Muscarinic Acetylcholine Receptors Modulate HCN Channel Properties in Vestibular Ganglion Neurons.

Efferent modulation of vestibular afferent excitability is linked to muscarinic signaling cascades that close low-voltage-gated potassium channels (i.e., KCNQ). Here, we show that muscarinic signaling cascades also depolarize the activation range of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels. We compared the voltage activation range and kinetics of HCN channels and induced firing patterns before and after administering the muscarinic acetylcholine receptor (mAChR) agonist oxotremorine-M (Oxo-M) in dissociated vestibular ganglion neurons (VGNs) from rats of either sex using perforated whole-cell patch-clamp methods. Oxo-M depolarized HCN channels' half-activation voltage (V 1/2) and sped up the rate of activation near resting potential twofold. HCN channels in large-diameter and/or transient firing VGN (putative cell bodies of irregular firing neuron from central epithelial zones) had relatively depolarized V 1/2 in control solution and were less sensitive to mAChR activation than those found in small-diameter VGN with sustained firing patterns (putatively belonging to regular firing afferents). The impact of mAChR on HCN channels is not a direct consequence of closing KCNQ channels since pretreating the cells with Linopirdine, a KCNQ channel blocker, did not prevent HCN channel depolarization by Oxo-M. Efferent signaling promoted ion channel configurations that were favorable to highly regular spiking in some VGN, but not others. This is consistent with previous observations that low-voltage gated potassium currents in VGN are conducted by mAChR agonist-sensitive and -insensitive channels. Connecting efferent signaling to HCN channels is significant because of the channel's impact on spike-timing regularity and nonchemical transmission between Type I hair cells and vestibular afferents.SIGNIFICANCE STATEMENT Vestibular afferents express a diverse complement of ion channels. In vitro studies identified low-voltage activated potassium channels and hyperpolarization-activated cyclic-nucleotide gated (HCN) channels as crucial for shaping the timing and sensitivity of afferent responses. Moreover, a network of acetylcholine-releasing efferent neurons controls afferent excitability by closing a subgroup of low-voltage activated potassium channels on the afferent neuron. This work shows that these efferent signaling cascades also enhance the activation of HCN channels by depolarizing their voltage activation range. The size of this effect varies depending on the endogenous properties of the HCN channel and on cell type (as determined by discharge patterns and cell size). Simultaneously controlling two ion-channel groups gives the vestibular efferent system exquisite control over afferent neuron activity.

Neuroprotective and Antioxidant Role of Oxotremorine-M, a Non-selective Muscarinic Acetylcholine Receptors Agonist, in a Cellular Model of Alzheimer Disease.

Alzheimer disease (AD) is a multifactorial and age-dependent neurodegenerative disorder, whose pathogenesis, classically associated with the formation of senile plaques and neurofibrillary tangles, is also dependent on oxidative stress and neuroinflammation chronicization. Currently, the standard symptomatic therapy, based on acetylcholinesterase inhibitors, showed a limited therapeutic potential, whereas disease-modifying treatment strategies are still under extensive research. Previous studies have demonstrated that Oxotremorine-M (Oxo), a non-selective muscarinic acetylcholine receptors agonist, exerts neurotrophic functions in primary neurons, and modulates oxidative stress and neuroinflammation phenomena in rat brain. In the light of these findings, in this study, we aimed to investigate the neuroprotective effects of Oxo treatment in an in vitro model of AD, represented by differentiated SH-SY5Y neuroblastoma cells exposed to Aß1-42 peptide. The results demonstrated that Oxo treatment enhances cell survival, increases neurite length, and counteracts DNA fragmentation induced by Aß1-42 peptide. The same treatment was also able to block oxidative stress and mitochondria morphological/functional impairment associated with Aß1-42 cell exposure. Overall, these results suggest that Oxo, by modulating cholinergic neurotransmission, survival, oxidative stress response, and mitochondria functionality, may represent a novel multi-target drug able to achieve a therapeutic synergy in AD. Illustration of the main pathological hallmarks and mechanisms underlying AD pathogenesis, including neurodegeneration and oxidative stress, efficiently counteracted by treatment with Oxo, which may represent a promising therapeutic molecule. Created with BioRender.com under academic license.

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.

Development of KVO treatment strategies for chronic pain in a rat model of Gulf War Illness.

We examined whether combinations of Kv7 channel openers could be effective modifiers of deep tissue nociceptor activity; and whether such combinations could then be optimized for use as safe analgesics for pain-like signs that developed in a rat model of GWI (Gulf War Illness) pain. Voltage clamp experiments were performed on subclassified nociceptors isolated from rat DRG (dorsal root ganglion). A stepped voltage protocol was applied (-55 to -40 mV; Vh = -60 mV; 1500 ms) and Kv7 evoked currents were subsequently isolated by linopirdine subtraction. Directly activated and voltage activated K+ currents were characterized in the presence and absence of Retigabine (5-100 µM) and/or Diclofenac (50-140 µM). Retigabine produced substantial voltage dependent effects and a maximal sustained current of 1.14 pA/pF ± 0.15 (ED50: 62.7 ± 3.18 µM). Diclofenac produced weak voltage dependent effects but a similar maximum sustained current of 1.01 ± 0.26 pA/pF (ED50: 93.2 ± 8.99 µM). Combinations of Retigabine and Diclofenac substantially amplified resting currents but had little effect on voltage dependence. Using a cholinergic challenge test (Oxotremorine, 10 µM) associated with our GWI rat model, combinations of Retigabine (5 uM) and Diclofenac (2.5, 20 and 50 µM) substantially reduced or totally abrogated action potential discharge to the cholinergic challenge. When combinations of Retigabine and Diclofenac were used to relieve pain-signs in our rat model of GWI, only those combinations associated with serious subacute side effects could relieve pain-like behaviors.

A highly selective bulk optode based on 6-{4-(2,4-dihydroxy-phenyl)diazenyl)phenyl}-2-oxo-4-phenyl-1,2-dihydro-pyridine-3-carbonitrile incorporating chromoionophore V for determination of nano levels of cadmium.

A novel optical sensor has been fabricated for highly accurate, simple and selective determination of nanomolar levels of cadmium ions. The sensor depends on the interaction of 6-{4-(2,4-dihydroxyphenyl)diazenyl)phenyl}-2-oxo-4-phenyl-1,2-dihydropyri-dine-3-carbonitrile (DDPODC) with Cd(II) in plasticized (2-nitrophenyloctyl ether) (o-NPOE) polyvinylchloride (PVC) membrane incorporating chromoionophore V as a lipophilic H+-selective indicator. It would seem that the higher Cd(II) concentration, the lower absorbance of chromoionophore V in the membrane at 668 nm, whereas the absorbance at 586 nm increased. The developed sensor at pH 4.7 has a linear range of 5.0 × 10-12 - 2.5 × 10-5 M with limits of detection and quantification of 1.62 × 10-12 and 4.95 × 10-12 M, respectively. The relative standard deviation (RSD) for eight determination of 1.0 × 10-7 M Cd(II) was 1.67%. Finally, the proposed sensor gives good results for applications in the direct determination of cadmium ions in water, food, and biological samples. Additionally, we compared the obtained results with the data obtained from the flame atomic absorption spectrometry (FAAS).

An efficient domino strategy for synthesis of 3-substituted 4-oxo-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine derivatives in water.

A strategy for catalyst-free domino reaction of 4-aminopyridin-2(1H)-ones, arylglyoxal hydrates and different 1,3-dicarbonyl compounds in water has been established. The mild and efficient procedure afforded pyrrolo[3,2-c]pyridine derivatives with 76-94% yields after simple crystallization. The present procedure shows promising characteristics, such as readily available starting materials, the use of water as reaction media, simple and efficient one-pot operation, and avoiding the need for any hazardous or expensive catalysts.

4-Oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-d]pyrimidine Derivatives: Design, Synthesis, Insecticidal Assay and Binding Mode Studies.

A series of 4-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-d]pyrimidine derivatives were designed and synthesized based on the fipronil low energy conformation by scaffold hopping strategy. Physicochemical properties calculation, insecticidal assay and binding mode studies were also performed. It was found that the target compounds displayed lower insecticidal activities than fipronil. The differences in binding modes between these compounds and fipronil may be the major reason for reduced insecticidal activities.

Role of prediction error and the cholinergic system on memory reconsolidation processes in mice.

The ability to make predictions based on stored information is a general coding strategy. A prediction error (PE) is a mismatch between expected and current events. Our memories, like ourselves, are subject to change. Thus, an acquired memory can become active and update its content or strength by a labilization-reconsolidation process. Within the reconsolidation framework, PE drives the updating of consolidated memories. In the past our lab has made key progresses showing that a blockade in the central cholinergic system during reconsolidation can cause memory impairment, while reinforcement of cholinergic activity enhances it. In the present work we determined that PE is a necessary condition for memory to reconsolidate in an inhibitory avoidance task using both male and female mice. Depending on the intensity of the unconditioned stimulus (US) used during training, a negative (higher US intensity) or positive (lower US intensity/no US) PE on a retrieval session modified the behavioral response on a subsequent testing session. Furthermore, we demonstrated that the cholinergic system modulates memory reconsolidation only when PE is detected. In this scenario administration of oxotremorine, scopolamine or nicotine after memory reactivation either enhanced or impaired memory reconsolidation in a sex-specific manner.

Critical role of hippocampal muscarinic acetylcholine receptors on memory reconsolidation in mice.

Over the years, experimental and clinical evidence has given support to the idea that acetylcholine (Ach) plays an essential role in mnemonic phenomena. On the other hand, the Hippocampus is already known to have a key role in learning and memory. What is yet unclear is how the Ach receptors may contribute to this brain region role during memory retrieval. The Ach receptors are divided into two broad subtypes: the ionotropic nicotinic acetylcholine receptors and the metabotropic muscarinic acetylcholine receptors. Back in 2010, we demonstrated for the first time the critical role of hippocampal α7 nicotinic acetylcholine receptors in memory reconsolidation process of an inhibitory avoidance response in mice. In the present work, we further investigate the possible implication of hippocampal muscarinic Ach receptors (mAchRs) in this process using a pharmacological approach. By specifically administrating agonists and antagonists of the different mAchRs subtypes in the hippocampus, we found that M1 and M2 but not M3 subtype may be involved in memory reconsolidation processes in mice.

Cholinergic-mediated coordination of rhythmic sympathetic and motor activities in the newborn rat spinal cord.

Here, we investigated intrinsic spinal cord mechanisms underlying the physiological requirement for autonomic and somatic motor system coupling. Using an in vitro spinal cord preparation from newborn rat, we demonstrate that the specific activation of muscarinic cholinergic receptors (mAchRs) (with oxotremorine) triggers a slow burst rhythm in thoracic spinal segments, thereby revealing a rhythmogenic capability in this cord region. Whereas axial motoneurons (MNs) were rhythmically activated during both locomotor activity and oxotremorine-induced bursting, intermediolateral sympathetic preganglionic neurons (IML SPNs) exhibited rhythmicity solely in the presence of oxotremorine. This somato-sympathetic synaptic drive shared by MNs and IML SPNs could both merge with and modulate the locomotor synaptic drive produced by the lumbar motor networks. This study thus sheds new light on the coupling between somatic and sympathetic systems and suggests that an intraspinal network that may be conditionally activated under propriospinal cholinergic control constitutes at least part of the synchronizing mechanism.

Cannabinoid receptor subtype influence on neuritogenesis in human SH-SY5Y cells.

Human SH-SY5Y neuroblastoma cells stably expressing exogenous CB1 (CB1XS) or CB2 (CB2XS) receptors were developed to investigate endocannabinoid signaling in the extension of neuronal projections. Expression of cannabinoid receptors did not alter proliferation rate, viability, or apoptosis relative to parental SH-SY5Y. Transcripts for endogenous cannabinoid system enzymes (diacylglycerol lipase, monoacylglycerol lipase, α/ß-hydrolase domain containing proteins 6 and 12, N-acyl phosphatidylethanolamine-phospholipase D, and fatty acid amide hydrolase) were not altered by CB1 or CB2 expression. Endocannabinoid ligands 2-arachidonoylglycerol (2-AG) and anandamide were quantitated in SH-SY5Y cells, and diacylglycerol lipase inhibitor tetrahydrolipstatin decreased 2-AG abundance by 90% but did not alter anandamide abundance. M3 muscarinic agonist oxotremorine M, and inhibitors of monoacylglycerol lipase and α/ß hydrolase domain containing proteins 6 &12 increased 2-AG abundance. CB1 receptor expression increased lengths of short (<30 µm) and long (>30 µm) projections, and this effect was significantly reduced by tetrahydrolipstatin, indicative of stimulation by endogenously produced 2-AG. Pertussis toxin, Gßγ inhibitor gallein, and ß-arrestin inhibitor barbadin did not significantly alter long projection length in CB1XS, but significantly reduced short projections, with gallein having the greatest inhibition. The rho kinase inhibitor Y27632 increased CB1 receptor-mediated long projection extension, indicative of actin cytoskeleton involvement. CB1 receptor expression increased GAP43 and ST8SIA2 mRNA and decreased ITGA1 mRNA, whereas CB2 receptor expression increased NCAM and SYT mRNA. We propose that basal endogenous production of 2-AG provides autocrine stimulation of CB1 receptor signaling through Gi/o, Gßγ, and ß-arrestin mechanisms to promote neuritogenesis, and rho kinase influences process extension.

Fusion with Promiscuous Gα16 Subunit Reveals Signaling Bias at Muscarinic Receptors.

A complex evaluation of agonist bias at G-protein coupled receptors at the level of G-protein classes and isoforms including non-preferential ones is essential for advanced agonist screening and drug development. Molecular crosstalk in downstream signaling and a lack of sufficiently sensitive and selective methods to study direct coupling with G-protein of interest complicates this analysis. We performed binding and functional analysis of 11 structurally different agonists on prepared fusion proteins of individual subtypes of muscarinic receptors and non-canonical promiscuous α-subunit of G16 protein to study agonist bias. We have demonstrated that fusion of muscarinic receptors with Gα16 limits access of other competitive Gα subunits to the receptor, and thus enables us to study activation of Gα16 mediated pathway more specifically. Our data demonstrated agonist-specific activation of G16 pathway among individual subtypes of muscarinic receptors and revealed signaling bias of oxotremorine towards Gα16 pathway at the M2 receptor and at the same time impaired Gα16 signaling of iperoxo at M5 receptors. Our data have shown that fusion proteins of muscarinic receptors with α-subunit of G-proteins can serve as a suitable tool for studying agonist bias, especially at non-preferential pathways.

Graded Transmission without Action Potentials Sustains Rhythmic Activity in Some But Not All Modulators That Activate the Same Current.

Neurons in the central pattern-generating circuits in the crustacean stomatogastric ganglion (STG) release neurotransmitter both as a graded function of presynaptic membrane potential that persists in TTX and in response to action potentials. In the STG of the male crab Cancer borealis, the modulators oxotremorine, C. borealis tachykinin-related peptide Ia (CabTRP1a), red pigment concentrating hormone (RPCH), proctolin, TNRNFLRFamide, and crustacean cardioactive peptide (CCAP) produce and sustain robust pyloric rhythms by activating the same modulatory current (IMI), albeit on different subsets of pyloric network targets. The muscarinic agonist oxotremorine, and the peptides CabTRP1a and RPCH elicited rhythmic triphasic intracellular alternating fluctuations of activity in the presence of TTX. Intracellular waveforms of pyloric neurons in oxotremorine and CabTRP1a in TTX were similar to those in the intact rhythm, and phase relationships among neurons were conserved. Although cycle frequency was conserved in oxotremorine and TTX, it was altered in CabTRP1a in the presence of TTX. Both rhythms were primarily driven by the pacemaker kernel consisting of the Anterior Burster and Pyloric Dilator neurons. In contrast, in TTX the circuit remained silent in proctolin, TNRNFLRFamide, and CCAP. These experiments show that graded synaptic transmission in the absence of voltage-gated Na+ current is sufficient to sustain rhythmic motor activity in some, but not other, modulatory conditions, even when each modulator activates the same ionic current. This further demonstrates that similar rhythmic motor patterns can be produced by qualitatively different mechanisms, one that depends on the activity of voltage-gated Na+ channels, and one that can persist in their absence.SIGNIFICANCE STATEMENT The pyloric rhythm of the crab stomatogastric ganglion depends both on spike-mediated and graded synaptic transmission. We activate the pyloric rhythm with a wide variety of different neuromodulators, all of which converge on the same voltage-dependent inward current. Interestingly, when action potentials and spike-mediated transmission are blocked using TTX, we find that the muscarinic agonist oxotremorine and the neuropeptide CabTRP1a sustain rhythmic alternations and appropriate phases of activity in the absence of action potentials. In contrast, TTX blocks rhythmic activity in the presence of other modulators. This demonstrates fundamental differences in the burst-generation mechanisms in different modulators that would not be suspected on the basis of their cellular actions at the level of the targeted current.

Heat shock protein (Hsp) regulation by muscarinic acetylcholine receptor (mAChR) activation in the rat hippocampus.

The cholinergic system plays a crucial role in modulating in the central nervous system physiological responses such as neurogenesis, neuronal differentiation, synaptic plasticity, and neuroprotection. In a recent study, we showed that Oxotremorine-M, a non-selective muscarinic acetylcholine receptor agonist, is able to transactivate the fibroblast growth factor receptor and to produce a significant increase in the hippocampal primary neurite outgrowth. In the present study we aimed to explore in the rat hippocampus the possible effect of acute or chronic treatment with Oxotremorine-M on some heat shock proteins (Hsp60, Hsp70, Hsp90) and on activation of related transcription factor heat shock factor 1 (HSF1). Following single injection of Oxotremorine-M (0.4 mg/kg) all Hsps examined were significantly increased in at least one of the time points studied (24, 48, and 72 hr). Treatment with Oxotremorine-M significantly increased the level of phosphorylated HSF1 in all time points studied, without change of protein levels. Similar pattern of Hsps changes was obtained following chronic Oxotremorine-M treatment (0.2 mg/kg) for 5 days. Surprisingly, following chronic treatment for 10 days no changes were observed in Hsps. The muscarinic acetylcholine receptor antagonist scopolamine (1 mg/kg) was able to completely block Oxotremorine-M effects on Hsps. In conclusion, considering the function of Hsps in protecting neuronal cells from deleterious proteotoxic stress, for example, protein mis-folding and aggregation, the results obtained indicate that muscarinic acetylcholine receptor activation may have implications in potential treatment of neurodegenerative disorders linked to protein aggregation, such as Alzheimer disease.

Pharmacological manipulation of the olfactory bulb modulates beta oscillations: testing model predictions.

The mammalian olfactory bulb (OB) generates gamma (40-100 Hz) and beta (15-30 Hz) local field potential (LFP) oscillations. Gamma oscillations arise at the peak of inhalation supported by dendrodendritic interactions between glutamatergic mitral cells (MCs) and GABAergic granule cells (GCs). Beta oscillations are induced by odorants in learning or odor sensitization paradigms, but their mechanism and function are still poorly understood. When centrifugal OB inputs are blocked, beta oscillations disappear, but gamma oscillations persist. Centrifugal inputs target primarily GABAergic interneurons in the GC layer (GCL) and regulate GC excitability, suggesting a causal link between beta oscillations and GC excitability. Our previous modeling work predicted that convergence of excitatory/inhibitory inputs onto MCs and centrifugal inputs onto GCs increase GC excitability sufficiently to produce beta oscillations primarily through voltage dependent calcium channel-mediated GABA release, independently of NMDA channels. We test some of the predictions of this model by examining the influence of NMDA and muscarinic acetylcholine (ACh) receptors, which affect GC excitability in different ways, on beta oscillations. A few minutes after intrabulbar infusion, scopolamine (muscarinic antagonist) suppressed odor-evoked beta in response to a strong stimulus but increased beta power in response to a weak stimulus, as predicted by our model. Pyriform cortex (PC) beta power was unchanged. Oxotremorine (muscarinic agonist) suppressed all oscillations, likely from overinhibition. APV, an NMDA receptor antagonist, suppressed gamma oscillations selectively (in OB and PC), lending support to the model's prediction that beta oscillations can be supported independently of NMDA receptors. NEW & NOTEWORTHY Olfactory bulb local field potential beta oscillations appear to be gated by GABAergic granule cell excitability. Reducing excitability with scopolamine reduces beta induced by strong odors but increases beta induced by weak odors. Beta oscillations rely on the same synapse as gamma oscillations but, unlike gamma, can persist in the absence of NMDA receptor activation. Pyriform cortex beta oscillations maintain power when olfactory bulb beta power is low, and the system maintains beta band coherence.

Oxotremorine-M potentiates NMDA receptors by muscarinic receptor dependent and independent mechanisms.

Muscarinic acetylcholine M1 receptors play an important role in synaptic plasticity in the hippocampus and cortex. Potentiation of NMDA receptors as a consequence of muscarinic acetylcholine M1 receptor activation is a crucial event mediating the cholinergic modulation of synaptic plasticity, which is a cellular mechanism for learning and memory. In Alzheimer's disease, the cholinergic input to the hippocampus and cortex is severely degenerated, and agonists or positive allosteric modulators of M1 receptors are therefore thought to be of potential use to treat the deficits in cognitive functions in Alzheimer's disease. In this study we developed a simple system in which muscarinic modulation of NMDA receptors can be studied in vitro. Human M1 receptors and NR1/2B NMDA receptors were co-expressed in Xenopus oocytes and various muscarinic agonists were assessed for their modulatory effects on NMDA receptor-mediated responses. As expected, NMDA receptor-mediated responses were potentiated by oxotremorine-M, oxotremorine or xanomeline when the drugs were applied between subsequent NMDA responses, an effect which was fully blocked by the muscarinic receptor antagonist atropine. However, in oocytes expressing NR1/2B NMDA receptors but not muscarinic M1 receptors, oxotremorine-M co-applied with NMDA also resulted in a potentiation of NMDA currents and this effect was not blocked by atropine, demonstrating that oxotremorine-M is able to directly potentiate NMDA receptors. Oxotremorine, which is a close analogue of oxotremorine-M, and xanomeline, a chemically distinct muscarinic agonist, did not potentiate NMDA receptors by this direct mechanism. Comparing the chemical structures of the three different muscarinic agonists used in this study suggests that the tri-methyl ammonium moiety present in oxotremorine-M is important for the compound's interaction with NMDA receptors.

Acetylcholine-dependent upregulation of TASK-1 channels in thalamic interneurons by a smooth muscle-like signalling pathway.

KEY POINTS: The ascending brainstem transmitter acetylcholine depolarizes thalamocortical relay neurons while it induces hyperpolarization in local circuit inhibitory interneurons. Sustained K+ currents are modulated in thalamic neurons to control their activity modes; for the interneurons the molecular nature of the underlying ion channels is as yet unknown. Activation of TASK-1 K+ channels results in hyperpolarization of interneurons and suppression of their action potential firing. The modulation cascade involves a non-receptor tyrosine kinase, c-Src. The present study identifies a novel pathway for the activation of TASK-1 channels in CNS neurons that resembles cholinergic signalling and TASK-1 current modulation during hypoxia in smooth muscle cells. ABSTRACT: The dorsal part of the lateral geniculate nucleus (dLGN) is the main thalamic site for state-dependent transmission of visual information. Non-retinal inputs from the ascending arousal system and inhibition provided by γ-aminobutyric acid (GABA)ergic local circuit interneurons (INs) control neuronal activity within the dLGN. In particular, acetylcholine (ACh) depolarizes thalamocortical relay neurons by inhibiting two-pore domain potassium (K2P ) channels. Conversely, ACh also hyperpolarizes INs via an as-yet-unknown mechanism. By using whole cell patch-clamp recordings in brain slices and appropriate pharmacological tools we here report that stimulation of type 2 muscarinic ACh receptors induces IN hyperpolarization by recruiting the G-protein ßγ subunit (Gßγ), class-1A phosphatidylinositol-4,5-bisphosphate 3-kinase, and cellular and sarcoma (c-Src) tyrosine kinase, leading to activation of two-pore domain weakly inwardly rectifying K+ channel (TWIK)-related acid-sensitive K+ (TASK)-1 channels. The latter was confirmed by the use of TASK-1-deficient mice. Furthermore inhibition of phospholipase Cß as well as an increase in the intracellular level of phosphatidylinositol-3,4,5-trisphosphate facilitated the muscarinic effect. Our results have uncovered a previously unknown role of c-Src tyrosine kinase in regulating IN function in the brain and identified a novel mechanism by which TASK-1 channels are activated in neurons.

Tonic inhibition of brown adipose tissue sympathetic nerve activity via muscarinic acetylcholine receptors in the rostral raphe pallidus.

KEY POINTS: A tonically active, muscarinic cholinergic inhibition of rostral raphe pallidus (rRPa) neurons influences thermogenesis of brown adipose tissue (BAT) independent of ambient temperature conditions. The tonically active cholinergic input to rRPa originates caudal to the hypothalamus. Muscarinic acetylcholine receptor (mAChR) activation in rRPa contributes to the inhibition of BAT sympathetic nerve activity (SNA) evoked by activation of neurons in the rostral ventrolateral medulla (RVLM). The RVLM is not the sole source of the muscarinic cholinergic input to rRPa. Activation of GABA receptors in rRPa does not mediate the cholinergic inhibition of BAT SNA. ABSTRACT: We sought to determine if body temperature and energy expenditure are influenced by a cholinergic input to neurons in the rostral raphe pallidus (rRPa), the site of sympathetic premotor neurons controlling thermogenesis of brown adipose tissue (BAT). Nanoinjections of the muscarinic acetylcholine receptor (mAChR) agonist, oxotremorine, or the cholinesterase inhibitor, neostigmine (NEOS), in the rRPa of anaesthetized rats decreased cold-evoked BAT sympathetic nerve activity (SNA, nadirs: -72 and -95%), BAT temperature (Tbat, -0.5 and -0.6°C), expired CO2 (Exp. CO2 , -0.3 and -0.5%) and heart rate (HR, -22 and -41 bpm). NEOS into rRPa reversed the increase in BAT SNA evoked by blockade of GABA receptors in rRPa. Nanoinjections of the mAChR antagonist, scopolamine (SCOP), in the rRPa of warm rats increased BAT SNA (peak: +1087%), Tbat (+1.8°C), Exp. CO2 (+0.7%), core temperature (Tcore, +0.5°C) and HR (+54 bpm). SCOP nanoinjections in rRPa produced similar activations of BAT during cold exposure, following a brain transection caudal to the hypothalamus, and during the blockade of glutamate receptors in rRPa. We conclude that a tonically active cholinergic input to the rRPa inhibits BAT SNA via activation of local mAChR. The inhibition of BAT SNA mediated by mAChR in rRPa does not depend on activation of GABA receptors in rRPa. The increase in BAT SNA following mAChR blockade in rRPa does not depend on the activity of neurons in the hypothalamus or on glutamate receptor activation in rRPa.

Existence of muscarinic acetylcholine receptor (mAChR) and fibroblast growth factor receptor (FGFR) heteroreceptor complexes and their enhancement of neurite outgrowth in neural hippocampal cultures.

BACKGROUND: Recently, it was demonstrated that G-protein-coupled receptors (GPCRs) can transactivate tyrosine kinase receptors in absence of their ligands. In this work, driven by the observation that mAChRs and fibroblast growth factor receptors (FGFRs) share signalling pathways and regulation of brain functions, it was decided to explore whether mAChRs activation may transactivate FGFRs and, if so, to characterize the related trophic effects in cultured hippocampal neurons. METHODS: Oxotremorine-M transactivation of FGFRs and related trophic effects were tested in primary hippocampal neurons. Western blotting and in situ proximity ligation assay (PLA) were used to detect FGFR phosphorylation (pFGFR) levels and M1R-FGFR1 heteroreceptor complexes, respectively. RESULTS: Oxotremorine-M, a non-selective mAChRs agonist, was able to transactivate FGFR and this transactivation was blocked by Src inhibitors. Oxotremorine-M treatment produced a significant increase in the primary neurite outgrowth that was blocked by pre-treatment with the pFGFR inhibitor SU5402 and Src inhibitors. This trophic effect was almost similar to that induced by fibroblast growth factor-2 (FGF-2). By using atropine as nonselective mAChRs or pirenzepine as selective antagonist for M1 receptor (M1R) we could show that mAChRs are involved in modulating the pFGFRs. Using PLA, M1R-FGFR1 heteroreceptor complexes were identified in the hippocampus and cerebral cortex. CONCLUSION: The current findings, by showing functional mAChR-FGFR interactions, will contribute to advance the understanding of the mechanisms involved in the actions of cholinergic drugs on neuronal plasticity. GENERAL SIGNIFICANT: Data may help to develop novel therapeutic strategies not only for neurodegenerative diseases but also for depression-induced atrophy of hippocampal neurons.

Voltage-Independent Inhibition of the Tetrodotoxin-Sensitive Sodium Currents by Oxotremorine and Angiotensin II in Rat Sympathetic Neurons.

Tetrodotoxin-sensitive Na(+) currents have been extensively studied because they play a major role in neuronal firing and bursting. In this study, we showed that voltage-dependent Na(+) currents are regulated in a slow manner by oxotremorine (oxo-M) and angiotensin II in rat sympathetic neurons. We found that these currents can be readily inhibited through a signaling pathway mediated by G proteins and phospholipase C (PLC) ß1. This inhibition is slowly established, pertussis toxin-insensitive, partially reversed within tens of seconds after oxo-M washout, and not relieved by a strong depolarization, suggesting a voltage-insensitive mechanism of inhibition. Specificity of the M1 receptor was tested by the MT-7 toxin. Activation and inactivation curves showed no shift in the voltage dependency under the inhibition by oxo-M. This inhibition is blocked by a PLC inhibitor (U73122, 1-(6-{[(17ß)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione), and recovery from inhibition is prevented by wortmannin, a PI3/4 kinase inhibitor. Hence, the pathway involves Gq/11 and is mediated by a diffusible second messenger. Oxo-M inhibition is occluded by screening phosphatidylinositol 4,5-bisphosphate (PIP2)-negative charges with poly-l-lysine and prevented by intracellular dialysis with a PIP2 analog. In addition, bisindolylmaleimide I, a specific ATP-competitive protein kinase C (PKC) inhibitor, rules out that this inhibition may be mediated by this protein kinase. Furthermore, oxo-M-induced suppression of Na(+) currents remains unchanged when neurons are treated with calphostin C, a PKC inhibitor that targets the diacylglycerol-binding site of the kinase. These results support a general mechanism of Na(+) current inhibition that is widely present in excitable cells through modulation of ion channels by specific G protein-coupled receptors.