Application Of Acetylcholine Research Articles

ON and OFF starburst amacrine cells are controlled by distinct cholinergic pathways.

Cholinergic signaling in the retina is mediated by acetylcholine (ACh) released from starburst amacrine cells (SACs), which are key neurons for motion detection. SACs comprise ON and OFF subtypes, which morphologically show mirror symmetry to each other. Although many physiological studies on SACs have targeted ON cells only, the synaptic computation of ON and OFF SACs is assumed to be similar. Recent studies demonstrated that gene expression patterns and receptor types differed between ON and OFF SACs, suggesting differences in their functions. Here, we compared cholinergic signaling pathways between ON and OFF SACs in the mouse retina using the patch clamp technique. The application of ACh increased GABAergic feedback, observed as postsynaptic currents to SACs, in both ON and OFF SACs; however, the mode of GABAergic feedback differed. Nicotinic receptors mediated GABAergic feedback in both ON and OFF SACs, while muscarinic receptors mediated GABAergic feedback in ON SACs only in adults. Neither tetrodotoxin, which blocked action potentials, nor LY354740, which blocked neurotransmitter release from SACs, eliminated ACh-induced GABAergic feedback in SACs. These results suggest that ACh-induced GABAergic feedback in ON and OFF SACs is regulated by different feedback mechanisms in adults and mediated by non-spiking amacrine cells other than SACs.

Noninvasive assessment of human microvascular function in health and disease using incident dark-field microscopy.

Reduced peripheral microvascular reactivity is associated with an increased risk for major adverse cardiac events (MACEs). Tools for noninvasive assessment of peripheral microvascular function are limited, and existing technology is poorly validated in both healthy populations and patients with cardiovascular disease (CVD). Here, we used a handheld incident dark-field imaging tool (CytoCam) to test the hypothesis that, compared with healthy individuals (no risk factors for CVD), subjects formally diagnosed with coronary artery disease (CAD) or those with ≥2 risk factors for CAD (at risk) would exhibit impaired peripheral microvascular reactivity. A total of 17 participants (11 healthy, 6 at risk) were included in this pilot study. CytoCam was used to measure sublingual microvascular total vessel density (TVD), perfused vessel density (PVD), and microvascular flow index (MFI) in response to the topical application of acetylcholine (ACh) and sublingual administration of nitroglycerin (NTG). Baseline MFI and PVD were significantly reduced in the at-risk cohort compared with healthy individuals. Surprisingly, following the application of acetylcholine and nitroglycerin, both groups showed a significant improvement in all three microvascular perfusion parameters. These results suggest that, despite baseline reductions in both microvascular density and perfusion, human in vivo peripheral microvascular reactivity to both endothelial-dependent and -independent vasoactive agents remains intact in individuals with CAD or multiple risk factors for disease.NEW & NOTEWORTHY To our knowledge, this is the first study to comprehensively characterize in vivo sublingual microvascular structure and function (endothelium-dependent and -independent) in healthy patients and those with CVD. Importantly, we used an easy-to-use handheld device that can be easily translated to clinical settings. Our results indicate that baseline microvascular impairments in structure and function can be detected using the CytoCam technology, although reactivity to acetylcholine may be maintained even during disease in the peripheral microcirculation.

Combined application of acetylcholine (ACh) and gamma-aminobutyric acid (GABA) mitigates the effects of water deficit in soybean

Bioregulatory substances, such as acetylcholine (ACh) and γ-aminobutyric acid (GABA), have shown promise in mitigating water deficit effects on plants. Their action on plant physiology, particularly in stomatal regulation and osmoprotection, contributes to enhanced agricultural productivity. Regarding soybean plants, changes in the metabolic profile that could justify the improvement in physiological performance and growth are still unknown. This study investigated the impact of combined ACh:GABA application during soybean V4 and R2 stages using physiological assessments and NMR-based metabolomics. Two equimolar doses (2 and 8 mM) of ACh:GABA were applied to soybean plants subjected to two water regimes: 100 % of field capacity (continuously irrigated) and water deficit (water suspension). Results demonstrated enhanced gas exchange, biomass, and chlorophyll content, alongside favorable enzymatic activity. NMR analysis revealed metabolites countering oxidative stress, supporting osmoprotection, and contributing to soybean development and energy supply. The 8 mM ACh:GABA application notably improved plant performance, showcasing potential for water deficit mitigation in soybeans.

New promising agents against COPD and asthma among the amides of 1-oxo-3-phenyl-isochroman-6-carboxylic acid

Background: Bronchodilators, which are compounds that can relax airway smooth muscle, are perhaps the most important component of combination therapy for chronic obstructive pulmonary disease, one of the most common non-communicable diseases in the world, which is the second most lethal disease after cardiovascular disease. Unfortunately, current clinical bronchodilators, whose activity is mediated by their interaction with muscarinic acetylcholine receptors, have side effects (up to myocardial infarction) due to their cross-affinity for different types of these receptors, including those prevalent in the heart muscle. Objectives: The aim of this work is to search/develop compounds — effective bronchodilators capable of selectively inhibiting type 3 muscarinic acetylcholine receptors (M3 receptors), predominantly present in smooth muscles and not characteristic of cardiomyocytes. Materials and Methods: High-throughput virtual screening of a collection of 150,000 compounds was conducted on the spatial structure of the M3 receptor, reconstructed in our previous studies. The effect of substances on contractile activity was investigated using tensometry in isometric mode on multicellular tracheal preparations. Antagonistic activity and type of inhibition were determined against the background of acetylcholine application (concentration range 10-10–10-3 M). To establish the affinity value of the compound-antagonist, the Schild regression equation was used. Results: Based on virtual screening data, a series of compounds — amides of 1-oxo-3-phenyl-iso-chroman-6-carboxylic acid — were selected for biological testing. For two of these compounds (Compounds 1 and 7), the ability to selectively inhibit M3 receptors was demonstrated. Specifically, the affinity value pKB for Compound 1 was 7.28 ± 0.70, with an IC50 of 5.25·10-8 M. A critically important advantage of this compound is its ability, at equal concentrations, to more effectively inhibit signal transmission through M3 receptors compared to ipratropium bromide — a clinical cholinergic receptor inhibitor. Conclusions: The sufficient effectiveness of inhibition and significantly increased selectivity of the studied compounds specifically towards M3 receptors provide strong grounds to consider these compounds as promising precursors of new generation cholinolytic drugs with targeted action on M3-type cholinergic receptors.

A new affine inhibitor of sodium pump thiacalix[4]arene С-1193 increases the intracellular concentration of Ca ions and modifies myometrium contractility

The methods of enzymatic and kinetic analysis were used to demonstrate that thiacalix[4]arene-bis-hydroxymethylphosphonic acid С-1193 had the inhibitory effect (І0.5 = 42.1 ± 0.6 nM) on Na+,K+-ATPase activity in the plasma membrane of myometrium cells with no effect on the relative activity of other ATPases localized in this subcellular structure. The method of confocal microscopy and Са2+-sensitive fluorescent probe fluo-4 were used to demonstrate that thiacalix[4]arene С-1193 increased the intracellular concentration of Ca ions in the immobilized uterine myocytes. The tenzometric studies proved that С-1193 (10 and 100 μМ) increased the isometric phasic contractions, induced via the paths of both electromechanical (depolarization with high-potassium solution) and pharmacomechanical (application of uterotonic hormone oxytocin, neurotransmitter acetylcholine or selective agonist of muscarinic acetylcholine receptors cevimeline) coupling. Application of thiacalix[4]arene С-1193 as a selective and effective inhibitor of Nа+,K+-ATPase may be useful both for studyng the regulation of ion homeostasis in smooth muscle cells and creation of new uterotonics based on the calixarene core. Keywords: contractility mechanokinetics, myometrium, Na+‚K+-АТРase, thiacalix[4]arene

Methyl Palmitate Modulated NMDA-Induced Cerebral Hyperemia in Hypertensive Rats

N-methyl-D-aspartate (NMDA) receptors were found to be dysfunctional in hypertensive rats. Methyl palmitate (MP) has been shown to diminish the nicotine-induced increase in blood flow in the brainstem. The aim of this study was to determine how MP modulated NMDA-induced increased regional cerebral blood flow (rCBF) in normotensive (WKY), spontaneously hypertensive (SHR), and renovascular hypertensive (RHR) rats. The increase in rCBF after the topical application of experimental drugs was measured using laser Doppler flowmetry. Topical NMDA application induced an MK-801-sensitive increase in rCBF in anesthetized WKY rats, which was inhibited by MP pretreatments. This inhibition was prevented by pretreatment with chelerythrine (a PKC inhibitor). The NMDA-induced increase in rCBF was also inhibited by the PKC activator in a concentration-dependent manner. Neither MP nor MK-801 affected the increase in rCBF induced by the topical application of acetylcholine or sodium nitroprusside. Topical application of MP to the parietal cortex of SHRs, on the other hand, increased basal rCBF slightly but significantly. MP enhanced the NMDA-induced increase in rCBF in SHRs and RHRs. These results suggested that MP had a dual effect on the modulation of rCBF. MP appears to play a significant physiological role in CBF regulation.

Diclofenac and dexamethasone modulate the effect of cannabidiol on the rat colon motility ex vivo.

Due to the growing interest in the use of cannabinoids in supportive therapies, they are increasingly used together with anti-inflammatory drugs. Cannabinoids inhibit gastrointestinal motility, while steroidal and nonsteroidal anti-inflammatory drugs influence motility in other ways. The aim of the research was to study the interactions between cannabidiol (CBD) and these two classes of anti-inflammatory drugs in the context of gastrointestinal motility. Dexamethasone (DEX) was selected as a steroidal drug and diclofenac (DCF) as a nonsteroidal counterpart. The experiments were performed on isolated rat colon strips in isometric conditions. The contractile response to acetylcholine (ACh) (1 μM) was measured with no substance applied as a control value and was measured after application of CBD (80 μM), DEX (100 μM), DCF (100 μM), or a combination of these substances. Cannabidiol strongly inhibited intestinal motility mediated by ACh application, DCF inhibited it non-significantly, while DEX intensified it. When CBD was co-administered with DEX, the combination inhibited intestinal motility non-significantly relative to the ACh-only control. Co-administration of CBD with DCF inhibited motility more than when these substances were administered separately. Inhibition of the intestinal response to ACh is likely due to the synergistic effect of CBD and endogenous cannabinoids. Dexamethasone lessened the inhibitory effect of CBD, likely because of diminished availability of the arachidonic acid necessary for endogenous cannabinoid synthesis. However, diclofenac may increase endogenous cannabinoid synthesis, because of the greater availability of arachidonic acid caused by DCF blocking the cyclooxygenation pathway.

Acetylcholine Inhibits Spontaneous Firing Activity of Terminal Nerve GnRH Neurons in Medaka.

Vertebrates generally possess hypophysiotropic and non-hypophysiotropic gonadotropin releasing hormone (GnRH) neurons. The terminal nerve (TN) GnRH neurons are known to belong to the non-hypophysiotropic neurons and have been suggested to modulate sexual behaviors. These neurons show spontaneous pacemaker firing activity and release neuropeptides GnRH and neuropeptide FF. Since the spontaneous firing activities of peptidergic neurons, including GnRH neurons, are believed to play important roles in the release of neuropeptides, understanding the regulatory mechanisms of these spontaneous firing activities is important. Here, we analyzed firing activities of the TN-GnRH neurons in medaka during application of acetylcholine (ACh), which is one of the essential neuromodulators in the brain. Whole cell patch clamp recording of TN-GnRH neurons demonstrated that ACh induces hyperpolarization and inhibits their pacemaker firing. Electrophysiological analysis using an antagonist for acetylcholine receptors and in situ hybridization analysis showed that firing of TN-GnRH neurons is inhibited via M2-type muscarinic acetylcholine receptor. These findings, taken together with literature from several other fish species (including teleosts and elasmobranchs), indicate that ACh may generally play an inhibitory role in modulating spontaneous activities of TN-GnRH neurons and thereby sexual behaviors in fish.

Parkin Knockdown Modulates Dopamine Release in the Central Complex, but Not the Mushroom Body Heel, of Aging Drosophila.

Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic neurons leading to reduced locomotion. Mutations of parkin gene in Drosophila produce the same phenotypes as vertebrate models, but the effect of parkin knockdown on dopamine release is not known. Here, we report age-dependent, spatial variation of dopamine release in the brain of parkin-RNAi adult Drosophila. Dopamine was repetitively stimulated by local application of acetylcholine and quantified by fast-scan cyclic voltammetry in the central complex or mushroom body heel. In the central complex, the main area controlling locomotor function, dopamine release is maintained for repeated stimulations in aged control flies, but lower concentrations of dopamine are released in the central complex of aged parkin-RNAi flies. In the mushroom body heel, the dopamine release decrease in older parkin-RNAi flies is similar to controls. There is not significant dopaminergic neuronal loss even in older parkin knockdown flies, which indicates that the changes in stimulated dopamine release are due to alterations of neuronal function. In young parkin-RNAi flies, locomotion is inhibited by 30%, while in older parkin-RNAi flies it is inhibited by 85%. Overall, stimulated dopamine release is modulated by parkin in an age and brain region dependent manner. Correlating the functional state of the dopaminergic system with behavioral phenotypes provides unique insights into the PD mechanism. Drosophila can be used to study dopamine functionality in PD, elucidate how genetics influence dopamine, and test potential therapies to maintain dopamine release.

Red blood cells induce endothelial dysfunction in patients with ST-elevation myocardial infarction and elevated C-reactive protein

Abstract Background The important role of inflammation in atherosclerotic plaque progression and instability leading to myocardial infarction has been widely demonstrated. C-reactive protein (CRP) has been shown to be of predictive value in atherosclerotic cardiovascular disease. The red blood cell (RBC) is an important regulator of cardiovascular function through nitric oxide bioactivity and oxidative stress in ischemic heart disease. Also, arginase-1 has been shown to greatly influence nitric oxide bioactivity in RBCs and to cause endothelial dysfunction. However, the mechanisms by which RBCs regulate vascular function in patients with myocardial infarction and its relation to inflammation and arginase-1 remain unknown. Objective The study aimed to investigate the effect of RBCs on endothelial function in patients with ST-elevation myocardial infarction (STEMI) and its possible association with systemic inflammation and arginase-1. Material and methods Blood samples were collected from patients with STEMI within 36 h after admission and from age-matched healthy controls. RBCs were incubated with isolated rat aortic segments for 18 h after which the aortas were assessed for endothelium-dependent and endothelium-independent relaxations using wire myographs and application of acetylcholine and nitroprusside, respectively. The vascular response was evaluated in relation to the level of inflammation defined as CRP <2 and ≥2 mg/L at admission. The levels of 4-hydroxynonenal (4-HNE; a marker of oxidative stress formed by lipid peroxidation) and the expression of arginase-1 were visualized in rat aortas following incubation with RBCs by immunohistochemistry. All handling and procedures regarding human subjects and sampling were performed according to the Declaration of Helsinki. All animal experiments and procedures were performed according to the guidelines by the U.S National Institutes of Health (NIH publication no 85–23, revised 1996). Results RBCs from patients with STEMI and elevated CRP (≥2 mg/L, mean of 9.6 mg/L) induced significant impairment of endothelium-dependent relaxation compared to RBCs from patients with STEMI and low CRP (<2 mg/L, mean of 1.0 mg/L) and to the healthy controls (Fig. 1). Endothelium-independent relaxations did not differ between the groups. Immunohistochemical staining of the aorta revealed that incubation with RBCs from patients with STEMI and high CRP increased the expression of 4-HNE and arginase-1 compared to incubation of RBCs from healthy controls and patients with STEMI and low CRP (Fig. 2). Conclusion RBCs from patients with STEMI and elevated CRP induce endothelial dysfunction and increase the expression of 4-HNE and arginase-1, indicating that inflammation is involved in the mechanism by which RBCs induce endothelial dysfunction in STEMI. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swedish Heart and Lung foundation

Pharmacological and molecular characterization of the A-type muscarinic acetylcholine receptor from Anopheles gambiae.

Muscarinic acetylcholine receptors (mAChRs) which are G protein-coupled receptors play key roles in insect physiology. Whereas vertebrate mAChRs are important targets for pharmaceutical drugs, insect mAChRs are under-exploited by the agro-chemical industry. Moreover, insect mAChRs have been less well studied than their vertebrate counterparts. Their critical functions mean that a better knowledge of the insect mAChRs is crucial for the effort to develop a new molecular-level strategy for insect pest management. Almost all insects possess three mAChRs named A, B and C which differ according to their coupling effector systems and their pharmacological profile. The aim of this study was to characterize the A-type mAChR (mAChR-A) from Anopheles gambiae which is the major vector of malaria in order to develop new strategies in pest management. In this paper, we reported that mAChR-A is more expressed in adult mosquitoes than in larvae. Furthermore, using calcium imaging recordings, we found that the An. gambiae mAChR-A expressed in Sf9 cells is activated by specific muscarinic agonists acetylcholine, muscarine and oxotremorine M and blocked by several mAChR antagonists. Moreover, using inhibitors of phosphoinositide pathway such as Gαq/11 protein blocker, we have shown that an increased intracellular calcium concentration elicited by the acetylcholine application was mediated by PLC/IP3R pathway. As a rise in intracellular calcium concentration could lead to an increase in the insecticide target sensitivity, these results suggest that An. gambiae mAChR-A should not be only considered as a potential target for new molecules but also as a key element to optimize the efficacy of insecticide in vector control.

Nicotinic agonists promoted the activation of nicotinic acetylcholine α7 receptors (α7 nAChR) in neurons, but failed to activate these receptors in mouse peritoneal macrophages

Nicotinic acetylcholine receptor (nAChR) of subtypes said "neuronal" are expressed in epithelial and immune system cells and participate in acetylcholine signaling by neural or non-neural pathways. It has been shown in macrophages that activation of type a7 nAChRs inhibits the release of pro-inflammatory cytokines, but the ion channel function has not been recorded in these cells. The objective of this work was to clarify what are the molecular mechanisms of transduction of a7 nAChRs in macrophages. To this end, RAW 264.7 cells, mouse peritoneal macrophages and rat hippocampal neurons were used. Cells were submitted to electrophysiological studies and stimulated with brief applications of the agonists acetylcholine, choline and nicotine, associated or not with the allosteric modulator PNU-120596. Responses to ATP were recorded as a reference. Furthermore, macrophages were submitted to cytokine quantitation. The electrophysiological results showed that macrophages responded to ATP but did not show whole-cell current by stimulation with nicotinic agonists. However, hippocampal neurons stimulated in the same pharmacological conditions of the macrophages showed ionic currents typical of the a7 nicotinic receptors. No effect of nicotine was observed in the lipopolysaccharide-induced TNF-a release. These results suggest that the a7 nAChR in macrophages do not work as ion channels similar to those expressed in neurons.

Neurotransmitters Affect Larval Development by Regulating the Activity of Prothoracicotropic Hormone-Releasing Neurons in Drosophila melanogaster.

Ecdysone, an essential insect steroid hormone, promotes larval metamorphosis by coordinating growth and maturation. In Drosophila melanogaster, prothoracicotropic hormone (PTTH)-releasing neurons are considered to be the primary promoting factor in ecdysone biosynthesis. Recently, studies have reported that the regulatory mechanisms of PTTH release in Drosophila larvae are controlled by different neuropeptides, including allatostatin A and corazonin. However, it remains unclear whether neurotransmitters provide input to PTTH neurons and control the metamorphosis in Drosophila larvae. Here, we report that the neurotransmitters acetylcholine (ACh) affect larval development by modulating the activity of PTTH neurons. By downregulating the expression of different subunits of nicotinic ACh receptors in PTTH neurons, pupal volume was significantly increased, whereas pupariation timing was relatively unchanged. We also identified that PTTH neurons were excited by ACh application ex vivo in a dose-dependent manner via ionotropic nicotinic ACh receptors. Moreover, in our Ca2+ imaging experiments, relatively low doses of OA caused increased Ca2+ levels in PTTH neurons, whereas higher doses led to decreased Ca2+ levels. We also demonstrated that a low dose of OA was conveyed through OA β-type receptors. Additionally, our electrophysiological experiments revealed that PTTH neurons produced spontaneous activity in vivo, which provides the possibility of the bidirectional regulation, coming from neurons upstream of PTTH cells in Drosophila larvae. In summary, our findings indicate that several different neurotransmitters are involved in the regulation of larval metamorphosis by altering the activity of PTTH neurons in Drosophila.

Efferent neurons control hearing sensitivity and protect hearing from noise through the regulation of gap junctions between cochlear supporting cells.

It is critical for hearing that the descending cochlear efferent system provides a negative feedback to hair cells to regulate hearing sensitivity and protect hearing from noise. The medial olivocochlear (MOC) efferent nerves project to outer hair cells (OHCs) to regulate OHC electromotility, which is an active cochlear amplifier and can increase hearing sensitivity. Here, we report that the MOC efferent nerves also could innervate supporting cells (SCs) in the vicinity of OHCs to regulate hearing sensitivity. MOC nerve fibers are cholinergic, and acetylcholine (ACh) is a primary neurotransmitter. Immunofluorescent staining showed that MOC nerve endings, presynaptic vesicular acetylcholine transporters (VAChTs), and postsynaptic ACh receptors were visible at SCs and in the SC area. Application of ACh in SCs could evoke a typical inward current and reduce gap junctions (GJs) between them, which consequently enhanced the direct effect of ACh on OHCs to shift but not eliminate OHC electromotility. This indirect, GJ-mediated inhibition had a long-lasting influence. In vivo experiments further demonstrated that deficiency of this GJ-mediated efferent pathway decreased the regulation of active cochlear amplification and compromised the protection against noise. In particular, distortion product otoacoustic emission (DPOAE) showed a delayed reduction after noise exposure. Our findings reveal a new pathway for the MOC efferent system via innervating SCs to control active cochlear amplification and hearing sensitivity. These data also suggest that this SC GJ-mediated efferent pathway may play a critical role in long-term efferent inhibition and is required for protection of hearing from noise trauma.NEW & NOTEWORTHY The cochlear efferent system provides a negative feedback to control hair cell activity and hearing sensitivity and plays a critical role in noise protection. We reveal a new efferent control pathway in which medial olivocochlear efferent fibers have innervations with cochlear supporting cells to control their gap junctions, therefore regulating outer hair cell electromotility and hearing sensitivity. This supporting cell gap junction-mediated efferent control pathway is required for the protection of hearing from noise.

The Effectiveness in Activating M-Type K+ Current Produced by Solifenacin ([(3R)-1-azabicyclo[2.2.2]octan-3-yl] (1S)-1-phenyl-3,4-dihydro-1H-isoquinoline-2-carboxylate): Independent of Its Antimuscarinic Action.

Solifenacin (Vesicare®, SOL), known to be a member of isoquinolines, is a muscarinic antagonist that has anticholinergic effect, and it has been beneficial in treating urinary incontinence and neurogenic detrusor overactivity. However, the information regarding the effects of SOL on membrane ionic currents is largely uncertain, despite its clinically wide use in patients with those disorders. In this study, the whole-cell current recordings revealed that upon membrane depolarization in pituitary GH3 cells, the exposure to SOL concentration-dependently increased the amplitude of M-type K+ current (IK(M)) with effective EC50 value of 0.34 μM. The activation time constant of IK(M) was concurrently shortened in the SOL presence, hence yielding the KD value of 0.55 μM based on minimal reaction scheme. As cells were exposed to SOL, the steady-state activation curve of IK(M) was shifted along the voltage axis to the left with no change in the gating charge of the current. Upon an isosceles-triangular ramp pulse, the hysteretic area of IK(M) was increased by adding SOL. As cells were continually exposed to SOL, further application of acetylcholine (1 μM) failed to modify SOL-stimulated IK(M); however, subsequent addition of thyrotropin releasing hormone (TRH, 1 μM) was able to counteract SOL-induced increase in IK(M) amplitude. In cell-attached single-channel current recordings, bath addition of SOL led to an increase in the activity of M-type K+ (KM) channels with no change in the single channel conductance; the mean open time of the channel became lengthened. In whole-cell current-clamp recordings, the SOL application reduced the firing of action potentials (APs) in GH3 cells; however, either subsequent addition of TRH or linopirdine was able to reverse SOL-mediated decrease in AP firing. In hippocampal mHippoE-14 neurons, the IK(M) was also stimulated by adding SOL. Altogether, findings from this study disclosed for the first time the effectiveness of SOL in interacting with KM channels and hence in stimulating IK(M) in electrically excitable cells, and this noticeable action appears to be independent of its antagonistic activity on the canonical binding to muscarinic receptors expressed in GH3 or mHippoE-14 cells.

Ethanol interaction with α3β4 nicotinic acetylcholine receptors in neurons of the laterodorsal tegmentum.

Nicotinic acetylcholine receptors (nAChRs) play a key role in the rewarding effects of ethanol (EtOH), and while several nAChR subtypes have been implicated, attention has recently shifted to a role for the α3β4 nAChR. The laterodorsal tegmental nucleus (LDTg), a brainstem cholinergic nucleus that sends excitatory projections to the ventral tegmental area, is an Integral part of the brain reward pathway. Here we investigate a potential role for LDTg α3β4 nAChRs in EtOH self-administration and reward. Sprague-Dawley rats were given ad libitum access to a 20% EtOH solution, as part of a two-bottle choice paradigm. Approximately 1week after removal of EtOH access, we measured LDTg α3β4 nAChR current responses to focal application of acetylcholine (ACh), using whole-cell patch clamp electrophysiology recordings in acute brain slices. In addition, we used whole-cell electrophysiology to assess the acute effects of EtOH on the sensitivity of LDTg α3β4 nAChRs. Focal application of ACh onto LDTg neurons resulted in large α3β4 nAChR-mediated inward currents, the magnitude of which showed a positive correlation with levels of EtOH self-administration. In addition, using brain slices taken from EtOH-naïve rats, bath application of EtOH resulted in a moderate potentiation of LDTg α3β4 nAChR sensitivity. Using a rat model, increased α3β4 nAChR function was associated with greater EtOH self-administration, with α3β4 nAChR function also acutely potentiated by EtOH. Assuming that similar findings apply to humans, the α3β4 nAChR could be a therapeutic target in the treatment of EtOH use disorder.

Neuron-specific responses to acetylcholine within the spinal dorsal horn circuits of rodent and primate

Excitatory and inhibitory neurotransmission within the spinal dorsal horn is tightly controlled to regulate transmission of nociceptive signals to the brain. One aspect of this control is modulation of neuronal activity through cholinergic signaling. Nociceptive neurons in the dorsal horn express both nicotinic and muscarinic cholinergic receptors and activation of these receptors reduces pain in humans, while inhibition leads to nociceptive hypersensitivity. At a cellular level, acetylcholine (ACh) has diverse effects on excitability which is dependent on the receptor and neuronal subtypes involved. In the present study we sought to characterize the electrophysiological responses of specific subsets of lamina II interneurons from rat and marmoset spinal cord. Neurons were grouped by morphology and by action potential firing properties. Whole-cell voltage-clamp recordings from lamina II dorsal horn neurons of adult rats showed that bath applied acetylcholine increased, decreased or had no effect on spontaneous synaptic current activity in a cell-type specific manner. ACh modulated inhibitory synaptic activity in 80% of neurons, whereas excitatory synaptic activity was affected in less than 50% of neurons. In whole-cell current clamp recordings, brief somatic application of ACh induced cell-type specific responses in 79% of rat lamina II neurons, which included: depolarization and action potential firing, subthreshold membrane depolarization, biphasic responses characterized by transient depolarization followed by hyperpolarization and membrane hyperpolarization alone. Similar responses were seen in marmoset lamina II neurons and the properties of each neuron group were consistent across species. ACh-induced hyperpolarization was blocked by the muscarinic antagonist atropine and all forms of acetylcholine-induced depolarization were blocked by the nicotinic antagonist mecamylamine. The cholinergic system plays an important role in regulating nociception and this study contributes to our understanding of how circuit activity is controlled by ACh at a cellular level in primate and rodent spinal cord.

Effects of acetylcholine on hypoglossal and C4 nerve activity in brainstem-spinal cord preparations from newborn rat

Effects of acetylcholine (ACh) on respiratory activity have been an intriguing theme especially in relation to central chemoreception and the control of hypoglossal nerve activity. We studied the effects of ACh on hypoglossal and phrenic (C4) nerve activities and inspiratory and pre-inspiratory neurons in the rostral ventrolateral medulla in brainstem-spinal cord preparations from newborn rats. ACh application increased respiratory rhythm, decreased inspiratory hypoglossal and C4 nerve burst amplitude, and enhanced pre-inspiratory hypoglossal activity. ACh induced membrane depolarization of pre-inspiratory neurons that might be involved in facilitation of respiratory rhythm by ACh. Effects of ACh on hypoglossal and C4 nerve activity were partially reversed by a nicotinic receptor blocker, mecamylamine. Further application of a muscarinic receptor antagonist, oxybutynin, resulted in slight increase of hypoglossal (but not C4) burst amplitude. Thus, ACh induced different effects on hypoglossal and C4 nerve activity in the brainstem-spinal cord preparation.

Edaravone attenuates sustained pial arteriolar vasoconstriction independently of endothelial function after unclamping of the abdominal aorta in rabbits.

BackgroundCerebral blood flow (CBF) has direct effects on neuronal function and neurocognitive disorders. Oxidative stress from abdominal aortic surgery is important in the pathophysiology of CBF impairment. We investigated the effect of edaravone on the pial arteriolar diameter changes induced by abdominal aortic surgery and the involvement of the endothelium in the changes.MethodsThe closed cranial window technique was used in rabbits to measure changes in pial arteriolar diameter after the unclamping of abdominal aortic cross-clamping with an intravenous free radical scavenger, edaravone (control group [n = 6], edaravone 10 μg/kg/ min [n = 6], 100 μg/kg/min [n = 6]). Pial vasodilatory responses to topical application of acetylcholine (ACh) into the cranial window were investigated before abdominal aortic cross-clamping and after unclamping with intravenous administration of edaravone (control group [n = 6], edaravone 100 μg/kg/min [n = 6]).ResultsAortic unclamping-induced vasoconstriction was significantly attenuated by continuous infusion of edaravone at 100 μg/kg/min. Topical ACh after unclamping did not produce any changes in pial arteriolar responses in comparison to before aortic cross-clamping in the control or edaravone groups. The changes in the response to topical ACh after unclamping in the saline and edaravone groups did not differ significantly.ConclusionsFree radicals during abdominal aortic surgery might have contracted cerebral blood vessels independently of endothelial function in rabbits. Suppression of free radicals attenuated the sustained pial arteriolar vasoconstriction after aortic unclamping. Thus, the free radical scavenger might have some brain protective effect that maintains CBF independently of endothelial function.

Levosimendan as a therapeutic strategy to prevent neuroinflammation after aneurysmal subarachnoid hemorrhage?

BackgroundPoor patient outcomes after aneurysmal subarachnoid hemorrhage (SAH) occur due to a multifactorial process, mainly involving cerebral inflammation (CI), delayed cerebral vasospasm (DCVS), and delayed cerebral ischemia, followed by neurodegeneration....