O above basal distension further increased ANP secretion proportionally in the atria from WKY-V, but the response was significantly suppressed in the atria from SHR-V. Cardiomyocyte ANP release, the first step of atrial ANP secretion, was suppressed in the atria from SHR-V compared to those from WKY-V (−8.02 ± 2.86, −15.86 ± 2.27, and −20.09 ± 3.62%; n = 8, for SHR-V vs. 8.59 ± 2.81, 15.65 ± 7.14, and 38.12 ± 8.28%; n = 8, for WKY-V; p < 0.001 for all stepwise distension, respectively). Chronic treatment with ORS reversed the suppressed ANP release in atria from SHR-ORS group (6.76 ± 3.92, 9.12 ± 2.85, and 28.79 ± 1.79% for SHR-ORS; n = 5 vs. SHR-V; n = 8; p = 0.01, p < 0.001, p < 0.001, respectively). The effects of ORS were comparable to those of losartan. Trans-endocardial translocation of ISF, the second step of atrial ANP secretion was similar in the atria from the hypertensive SHR-V and normotensive WKY-V. ACh-induced ANP secretion and cardiomyocyte ANP release were also suppressed in the atria from SHR-V compared to WKY-V and ORS reversed the suppression. These findings were accompanied with accentuation of the AT1 receptor expression and suppression of the AT2/Mas receptor, M2 mACh receptor and GIRK4, a molecular component of KACh channel, expression in the atria from SHR-V. Further, treatment with ORS or losartan reversed the expressions in the groups of SHR-ORS and SHR-LOS. These results show that ANP secretion is suppressed in the atria from SHR in association with accentuation of AT1 receptor and suppression of AT2/Mas receptor and KACh channel expression. Treatment with ORS ameliorates impaired ANP secretion through improving cardiomyocyte ANP release with modulation of the cardiac RAS and muscarinic signaling. These findings provide experimental evidence which supports the effect of ORS on the regulation of atrial ANP secretion in the atria from SHR.

The cholinergic system modulates negative BOLD responses in the prefrontal cortex once electrical perforant pathway stimulation triggers neuronal afterdischarges in the hippocampus

Repeated high-frequency pulse-burst stimulations of the rat perforant pathway elicited positive BOLD responses in the right hippocampus, septum and prefrontal cortex. However, when the first stimulation period also triggered neuronal afterdischarges in the hippocampus, then a delayed negative BOLD response in the prefrontal cortex was generated. While neuronal activity and cerebral blood volume (CBV) increased in the hippocampus during the period of hippocampal neuronal afterdischarges (h-nAD), CBV decreased in the prefrontal cortex, although neuronal activity did not decrease. Only after termination of h-nAD did CBV in the prefrontal cortex increase again. Thus, h-nAD triggered neuronal activity in the prefrontal cortex that counteracted the usual neuronal activity-related functional hyperemia. This process was significantly enhanced by pilocarpine, a mACh receptor agonist, and completely blocked when pilocarpine was co-administered with scopolamine, a mACh receptor antagonist. Scopolamine did not prevent the formation of the negative BOLD response, thus mACh receptors modulate the strength of the negative BOLD response.

Modulation of spatial memory and expression of hippocampal neurotransmitter receptors by selective lesion of medial septal cholinergic and GABAergic neurons.

The medial septum (MS) is an important modulator of hippocampal function. The degree of damage in which the particular set of septo-hippocampal projections contributes to the deficits of spatial memory with concomitant changes of hippocampal receptors expression has not been studied till present. Therefore, we investigated spatial memory and the expression level of cholinergic (α7 nACh and M1), GABAergic (α1 subunit of GABAA) and glutamatergic (NR2B subunit of NMDA and GluR 1 subunit of AMPA) receptors in the hippocampus following selective lesions of cholinergic and GABAergic septo-hippocampal projection. Learning process and long-term spatial memory were assessed using a Morris water maze. The obtained results revealed that in contrast to cholinergic lesions, rats with MS GABAergic lesions exhibit a retention deficit in 3days after training. Western blot analyses revealed the MS cholinergic lesions have significant effect on the expression level of the M1 mACh receptors, while MS GABAergic lesions induce dramatic modulations of hippocampal glutamatergic, cholinergic and GABAergic receptors expression. These results for the first time demonstrated thatselective lesions of MS cholinergic and GABAergic neurons differentially affect long-term spatial memory andthe memory deficit after MS GABAergic lesion is paralleled with significant changes of hippocampal glutamate, GABA and acetylcholine receptors expression.

Levetiracetam induction of theta frequency oscillations in rodent hippocampus in vitro.

Levetiracetam (LEV) has been demonstrated to improve cognitive function. Hippocampal theta rhythm (4-12 Hz) is associated with a variety of cognitively related behaviors, such as exploration in both humans and animal models. We investigated the effects of LEV on the theta rhythm in the rat hippocampal CA3 in hippocampal slices in vitro. We found that LEV increased the theta power in a dose-dependent manner. The increase in theta power can be blocked by GABAA receptor (GABAAR) or NMDA receptor (NMDAR) antagonists but not by AMPA receptor antagonist, indicating the involvement of GABAAR and NMDAR in the induction of theta activity. Interestingly, LEV enhancement of theta power can be also blocked by taurine or GABA-A agonist THIP, indicating that LEV induction of theta may be related to the indirect boosting of GABA action via reduction of extrasynaptic GABAAR activation. Furthermore, the increased theta power can be partially reduced by the mACh receptor (mAChR) antagonist atropine but not by nACh receptor antagonists, suggesting that mAChR activation provides excitatory input into local network responsible for LEV-induced theta. Our study demonstrated that LEV induced a novel theta oscillation in vitro, which may have implications in the treatment of the neuronal disorders with impaired theta oscillation and cognitive function.

Impairment of cholinergic bladder contractility in rat model of type I diabetes complicated by cystitis: Contribution of neurotransmitter-degrading ectoenzymes

Parasympathetic regulation of urinary bladder contractions primarily involves acetylcholine release and activation of detrusor smooth muscle (DSM) muscarinic acetylcholine (mACh) receptors. Co-release of ATP and activation of DSM purinergic P2X1-receptors may participate as well in some species. Both types of neuromuscular transmission (NMT) are impaired in diabetes, however, which factors may contribute to such impairment remains poorly understood. Here by using rats with streptozotocin(STZ)-induced type I diabetes (8th week after induction) we show that contribution of atropine-sensitive m-cholinergic component to the contractions of urothelium-denuded DSM strips evoked by electric field stimulation (EFS) greatly increased when diabetic bladders presented overt signs of accompanying cystitis. Modeling of hemorrhagic cystitis alone in control rats by cyclophosphamide injection only modestly increased m-cholinergic component of EFS-contractions. However, exposure of DSM strips from control animals to acetylcholinesterase (AChE) inhibitor, neostigmine (1–10 μM) largely reproduced alterations in EFS contractions observed in diabetic DSM complicated by cystitis. Ellman's assay revealed statistically significant 31% decrease of AChE activities in diabetic vs. control DSM. Changes in purinergic contractility of diabetic DSM were consistent with altered P2X1-receptor desensitization and re-sensitization. They could be mimicked by pharmacological inhibition of ATP-degrading ecto-ATPases with ARL 67156 (50 μM), pointing to compromised extracellular ATP clearance as underlying reason. We conclude that decreased AChE activities associated with diabetes and likely cystitis provide complementary factor to the described in literature altered expression of mACh receptor subtypes linked to diabetes as well as to cystitis to produce dramatic modification of cholinergic NMT.

Muscarinic Acetylcholine Receptor-2 in the Cerebellar Fastigial Nucleus is Involved in Acetylcholine-Mediated Blood Pressure Regulation in Rats

Our previous investigations have revealed that microinjection of acetylcholine (ACh) into the cerebellar fastigial nucleus (FN) induces a systemic blood pressure (BP) depressor response and that the muscarinic ACh (mACh) receptor contributes substantially to this process. This study was determined to explore the role of muscarinic ACh receptor-2 (M2 receptor) in ACh-mediated BP regulation and its underlying electrophysiological mechanism. ACh mixed with methoctramine hydrate (MCT, a selective M2 receptor antagonist) and normal saline (0.9% NaCl) were separately microinjected into the FN of anaesthetized rats, and the mean arterial pressure during the reactive time (the duration for BP to return to its basal level) was calculated; the responses of cholinergic reagents to the inward currents in FN neurons were also examined via patch-clamp recording in vitro; in addition, the receptor expression in the FN was confirmed by immunohistochemistry. The results demonstrated that MCT effectively blocked the ACh-mediated BP depressor response and the ACh-induced inward currents in FN neurons; moreover, M2 receptors were unequivocally distributed in the FN. Taken together, the results indicate that M2 receptors in the FN may be involved in ACh-mediated BP down-regulation by tuning neuronal excitation.

Effects of Cortisol-Induced Acetylcholine Accumulation on Tissue Damage and Regeneration in Steatotic Livers in the Context of Partial Hepatectomy Under Vascular Occlusion

Introduction Steatosis is a known risk factor in hepatic surgery and it is associated with postoperative morbidity and mortality. Acetylcholine (ACh) is the major neurotransmitter of vagus nerve and it is released as a response to stressful events like trauma or infection. Stress can also activate the HPA axis that drives the production of cortisol. Both cortisol and ACh have been proved beneficial in experimental models of sepsis, liver transplantation or myocardial ischemia, among other pathologies. In this study, we will study the relationship between cortisol and ACh and their effects on regeneration and injury in seatotic livers submitted to partial hepatectomy (PH) under vascular occlusion, a common technique used in hepatic surgery to avoid bleeding. Materials & Methods We determined hepatic levels of ACh and its receptors in obese Zucker rats that underwent partial hepatectomy under vascular occlusion. The ACTH-cortisol pathway was pharmacologically modulated and the effects on hepatic injury and regeneration, as well as its underlying mechanisms of action, were evaluated. Results There is a significant ACh accumulation driven by the administration of cortisol in steatotic livers submitted to PH under vascular occlusion, that consequently leads to an overexpression of a7 nicotinic and M3 muscarinic ACh receptors. However, administration of ACh prior to surgery increases injury and impairs regeneration via M3 mACh receptor. Interestingly, M3 mAChR antagonist, but not a7 nAChR, alone or in combination with cortisol, did confer eprotection against liver damage caused by the surgical procedure and enhanced regeneration. Moreover, administration of ACh reduced PI3K/Akt hepatic expression and consequently reduced levels of markers of oxidative stress, neutrophil accumulation and edema. In consonance with the results presented above, treatment with an M3 mAChR antagonist, but not a7 nAChR, alone or in combination with cortisol, reduced the hepatic levels of these harmful markers as a result of an overexpression of PI3K and pAKT in these steatotic livers. Thus, this indicates a lesser role of a7 nAChR. Conclusions The reported benefits of cortisol in PH cannot be extrapolated in situations of PH under vascular occlusion in steatotic livers. Cortisol is responsible of the accumulation of acetylcholine in these livers submitted to partial hepatectomy with vascular occlusion. ACh exerts its action through the M3 ACh receptor, as it exacerbates liver damage and oxidative stress and impairs regeneration. Strategies aimed to block cortisol activity in order to block ACh accumulation and/or blockade of M3 mAChR are probably worthwhile to protect steatotic livers and reduce the incidence of postoperative complications following PH under vascular occlusion.

The strength of reward-related learning depends on the degree of activation of ventral tegmental area dopamine neurons

We tested whether (1) the capacity of a reward-associated conditioned stimulus (CS) to cause conditioned activation of ventral tegmental area (VTA) dopamine (DA) neurons is associated with its capacity to elicit conditioned approach responses and (2) whether the acquisition of these capacities by a CS requires N-methyl-D-aspartate (NMDA) and muscarinic acetylcholine (mACh) receptor stimulation. Rats were trained to emit a conditioned approach response to a light CS that was previously paired with food and were treated systemically with scopolamine (a mACh receptor antagonist) or MK-801 (an NMDA receptor antagonist) either prior to each conditioning session (during which animals experienced paired CS and food presentations) or prior to the conditioned approach (CS-only) test. Brains were harvested after the CS-only test and processed for tyrosine hydroxylase (TH) (DA cells) and c-fos in the VTA. When animals received scopolamine or MK-801 treatment prior to conditioning sessions we observed significantly fewer TH-labeled (i.e., DA) cells in the VTA that expressed c-fos and significantly less conditioned approach responding during the CS-only test. Further analysis showed a correlation between the number of VTA DA cells activated and the number of conditioned approach responses. Treatments made prior to the CS-only test did not affect responding. Altogether these results suggest that the degree to which a CS elicits conditioned approach depends partially on the degree to which the CS activates VTA DA cells and that the acquisition of both of these capacities by a CS requires mACh and NMDA receptor stimulation.

Inhibition of basal and amphetamine-stimulated extracellular signal-regulated kinase (ERK) phosphorylation in the rat forebrain by muscarinic acetylcholine M4 receptors

The mitogen-activated protein kinase (MAPK), especially its extracellular signal-regulated kinase (ERK) subfamily, is a group of kinases enriched in the mammalian brain. While ERK is central to cell signaling and neural activities, the regulation of ERK by transmitters is poorly understood. In this study, the role of acetylcholine in the regulation of ERK was investigated in adult rat striatum in vivo. We focused on muscarinic M1 and M4 receptors, two principal muscarinic acetylcholine (mACh) receptor subtypes in the striatum. A systemic injection of the M1-preferring antagonist telenzepine did not alter ERK phosphorylation in the two subdivisions of the striatum, the caudate putamen and nucleus accumbens. Similarly, telenzepine did not affect ERK phosphorylation in the medial prefrontal cortex (mPFC), hippocampus, and cerebellum. Moreover, telenzepine had no effect on the ERK phosphorylation induced by dopamine stimulation with the psychostimulant amphetamine. In contrast to telenzepine, the M4-preferring antagonist tropicamide consistently increased ERK phosphorylation in the striatum and mPFC. This increase was rapid and transient. Tropicamide and amphetamine when coadministered at subthreshold doses induced a significant increase in ERK phosphorylation. These results demonstrate that mACh receptors exert a subtype-specific modulation of ERK in striatal and mPFC neurons. While the M1 receptor antagonist has no effect on ERK phosphorylation, M4 receptors inhibit constitutive and dopamine-stimulated ERK phosphorylation in these dopamine-innervated brain regions.

Muscarinic Acetylcholine Receptors Inhibit Fyn Activity in the Rat Striatum In Vivo.

The Src family kinase (SFK) is a subfamily of non-receptor tyrosine kinases. SFK members, Src and especially Fyn, are expressed in the striatum. These SFK members are involved in the regulation of neuronal and synaptic activities and are linked to the pathogenesis of a variety of neuropsychiatric and neurodegenerative disorders. Given the fact that muscarinic acetylcholine (mACh) receptors are highly expressed in striatal neurons and are critical for the regulation of striatal function, we investigated the role of mACh receptors in the regulation of SFKs in the adult rat striatum in vivo. We found that pharmacological blockade of mACh receptors by systemic administration of the mACh antagonist scopolamine induced a marked increase in phosphorylation of SFKs in the striatum of male and female rats. This scopolamine-induced increase in SFK phosphorylation occurred in the two subdivisions of the striatum (caudate putamen and nucleus accumbens) and was time-dependent and reversible. Another mACh antagonist atropine was also effective in stimulating SFK phosphorylation. Coadministration of subthreshold doses of scopolamine and a dopamine D1 receptor agonist SKF81297 enhanced striatal SFK phosphorylation. Between Fyn and Src proteins immunoprecipitated from striatal tissue, scopolamine selectively increased phosphorylation of Fyn. The increase in Fyn phosphorylation was accompanied by an increase in Fyn kinase activity in response to scopolamine. These results reveal a significant role of mACh receptors in the regulation of SFKs (mainly Fyn) in striatal neurons. Under normal conditions, endogenous mACh receptors appear to exert an inhibitory effect on Fyn activity.

Blockade of muscarinic acetylcholine receptors in the ventral tegmental area blocks the acquisition of reward-related learning

In the present study we investigated whether stimulation of muscarinic acetylcholine (mACh) receptors in the ventral tegmental area (VTA) plays a role in the acquisition of food-based conditioned approach learning. Rats were exposed to 3 (in Experiment 1) or 7 (in Experiment 2) conditioning sessions in which 30, randomly presented light (CS) presentations were paired with delivery of food pellets (US), followed by one session with no light or food and finally one CS-only test session with only light stimulus presentations. Bilateral microinjections of scopolamine (a mACh receptor antagonist) were made either prior to each conditioning session (Experiment 1; to test effects on acquisition) or prior to the CS-only test (Experiment 2; to test effects on performance of the learned response). Scopolamine produced a dose-related significant reduction in the acquisition of conditioned approach but had no effect on its performance. These results suggest that mACh receptor stimulation in the VTA plays a necessary role in the acquisition of reward-related learning.

Molecular and Cellular Mechanisms of Rapid-Acting Antidepressants Ketamine and Scopolamine

Major depressive disorder (MDD) is a prevalent neuropsychiatric disease that causes profound social and economic burdens. The impact of MDD is compounded by the limited therapeutic efficacy and delay of weeks to months of currently available medications. These issues highlight the need for more efficacious and faster-acting treatments to alleviate the burdens of MDD. Recent breakthroughs demonstrate that certain drugs, including ketamine and scopolamine, produce rapid and long-lasting antidepressant effects in MDD patients. Moreover, preclinical work has shown that the antidepressant actions of ketamine and scopolamine in rodent models are caused by an increase of extracellular glutamate, elevated BDNF, activation of the mammalian target of rapamycin complex 1 (mTORC1) cascade, and increased number and function of spine synapses in the prefrontal cortex (PFC). Here we review studies showing that both ketamine and scopolamine elicit rapid antidepressant effects through converging molecular and cellular mechanisms in the PFC. In addition, we discuss evidence that selective antagonists of NMDA and muscarinic acetylcholine (mACh) receptor subtypes (i.e., NR2B and M1-AChR) in the PFC produce comparable antidepressant responses. Furthermore, we discuss evidence that ketamine and scopolamine antagonize inhibitory interneurons in the PFC leading to disinhibition of pyramidal neurons and increased extracellular glutamate that promotes the rapid antidepressant responses to these agents. Collectively, these studies indicate that specific NMDA and mACh receptor subtypes on GABAergic interneurons are promising targets for novel rapid-acting antidepressant therapies.

Lateral Habenula Involvement in Impulsive Cocaine Seeking.

The lateral habenula (LHb) is a brain structure receiving inputs from limbic forebrain areas and innervating major midbrain monoaminergic nuclei. Evidence indicates LHb involvement in sleep control, reward-based decision making, avoidance of punishment, and responses to stress. Additional work has established that the LHb mediates negative feedback in response to aversive events. As a hallmark of drug addiction is the inability to limit drug use despite negative consequences, we hypothesize that LHb dysfunction may have a role in the lack of control over drug seeking. Here we examine the effects of LHb inactivation in control over drug seeking in several cocaine self-administration (SA) paradigms in rats. We find that inhibition of the LHb with GABAergic agonists did not alter cocaine SA under progressive ratio or seeking/taking chained reinforcement schedules, or during punishment-induced suppression of cocaine-reinforced responding. In contrast, LHb inhibition increased cocaine seeking when the drug was not available in rats trained to discriminate its presence using an environmental cue. This effect of LHb inhibition was selective for cocaine, as it did not impair responding for sucrose reinforcement. The effect of LHb injection of GABA agonists was mimicked by intra-LHb muscarinic cholinergic (mACh) antagonist injection, and activation of mACh receptors excited a majority of LHb neurons in in vitro electrophysiology experiments. These results indicate that the LHb participates in the suppression of impulsive responding for cocaine through the activation of a cholinergic circuit, and they suggest that LHb dysfunction may contribute to impaired impulse control associated with drug addiction.

Microinjection of acetylcholine into cerebellar fastigial nucleus induces blood depressor response in anesthetized rats

It is well known that the cerebellar fastigial nucleus (FN) is involved in cardiovascular modulation, and has direct evidence of cholinergic activity; however, whether and how acetylcholine (ACh) in the FN modulates blood pressure has not been investigated. In this study, we analyzed mean arterial pressure, maximal change in mean arterial pressure, and the reaction time of blood pressure changes after microinjection of cholinergic reagents into the FN in anesthetized rats. The results showed that ACh evoked a concentration-dependent (10, 30 and 100mM) effect on blood pressure down-regulation. The muscarinic ACh (mACh) receptor antagonist atropine, but not the nicotinic ACh (nACh) receptor antagonist mecamylamine, blocked the ACh-mediated depressor response. The mACh receptor agonist oxotremorine M, rather than nACh receptor agonist nicotine, mimicked the ACh-mediated blood pressure decrease in a dose-dependent manner (10, 30 and 100mM). These results indicate that cholinergic input in the cerebellar FN exerts a depressor effect on systemic blood pressure regulation, and such effects are substantially contributed by mACh rather than nACh receptors, although the precise mechanism concerning the role of mACh receptor in FN-mediated blood pressure modulation remains to be elucidated.