Isolation and electrophysiological recording of Ixodes ricinus synganglion neurons.

The central nervous system of hard ticks (Ixodidae) consists of a concentrated merged nerve mass known as the synganglion. Although knowledge of tick neurobiology has dramatically improved over the last two decades, this is the first time that isolation and electrophysiological recordings have been carried out on tick neurons from the synganglion. Method: We developed a simple protocol for synganglion neuron isolation and used a whole-cell patch clamp to measure ionic currents induced by acetylcholine, nicotine and muscarine. Relatively large neurons (∼ 25 µm and âˆ¼ 35 µm) were isolated and 1 mM acetylcholine was used to induce strong inward currents of -0.38 ± 0.1 nA and - 1.04 ± 0.1 nA, respectively, with the corresponding cell capacitances being at around 142 pF and 188 pF. In addition, successive application of 1 mM acetylcholine through ∼25 µm and âˆ¼ 35 µm cells for increasing amounts of time resulted in a rapid reduction in current amplitudes. We also found that acetylcholine-evoked currents were associated with a reversible increase in intracellular calcium levels for each neuronal type. In contrast, 1 mM muscarine and nicotine induced a strong and non-reversible increase in intracellular calcium levels. This study serves as a proof of concept for the mechanical isolation of tick synganglion neurons followed by their electrophysiological recording. This approach will aid investigations into the pharmacological properties of tick neurons and provides the tools needed for the identification of drug-targeted sites and effective tick control measures.

A muscarinic, GIRK channel-mediated inhibition of inspiratory-related XII nerve motor output emerges in early postnatal development in mice.

In neonatal rhythmic medullary slices, muscarinic acetylcholine receptor (mAChR) activation of hypoglossal (XII) motoneurons that innervate the tongue has a net excitatory effect on XII inspiratory motor output. Conversely, during rapid eye movement sleep in adult rodents, XII motoneurons experience a loss of excitability partly due to activation of mAChRs. This may be mediated by activation of G-protein-coupled inwardly rectifying potassium (GIRK) channels. Therefore, this study was designed to evaluate whether muscarinic modulation of XII inspiratory motor output in mouse rhythmic medullary slices includes GIRK channel-mediated inhibition and, if so, when this inhibitory mechanism emerges. Local pressure injection of the mAChR agonist muscarine potentiated inspiratory bursting by 150 ± 28% in postnatal day (P)0-P5 rhythmic medullary slice preparations. In the absence of muscarine, pharmacological GIRK channel block by Tertiapin-Q did not affect inspiratory burst parameters, whereas activation with ML297 decreased inspiratory burst area. Blocking GIRK channels by local preapplication of Tertiapin-Q revealed a developmental change in muscarinic modulation of inspiratory bursting. In P0-P2 rhythmic medullary slices, Tertiapin-Q preapplication had no significant effect on muscarinic potentiation of inspiratory bursting (a negligible 6% decrease). However, preapplication of Tertiapin-Q to P3-P5 rhythmic medullary slices caused a 19% increase in muscarinic potentiation of XII inspiratory burst amplitude. Immunofluorescence experiments revealed expression of GIRK 1 and 2 subunits and M1, M2, M3, and M5 mAChRs from P0 to P5. Overall, these data support that mechanisms underlying muscarinic modulation of inspiratory burst activity change postnatally and that potent GIRK-mediated inhibition described in adults emerges early in postnatal life.NEW & NOTEWORTHY Muscarinic modulation of inspiratory bursting at hypoglossal motoneurons has a net excitatory effect in neonatal rhythmic medullary slice preparations and a net inhibitory effect in adult animals. We demonstrate that muscarinic modulation of inspiratory bursting undergoes maturational changes from postnatal days 0 to 5 that include emergence of an inhibitory component mediated by G-protein-coupled inwardly rectifying potassium channels after postnatal day 3 in neonatal mouse rhythmic medullary slice preparations.

M4 muscarinic receptors mediate acetylcholine-induced suppressant effects on the cough reflex in the caudal nucleus tractus solitarii of the rabbit.

It has been shown that muscarinic acetylcholine receptors (mAChRs) located within the caudal nucleus tractus solitarii (cNTS) mediate a cholinergic inhibitory control mechanism of the cough reflex. Thus, identification of the involved mAChR subtypes could be of considerable interest for novel therapeutic strategies. In pentobarbital sodium-anesthetized, spontaneously breathing rabbits we investigated the contribution of different mAChR subtypes in the modulation of mechanically and chemically induced cough reflex. Bilateral microinjections of 1 mM muscarine into the cNTS increased respiratory frequency and decreased expiratory activity even to complete suppression. Interestingly, muscarine induced strong cough-suppressant effects up to the complete abolition of the reflex. Microinjections of specific mAChR subtype antagonists (M1-M5) into the cNTS were performed. Only microinjections of the M4 antagonist tropicamide (1 mM) prevented muscarine-induced changes in both respiratory activity and cough reflex. The results are discussed in light of the notion that cough involves the activation of the nociceptive system. They also suggest that M4 receptor agonists may have an important role in cough downregulation within the cNTS.

Hippocampal cholinergic receptors and the mTOR participation in fear-motivated inhibitory avoidance extinction memory.

Evidence has demonstrated the hippocampal cholinergic system and the mammalian target of rapamycin (mTOR) participation during the memory formation of aversive events. This study assessed the role of these systems in the hippocampus for the extinction memory process by submitting male Wistar rats to fear-motivated step-down inhibitory avoidance (IA). The post-extinction session administration of the nicotinic and muscarinic cholinergic receptor antagonists, mecamylamine and scopolamine, respectively, both at doses of 2 µg/µl/side, and rapamycin, an mTOR inhibitor (0.02 µg/µl/side), into the CA1 region of the dorsal hippocampus, impaired the IA extinction memory. Furthermore, the nicotinic and muscarinic cholinergic receptor agonists, nicotine and muscarine, respectively, had a dose-dependent effect on the IA extinction memory when administered intra-CA1, immediately after the extinction session. Nicotine (0.6 µg/µl/side) and muscarine (0.02 µg/µl/side), respectively, had no effect, while the higher doses (6 and 2 µg/µl/side, respectively) impaired the IA extinction memory. Interestingly, the co-administration of muscarine at the lower dose blocked the impairment that was induced by rapamycin. This effect was not observed when nicotine at the lower dose was co-administered. These results have demonstrated the participation of the cholinergic receptors and mTOR in the hippocampus for IA extinction, and that the cholinergic agonists had a dose-dependent effect on the IA extinction memory. This study provides insights related to the behavioural aspects and the neurobiological properties underlying the early stage of fear-motivated IA extinction memory consolidation and suggests that there is hippocampal muscarinic receptor participation independent of mTOR in this memory process.

Differential pharmacological and sex-specific effects of antimuscarinic agents at the hypoglossal motor nucleus in vivo in rats.

Successful cholinergic-noradrenergic pharmacotherapy for obstructive sleep apnea (OSA) is thought to be due to effects at the hypoglossal motor nucleus (HMN). Clinical efficacy varies with muscarinic-receptor (MR) subtype affinities. We hypothesized that oxybutynin (cholinergic agent in successful OSA pharmacotherapy) is an effective MR antagonist at the HMN and characterized its efficacy with other antagonists. We recorded tongue muscle activity of isoflurane anesthetized rats (121 males and 60 females, 7-13 per group across 13 protocols) in response to HMN microperfusion with MR antagonists with and without: (i) eserine-induced increased endogenous acetylcholine at the HMN and (ii) muscarine. Eserine-induced increased acetylcholine decreased tongue motor activity (p < 0.001) with lesser cholinergic suppression in females versus males (p = 0.017). Motor suppression was significantly attenuated by the MR antagonists atropine, oxybutynin, and omadacycline (MR2 antagonist), each p < 0.001, with similar residual activity between agents (p ≥ 0.089) suggesting similar efficacy at the HMN. Sex differences remained with atropine and oxybutynin (p < 0.001 to 0.05) but not omadacycline (p = 0.722). Muscarine at the HMN also decreased motor activity (p < 0.001) but this was not sex-specific (p = 0.849). These findings have translational relevance to antimuscarinic agents in OSA pharmacotherapy and understanding potential sex differences in HMN suppression with increased endogenous acetylcholine related to sparing nicotinic excitation.

Cholinergic modulation of persistent inward currents is mediated by activating muscarinic receptors of serotonergic neurons in the brainstem of ePet-EYFP mice.

Persistent inward currents (PICs) play important roles in regulating neural excitability. Results from our previous studies showed that serotonergic (5-HT) neurons of the brainstem expressed PICs. However, little is known about cholinergic (ACh) modulation of PICs in the 5-HT neurons. The whole-cell patch-clamp recordings were performed in the brainstem slices of ePet-EYFP mice to investigate the electrophysiological properties of PICs with cholinergic modulation. PICs in 5-HT neurons were activated at - 51.4 ± 3.7 mV with the amplitude of - 171.6 ± 48.9 pA (n = 71). Bath application of 20-25 µM ACh increased the amplitude by 79.1 ± 42.5 pA (n = 23, p < 0.001) and hyperpolarized the onset voltage by 2.2 ± 2.7 mV (n = 23, p < 0.01) and half-maximal activation by 3.6 ± 2.7 mV (n = 6, p < 0.01). Muscarine mimicked the effects of ACh on PICs, while bath application of nicotine (15-20 µM) did not induce substantial change in the PICs (n = 9). Muscarine enhanced the amplitude of PICs by 100.0 ± 27.4 pA (n = 28, p < 0.001) and lowered the onset voltage by 2.8 ± 1.2 mV (n = 28, p < 0.001) and the half-maximal activation by 2.9 ± 1.4 mV. ACh-induced increase of amplitude and hyperpolarization of onset voltage were blocked by 3-5 µM atropine. Furthermore, the muscarine-induced enhancement of the PICs was antagonized by 5 µM 4-DAMP, the antagonist of M3 receptor, while the antagonists of M1 (Telenzepine, 5 µM) and M5 (VU6008667, 5 µM) receptors did not significantly affect the PIC enhancement. This study suggested that ACh potentiated PICs in 5-HT neurons of the brainstem by activating muscarinic M3 receptor.

Mechanisms for pituitary adenylate cyclase-activating polypeptide-induced increase in excitability in guinea-pig and mouse adrenal medullary cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) acts on adrenal medullary (AM) cells as a neurotransmitter of the sympathetic preganglionic nerve. In guinea-pig AM cells, PACAP induces little catecholamine secretion, but enhances secretion evoked by stimulants, whereas in other animals, such as mouse, PACAP itself induces depolarization and/or catecholamine secretion. The present studies aim to explore the physiological implication of these species differences in PACAP actions, the ion channel mechanism for PACAP-induced depolarization, and the mechanism for facilitation of muscarinic receptor-mediated cation currents in mouse and guinea-pig AM cells. The perforated patch clamp technique was used to record the whole-cell current in isolated AM cells. The amplitudes of 3 nM PACAP-induced inward currents were significantly larger in mouse AM cells than guinea-pig, whereas 1 µM muscarine-induced currents were larger in guinea-pig AM cells than mouse. Exposure to PACAP consistently resulted in enhancement of muscarine-induced currents in guinea-pig AM cells and facilitation of cell membrane insertion of heteromeric TRPC1-TRPC4 channels in response to muscarine in PC12 cells. The PACAP-induced current was inhibited by 30 µM 9-phenanthrol, a specific TRPM4 channel inhibitor, and abolished by replacement of external Na+ with N-methyl D-glucamine. TRPM4-like immunoreactivity was located at the cell periphery in AM cells. The present results indicate that PACAP and muscarinic receptors are major metabotropic receptors mediating generation of depolarizing inward currents in mouse and guinea-pig AM cells, respectively. We conclude that PACAP activates TRPM4-like channels and enhance the muscarinic current through facilitating the membrane insertion of TRPC1-TRPC4 channels in AM cells.

Breakdown of phospholipids and the elevated nitric oxide are involved in M3 muscarinic regulation of acetylcholine secretion in the frog motor synapse.

Previously, we found that muscarine downregulates the acetylcholine release at the frog neuromuscular junction acting via M3 muscarinic receptors. Here, the molecular mechanisms underlying the inhibitory effect of muscarine on the quantal secretion of acetylcholine were studied. Inhibition of phospholipase C (with U-73122) prevented the reduction of evoked neurotransmitter release induced by muscarine. Interruption of synthesis of phosphatidylinositol 3-phosphate by the inhibitor of phosphoinositide-3-kinase (wortmannin) did not affect the depressant action of muscarine but precluded the restoration of secretion after removal of muscarine from the bathing solution. The effect of muscarine was not significantly modified by the blockade of endocannabinoid receptors (with AM 281), but it was abolished by the inhibitor of nitric oxide synthase (L-NAME) as well as extracellular nitric oxide (NO) chelator (hemoglobin). Moreover, muscarine increased NO-sensitive dye fluorescence in junctional region, which was prevented by the M3 receptor antagonist 4-DAMP. The data obtained indicate that the attenuation of acetylcholine release mediated by muscarine is associated with a change in the activity of both lipid-metabolizing enzymes and NO synthases.

Muscarinic receptor stimulation induces TASK1 channel endocytosis through a PKC-Pyk2-Src pathway in PC12 cells.

Muscarinic receptor stimulation or protein kinase C (PKC) activation in rat adrenal medullary and PC12 cells rapidly induces tyrosine phosphorylation of TWIK-related-acid-sensitive K+ 1 (TASK1) channels with the subsequent clathrin-dependent endocytosis. Our previous study suggested that the muscarinic signal is transmitted to the non-receptor tyrosine kinase Src through PKC and Pyk2. Although PKC activation is known to stimulate Pyk2 in certain types of cells, its molecular mechanism remains unclear. In this study, proximity ligation assay (PLA) and other molecular biological approaches were used to elucidate the details of this muscarinic signaling in PC12 cells. When green fluorescent protein (GFP)-TASK1 was expressed, the majority of GFP-TASK1 was located at the cell periphery. However, the simultaneous expression of GFP-TASK1 and PKCα, but not PKCδ, led to GFP-TASK1 internalization. Muscarinic receptor stimulation resulted in transient co-localization of Pyk2 and Src at the cell periphery, and expression of kinase dead (KD) Pyk2 and Src, but not Pyk2 and KD Src, resulted in GFP-TASK1 internalization. PLA analysis revealed that in response to muscarine, PKCαactivates Pyk2 through phosphorylating its serine residues. These results indicate that muscarinic receptor stimulation induces TASK1 channel endocytosis sequentially through PKCα, Pyk2, and Src, and PKCα activates Pyk2 through phosphorylation.

Balanced cholinergic modulation of spinal locomotor circuits via M2 and M3 muscarinic receptors.

Neuromodulation ensures that neural circuits produce output that is flexible whilst remaining within an optimal operational range. The neuromodulator acetylcholine is released during locomotion to regulate spinal motor circuits. However, the range of receptors and downstream mechanisms by which acetylcholine acts have yet to be fully elucidated. We therefore investigated metabotropic acetylcholine receptor-mediated modulation by using isolated spinal cord preparations from neonatal mice in which locomotor-related output can be induced pharmacologically. We report that M2 receptor blockade decreases the frequency and amplitude of locomotor-related activity, whilst reducing its variability. In contrast, M3 receptor blockade destabilizes locomotor-related bursting. Motoneuron recordings from spinal cord slices revealed that activation of M2 receptors induces an outward current, decreases rheobase, reduces the medium afterhyperpolarization, shortens spike duration and decreases synaptic inputs. In contrast, M3 receptor activation elicits an inward current, increases rheobase, extends action potential duration and increases synaptic inputs. Analysis of miniature postsynaptic currents support that M2 and M3 receptors modulate synaptic transmission via different mechanisms. In summary, we demonstrate that M2 and M3 receptors have opposing modulatory actions on locomotor circuit output, likely reflecting contrasting cellular mechanisms of action. Thus, intraspinal cholinergic systems mediate balanced, multimodal control of spinal motor output.

Subcellular distribution of human tyrosine hydroxylase isoforms 1 and 4 in SH-SY5Y cells.

Tyrosine hydroxylase (TH) is the key enzyme that controls the rate of synthesis of the catecholamines. SH-SY5Y cells with stable transfections of either human tyrosine hydroxylase isoform 1 (hTH1) or human tyrosine hydroxylase isoform 4 (hTH4) were used to determined the subcellular distribution of TH protein and phosphorylated TH, under basal conditions and after muscarine stimulation. Muscarine was previously shown to increase the phosphorylation of only serine 19 and serine 40 in hTH1 cells. Under basal conditions, the hTH1 and hTH4 proteins, their serine 19 phosphorylated forms and hTH1 phosphorylated at serine 40 were all similarly distributed; with ~80% in the cytosolic fraction, ~20% in the membrane fraction, and less than 1%, or not detectable, in the nuclear fraction. However, hTH4 phosphorylated at serine 71 had a significantly different distribution with ~65% cytosolic and ~35% membrane associated. Muscarine stimulation led to hTH1 being redistributed from the cytosol and nuclear fractions to the membrane fraction and hTH4 being redistributed from the cytosol to the nuclear fraction. These muscarine stimulated redistributions were not due to TH phosphorylation at serine 19, serine 40, or serine 71 and were most likely due to TH binding to proteins whose phosphorylation was increased by muscarine. This is the first study to show a difference in subcellular distribution between two human TH isoforms under basal and stimulated conditions.

Inhibitory muscarinic acetylcholine receptors enhance aversive olfactory learning in adult Drosophila.

Olfactory associative learning in Drosophila is mediated by synaptic plasticity between the Kenyon cells of the mushroom body and their output neurons. Both Kenyon cells and their inputs from projection neurons are cholinergic, yet little is known about the physiological function of muscarinic acetylcholine receptors in learning in adult flies. Here, we show that aversive olfactory learning in adult flies requires type A muscarinic acetylcholine receptors (mAChR-A), particularly in the gamma subtype of Kenyon cells. mAChR-A inhibits odor responses and is localized in Kenyon cell dendrites. Moreover, mAChR-A knockdown impairs the learning-associated depression of odor responses in a mushroom body output neuron. Our results suggest that mAChR-A function in Kenyon cell dendrites is required for synaptic plasticity between Kenyon cells and their output neurons.

Postnatal development of persistent inward currents in rat XII motoneurons and their modulation by serotonin, muscarine and noradrenaline.

KEY POINTS: Persistent inward currents (PICs) in spinal motoneurons are long-lasting, voltage-dependent currents that increase excitability; they are dramatically potentiated by serotonin, muscarine, and noradrenaline (norepinephrine). Loss of these modulators (and the PIC) during sleep is hypothesized as a major contributor to REM sleep atonia. Reduced excitability of XII motoneurons that drive airway muscles and maintain airway patency is causally implicated in obstructive sleep apnoea (OSA), but whether XII motoneurons possess a modulator-sensitive PIC that could be a factor in the reduced airway tone of sleep is unknown. Whole-cell recordings from rat XII motoneurons in brain slices indicate that PIC amplitude increases ∼50% between 1 and 23 days of age, when potentiation of the PIC by 5HT2 , muscarinic, or α1 noradrenergic agonists peaks at <50%, manyfold lower than the potentiation observed in spinal motoneurons. α1 noradrenergic receptor activation produced changes in XII motoneuron firing behaviour consistent with PIC involvement, but indicators of strong PIC activation were never observed; in vivo experiments are needed to determine the role of the modulator-sensitive PIC in sleep-dependent reductions in airway tone. ABSTRACT: Hypoglossal (XII) motoneurons play a key role in maintaining airway patency; reductions in their excitability during sleep through inhibition and disfacilitation, i.e. loss of excitatory modulation, is implicated in obstructive sleep apnoea. In spinal motoneurons, 5HT2 , muscarinic and α1 noradrenergic modulatory systems potentiate persistent inward currents (PICs) severalfold, dramatically increasing excitability. If the PICs in XII and spinal motoneurons are equally sensitive to modulation, loss of the PIC secondary to reduced modulatory tone during sleep could contribute to airway atonia. Modulatory systems also change developmentally. We therefore characterized developmental changes in magnitude of the XII motoneuron PIC and its sensitivity to modulation by comparing, in neonatal (P1-4) and juvenile (P14-23) rat brainstem slices, the PIC elicited by slow voltage ramps in the absence and presence of agonists for 5HT2 , muscarinic, and α1 noradrenergic receptors. XII motoneuron PIC amplitude increased developmentally (from -195 ± 12 to -304 ± 19 pA). In neonatal XII motoneurons, the PIC was only potentiated by α1 receptor activation (5 ± 4%). In contrast, all modulators potentiated the juvenile XII motoneurons PIC (5HT2 , 5 ± 5%; muscarine, 22 ± 11%; α1 , 18 ± 5%). These data suggest that the influence of the PIC and its modulation on XII motoneuron excitability will increase with postnatal development. Notably, the modulator-induced potentiation of the PIC in XII motoneurons was dramatically smaller than the 2- to 6-fold potentiation reported for spinal motoneurons. In vivo measurements are required to determine if the modulator-sensitive, XII motoneuron PIC is an important factor in sleep-state dependent reductions in airway tone.

Differences among muscarinic agonists in M1 receptor-mediated nonselective cation channel activation and TASK1 channel inhibition in adrenal medullary cells.

Muscarinic receptor stimulation induces depolarizing inward currents and catecholamine secretion in adrenal medullary (AM) cells from various mammals. In guinea-pig AM cells muscarine and oxotremorine at concentrations ≤ 1 µM produce activation of nonselective cation channels with a similar potency and efficacy, whereas muscarine at higher concentrations produces not only nonselective cation channel activation, but also TASK1 channel inhibition. In rat AM cells, the muscarinic M1 receptor is involved in TASK1 channel inhibition in response to muscarinic agonists, and the efficacy of oxotremorine is half that of muscarine. These pharmacological findings might indicate that different muscarinic receptor subtypes are responsible for the regulation of nonselective cation and TASK1 channel activities. The present study aimed to determine the muscarinic receptor subtypes involved in nonselective cation channel activation in guinea-pig and mouse AM cells. The inward current evoked by 1 µM muscarine was completely suppressed by 100 µM quinine, whereas 30 µM muscarine-induced inward currents were comprised of quinine-sensitive and -insensitive components. The electrophysiological and pharmacological properties of the muscarine-induced currents indicated that the quinine-sensitive and insensitive components are due to nonselective cation channel activation and TASK1 channel inhibition, respectively. Muscarine at 30 µM failed to induce any current in AM cells treated with muscarinic toxin 7 or genetically deleted of the M1 receptor. The KD value of VU0255035 against the muscarinic receptor mediating nonselective cation channel activation was 17.5 nM. These results indicate that the M1 receptor mediates nonselective cation channel activation as well as TASK1 channel inhibition.

RE1-silencing transcription factor controls the acute-to-chronic neuropathic pain transition and Chrm2 receptor gene expression in primary sensory neurons.

Neuropathic pain is associated with persistent changes in gene expression in primary sensory neurons, but the underlying epigenetic mechanisms that cause these changes remain unclear. The muscarinic cholinergic receptors (mAChRs), particularly the M2 subtype (encoded by the cholinergic receptor muscarinic 2 (Chrm2) gene), are critically involved in the regulation of spinal nociceptive transmission. However, little is known about how Chrm2 expression is transcriptionally regulated. Here we show that nerve injury persistently increased the expression of RE1-silencing transcription factor (REST, also known as neuron-restrictive silencing factor [NRSF]), a gene-silencing transcription factor, in the dorsal root ganglion (DRG). Remarkably, nerve injury-induced chronic but not acute pain hypersensitivity was attenuated in mice with Rest knockout in DRG neurons. Also, siRNA-mediated Rest knockdown reversed nerve injury-induced chronic pain hypersensitivity in rats. Nerve injury persistently reduced Chrm2 expression in the DRG and diminished the analgesic effect of muscarine. The RE1 binding site on the Chrm2 promoter is required for REST-mediated Chrm2 repression, and nerve injury increased the enrichment of REST in the Chrm2 promoter in the DRG. Furthermore, Rest knockdown or genetic ablation in DRG neurons normalized Chrm2 expression and augmented muscarine's analgesic effect on neuropathic pain and fully reversed the nerve injury-induced reduction in the inhibitory effect of muscarine on glutamatergic input to spinal dorsal horn neurons. Our findings indicate that nerve injury-induced REST up-regulation in DRG neurons plays an important role in the acute-to-chronic pain transition and is essential for the transcriptional repression of Chrm2 in neuropathic pain.

Caveolin-3 differentially orchestrates cholinergic and serotonergic constriction of murine airways.

The mechanisms of controlling airway smooth muscle (ASM) tone are of utmost clinical importance as inappropriate constriction is a hallmark in asthma and chronic obstructive pulmonary disease. Receptors for acetylcholine and serotonin, two relevant mediators in this context, appear to be incorporated in specialized, cholesterol-rich domains of the plasma membrane, termed caveolae due to their invaginated shape. The structural protein caveolin-1 partly accounts for anchoring of these receptors. We here determined the role of the other major caveolar protein, caveolin-3 (cav-3), in orchestrating cholinergic and serotonergic ASM responses, utilizing newly generated cav-3 deficient mice. Cav-3 deficiency fully abrogated serotonin-induced constriction of extrapulmonary airways in organ baths while leaving intrapulmonary airways unaffected, as assessed in precision cut lung slices. The selective expression of cav-3 in tracheal, but not intrapulmonary bronchial epithelial cells, revealed by immunohistochemistry, might explain the differential effects of cav-3 deficiency on serotonergic ASM constriction. The cholinergic response of extrapulmonary airways was not altered, whereas a considerable increase was observed in cav-3-/- intrapulmonary bronchi. Thus, cav-3 differentially organizes serotonergic and cholinergic signaling in ASM through mechanisms that are specific for airways of certain caliber and anatomical position. This may allow for selective and site-specific intervention in hyperreactive states.

Pre-synaptic Muscarinic Excitation Enhances the Discrimination of Looming Stimuli in a Collision-Detection Neuron.

Visual neurons that track objects on a collision course are often finely tuned to their target stimuli because this is critical for survival. The presynaptic neural networks converging on these neurons and their role in tuning them remain poorly understood. We took advantage of well-known characteristics of one such neuron in the grasshopper visual system to investigate the properties of its presynaptic input network. We find the structure more complex than hitherto realized. In addition to dynamic lateral inhibition used to filter out background motion, presynaptic circuits include normalizing inhibition and excitatory interactions mediated by muscarinic acetylcholine receptors. These interactions preferentially boost responses to coherently expanding visual stimuli generated by colliding objects, as opposed to spatially incoherent controls, helping to discriminate between them. Hence, in addition to active dendritic conductances within collision-detecting neurons, multiple layers of inhibitory and excitatory presynaptic connections are needed to finely tune neural circuits for collision detection.

Effects of cevimeline on excitability of parasympathetic preganglionic neurons in the superior salivatory nucleus of rats.

The superior salivatory nucleus (SSN) contains parasympathetic preganglionic neurons innervating the submandibular and sublingual salivary glands. Cevimeline, a muscarinic acetylcholine receptor (mAChR) agonist, is a sialogogue that possibly stimulates SSN neurons in addition to the salivary glands themselves because it can cross the blood-brain barrier (BBB). In the present study, we examined immunoreactivities for mAChR subtypes in SSN neurons retrogradely labeled with a fluorescent tracer in neonatal rats. Additionally, we examined the effects of cevimeline in labeled SSN neurons of brainstem slices using a whole-cell patch-clamp technique. Mainly M1 and M3 receptors were detected by immunohistochemical staining, with low-level detection of M4 and M5 receptors and absence of M2 receptors. Most (110 of 129) SSN neurons exhibited excitatory responses to application of cevimeline. In responding neurons, voltage-clamp recordings showed that 84% (101/120) of the neurons exhibited inward currents. In the neurons displaying inward currents, the effects of the mAChR antagonists were examined. A mixture of M1 and M3 receptor antagonists most effectively reduced the peak amplitude of inward currents, suggesting that the excitatory effects of cevimeline on SSN neurons were mainly mediated by M1 and M3 receptors. Current-clamp recordings showed that application of cevimeline induced membrane depolarization (9/9 neurons). These results suggest that most SSN neurons are excited by cevimeline via M1 and M3 muscarinic receptors.

Cyclic AMP Recruits a Discrete Intracellular Ca2+ Store by Unmasking Hypersensitive IP3 Receptors.

Inositol 1,4,5-trisphosphate (IP3) stimulates Ca2+ release from the endoplasmic reticulum (ER), and the response is potentiated by 3',5'-cyclic AMP (cAMP). We investigated this interaction in HEK293 cells using carbachol and parathyroid hormone (PTH) to stimulate formation of IP3 and cAMP, respectively. PTH alone had no effect on the cytosolic Ca2+ concentration, but it potentiated the Ca2+ signals evoked by carbachol. Surprisingly, however, the intracellular Ca2+ stores that respond to carbachol alone could be both emptied and refilled without affecting the subsequent response to PTH. We provide evidence that PTH unmasks high-affinity IP3 receptors within a discrete Ca2+ store. We conclude that Ca2+ stores within the ER that dynamically exchange Ca2+ with the cytosol maintain a functional independence that allows one store to be released by carbachol and another to be released by carbachol with PTH. Compartmentalization of ER Ca2+ stores adds versatility to IP3-evoked Ca2+ signals.

Electrophysiological characterization of the M-current in rat hypoglossal motoneurons.

The M-current (IM) is a voltage-dependent, persistent K+ current so termed because it is strongly inhibited by the cholinergic agonist muscarine. The IM main function is to limit neuronal excitability by contrasting action potential firing. Although motoneurons are sensitive to acetylcholine, the role of IM in modulating their excitability is still controversial. The aim of the present report was to examine the presence of IM in hypoglossal motoneurons (HMs) and its role in the modulation of firing properties using an in vitro model of rat brainstem slice. For this purpose, we employed the whole-cell patch-clamp technique to record HM responses upon stimulation with either a standard IM deactivation voltage protocol or depolarizing current steps. Voltage commands from depolarized potential induced inward relaxations with the common characteristics of IM, comprising inhibition by either muscarine (10µM) or the selective IM inhibitor linopirdine (30µM). IM was pharmacologically distinguished from the hyperpolarization-activated inward-rectifying current and, within the -20 to -50mV range, deactivated with >100-ms time constant. Current-clamp experiments demonstrated that IM strongly regulated HM action potential firing, since both muscarine and linopirdine increased spike frequency whereas the M-channel opener retigabine (20µM) reduced it. Conversely, IM seemed uninvolved in the generation of the medium afterhyperpolarizing potential. Our results suggest that HMs possess IM, whose pharmacological modulation is an important tool to up- or down-regulate excitability, to be explored in experimental models of neurodegeneration.