Differential regulation of prelimbic and thalamic transmission to the basolateral amygdala by acetylcholine receptors

The amygdalar anterior basolateral nucleus (BLa) plays a vital role in emotional behaviors. This region receives dense cholinergic projections from basal forebrain which are critical in regulating neuronal activity and synaptic transmission. Cholinergic signaling in BLa is thought to occur through both a slow mode of volume transmission as well as a rapid, phasic mode. However, the relative effect of each mode of signaling in BLa is not understood. Here, we used electrophysiology and optogenetics in mouse brain slices to compare regulation of afferent input from cortex and thalamus to the BLa by these two modes of transmission. Phasic ACh release evoked by single pulse stimulation of cholinergic terminals had a biphasic effect on glutamatergic transmission at cortical input, producing rapid nicotinic receptor-mediated facilitation followed by slower muscarinic receptor (mAChR)-mediated depression. In contrast, tonic elevation of ACh through application of the cholinesterase inhibitor physostigmine suppressed glutamatergic transmission at cortical inputs through mAChRs only. This suppression was not observed at thalamic inputs to BLa. In agreement with this pathway-specificity, the mAChR agonist, muscarine more potently suppressed transmission at inputs from prelimbic cortex (PL) than thalamus. Muscarinic inhibition at PL input was dependent on presynaptic M4 mAChRs, while at thalamic input it depended upon M3 mAChR-mediated stimulation of retrograde endocannabinoid signaling. Muscarinic inhibition at both pathways was frequency-dependent, allowing only high frequency activity to pass. These findings demonstrate complex cholinergic regulation of afferent input to BLa that depends upon the mode of ACh release and is both pathway specific and frequency dependent.

Activation of Neuronal Nicotinic Receptors Inhibits Acetylcholine Release in the Neuromuscular Junction by Increasing Ca2+ Flux through Cav1 Channels

Cholinergic neurotransmission is a key signal pathway in the peripheral nervous system and in several branches of the central nervous system. Despite the fact that it has been studied extensively for a long period of time, some aspects of its regulation still have not yet been established. One is the relationship between the nicotine-induced autoregulation of acetylcholine (ACh) release with changes in the concentration of presynaptic calcium levels. The mouse neuromuscular junction of m. Levator Auris Longus was chosen as the model of the cholinergic synapse. ACh release was assessed by electrophysiological methods. Changes in calcium transients were recorded using a calcium-sensitive dye. Nicotine hydrogen tartrate salt application (10 μM) decreased the amount of evoked ACh release, while the calcium transient increased in the motor nerve terminal. Both of these effects of nicotine were abolished by the neuronal ACh receptor antagonist dihydro-beta-erythroidine and Cav1 blockers, verapamil, and nitrendipine. These data allow us to suggest that neuronal nicotinic ACh receptor activation decreases the number of ACh quanta released by boosting calcium influx through Cav1 channels.

Disrupted choline clearance and sustained acetylcholine release in vivo by a common choline transporter coding variant associated with poor attentional control in humans

Transport of choline via the neuronal high-affinity choline transporter (CHT; SLC5A7) is essential for cholinergic terminals to synthesize and release acetylcholine (ACh). In humans, we previously demonstrated an association between a common CHT coding substitution (rs1013940; Ile89Val) and reduced attentional capacity as well as attenuated frontal cortex activation. Here, we used a CRISPR/Cas9 approach to generate mice expressing the I89V substitution and assessed, using in vivo cortical choline biosensing, CHT-mediated choline transport, and ACh release. CHT-mediated clearance of choline in mice expressing one or two Val89 alleles was reduced by over 7-fold relative to wild type (WT) mice, suggesting dominant-negative effects. Choline clearance in CHT Val89 mice was further reduced by neuronal inactivation. Deficits in ACh release, 5 and 10 min after repeated depolarization at a low, behaviorally relevant frequency, support an attenuated reloading capacity of cholinergic neurons in mutant mice. The density of CHTs in total synaptosomal lysates and neuronal plasma-membrane-enriched fractions was not impacted by the Val89 variant, indicating a selective impact on CHT function. Consistent with this hypothesis, structural modeling revealed that Val89 may attenuate choline transport by changing the ability of choline to induce conformational changes of CHT that support normal transport rates. Our findings suggest that diminished, sustained cholinergic signaling capacity in the frontal cortex underlies perturbed attentional performance in individuals expressing CHT Val89. Our work supports the utility of the CHT Val89 mouse model as a valuable model to study heritable risk for cognitive disorders arising from cholinergic dysfunction.

Activation of neuronal nicotinic receptors inhibits acetylcholine release in the neuromuscular junction by increasing Ca2+ flux through Cav1 channels

Background and Purpose: Cholinergic neurotransmission is a key signal pathway in the peripheral nervous system (PNS) and in several branches of the central nervous system (CNS). Despite the fact that it has been studied extensively for a long period of time, some aspects of its regulation still have not yet been established. One is relationship between nicotine-induced autoregulation of acetylcholine (ACh) release with changes in the concentration of presynaptic calcium levels. Experimental Approach: The mouse neuromuscular junction of m. Levator Auris Longus was chosen as the model of the cholinergic synapse. ACh release was assessed by electrophysiological methods. Changes in the calcium transients were recorded using a calcium-sensitive dye. Functional interaction between nicotinic ACh receptors and calcium channels was investigated pharmacologically using specific agonists and antagonists. Key Results: Nicotine hydrogen tartrate salt (considered as a stable form for potential therapeutic delivery of nicotine) effects on the parameters of ACh release from the nerve ending were analyzed. Nicotine application (10 μM) decrease the amount of evoked ACh release, while calcium transient increase in the motor nerve terminal. Both of these effects of nicotine were abolished by the neuronal ACh receptor antagonist dihydro-beta-erythroidine and Cav1 blockers, verapamil and nitrendipine. Conclusion and Implications: Neuronal nicotinic ACh receptors activation decreases the number of ACh quanta released by boosting calcium influx through Cav1 channels. Understanding of mechanisms of autoregulation of ACh release is important for the searching new approaches treat diseases associated with cholinergic dysfunction.

Contribution of afferent pathway to vagal nerve stimulation-induced myocardial interstitial acetylcholine release in rats

Vagal nerve stimulation (VNS) has been explored as a potential therapy for chronic heart failure. The contribution of the afferent pathway to myocardial interstitial acetylcholine (ACh) release during VNS has yet to be clarified. In seven anesthetized Wistar-Kyoto rats, we implanted microdialysis probes in the left ventricular free wall and measured the myocardial interstitial ACh release during right VNS with the following combinations of stimulation frequency (F in Hz) and voltage readout (V in volts): F0V0 (no stimulation), F5V3, F20V3, F5V10, and F20V10. F5V3 did not affect the ACh level. F20V3, F5V10, and F20V10 increased the ACh level to 2.83 ± 0.47 ( P < 0.01), 4.31 ± 1.09 ( P < 0.001), and 4.33 ± 0.82 ( P < 0.001) nM, respectively, compared with F0V0 (1.76 ± 0.22 nM). After right vagal afferent transection (rVAX), F20V3 and F20V10 increased the ACh level to 2.90 ± 0.53 ( P < 0.001) and 3.48 ± 0.63 ( P < 0.001) nM, respectively, compared with F0V0 (1.61 ± 0.19 nM), but F5V10 did not (2.11 ± 0.24 nM). The ratio of the ACh levels after rVAX relative to before was significantly <100% in F5V10 (59.4 ± 8.7%) but not in F20V3 (102.0 ± 8.7%). These results suggest that high-frequency and low-voltage stimulation (F20V3) evoked the ACh release mainly via direct activation of the vagal efferent pathway. By contrast, low-frequency and high-voltage stimulation (F5V10) evoked the ACh release in a manner dependent on the vagal afferent pathway.

Expression of cholinesterases and their anchoring proteins in rat heart

Acetylcholine (ACh)-mediated vagal transmission as well as nonneuronal ACh release are considered cardioprotective in pathological situations with increased sympathetic drive such as ischemia–reperfusion and cardiac remodeling. ACh action is terminated by hydrolysis by the cholinesterases (ChEs), acetylcholinesterase, and butyrylcholinesterase. Both ChEs exist in multiple molecular variants either soluble or anchored by specific anchoring proteins like collagen Q (ColQ) anchoring protein and proline-rich membrane anchoring protein (PRiMA). Here we assessed the expression of specific ChE molecular forms in different heart compartments using RT-qPCR. We show that both ChEs are expressed in all heart compartments but display different expression patterns. The acetylcholinesterase-T variant together with PRiMA and ColQ is predominantly expressed in rat atria. Butylcholinesterase is found in all heart compartments and is accompanied by both PRiMA and ColQ anchors. Its expression in the ventricular system suggests involvement in the nonneuronal cholinergic system. Additionally, two PRiMA variants are detected throughout the rat heart.

Interaction between Calcium Chelators and the Activity of P2X7 Receptors in Mouse Motor Synapses

The ability of P2X7 receptors to potentiate rhythmically evoked acetylcholine (ACh) release through Ca2+ entry via P2X7 receptors and via L-type voltage-dependent Ca2+ channels (VDCCs) was compared by loading Ca2+ chelators into motor nerve terminals. Neuromuscular preparations of the diaphragms of wild-type (WT) mice and pannexin-1 knockout (Panx1−/−) mice, in which ACh release is potentiated by the disinhibition of the L-type VDCCs upon the activation of P2X7 receptors, were used. Miniature end-plate potentials (MEPPs) and evoked end-plate potentials (EPPs) were recorded when the motor terminals were loaded with slow or fast Ca2+ chelators (EGTA-AM or BAPTA-AM, respectively, 50 μM). In WT and Panx1−/− mice, EGTA-AM did not change either spontaneous or evoked ACh release, while BAPTA-AM inhibited synaptic transmission by suppressing the quantal content of EPPs throughout the course of the short rhythmic train (50 Hz, 1 s). In the motor synapses of either WT or Panx1−/− mice in the presence of BAPTA-AM, the activation of P2X7 receptors by BzATP (30 μM) returned the EPP quantal content to the control level. In the neuromuscular junctions (NMJs) of Panx1−/− mice, EGTA-AM completely prevented the BzATP-induced increase in EPP quantal content. After Panx1−/− NMJs were treated with BAPTA-AM, BzATP lost its ability to enhance the EPP quantal content to above the control level. Nitrendipine (1 μM), an inhibitor of L-type VDCCs, was unable to prevent this BzATP-induced enhancement of EPP quantal content to the control level. We propose that the activation of P2X7 receptors may provide additional Ca2+ entry into motor nerve terminals, which, independent of the modulation of L-type VDCC activity, can partially reduce the buffering capacity of Ca2+ chelators, thereby providing sufficient Ca2+ signals for ACh secretion at the control level. However, the activity of both Ca2+ chelators was sufficient to eliminate Ca2+ entry via L-type VDCCs activated by P2X7 receptors and increase the EPP quantal content in the NMJs of Panx1−/− mice to above the control level.

A118 GUT MICROBIOTA TRANSPLANTATION FROM A PATIENT WITH SEVERE CONSTIPATION INDUCED CHANGES IN COLONIC FUNCTION AND STRUCTUR OF GNOTOBIOTIC MICE

Background Increasing evidence suggests that gut microbiota play a key role in gastrointestinal (GI) tract function. We have previously shown that fecal microbiota transplantation diarrhea predominant IBS patients into germ-free mice induces faster GI transit, increased permeability and innate immune activation. However, it is unknown whether gut dysfunction is induced by microbiota from patients with chronic constipation. Aims Here, we investigated the role of the intestinal microbiota in the expression of severe slow transit constipation in a patient with previous C difficile infection and extensive antibiotic exposure. Methods Germ-free (GF) mice (14 weeks old) were gavaged with diluted fecal content from the patient with constipation (PA) or a sex and age-matched healthy control (HC). 12 weeks later, we assessed gut motility and GI transit using videofluoroscopy and a bead expulsion test.. We then investigated intestinal and colonic smooth muscle isometric contraction in vitro using electric field stimulation (EFS), and acetylcholine (Ach) release was assessed by superfusion using [3H] choline. Histological changes were evaluated by H&E and immunohistochemistry. Results Mice with PA microbiota had faster whole GI transit (score 18.9 ± 0.9 (N=9) than mice with HC microbiota (15.4 ± 1.0, N=10, p=0.032), with markers located mainly in the distal small bowel and cecum. However, bead expulsion from the colon was significantly longer in PA mice (420.8 s ± 124.6 s, N=9) than in HC mice (82.6 s ± 20.0 s, N=10, p=0.026). This delayed colonic transit was likely due to colonic retroperistalsis visualized videofluoroscopically by retrograde flow of barium in the right colon of PA mice. There was no difference between the two groups in small intestinal or colonic tissues in Ach release or contractility induced by carbachol or KCl,. EFS caused transient biphasic relaxation and contraction in small intestine and colon, with the colonic contraction being stronger in the PA group. Microscopic tissue analysis showed disruption of the interstitial cells of Cajal (ICC) network and increased lymphocyte infiltration in colonic mucosa and submucosa in PA mice. Conclusions These results indicate that the microbiota is a driver of delayed colonic transit in a patient whose constipation started following extensive antibiotic exposure for C. difficile infection. The observed dysmotility pattern was not due to lower muscle contractility but likely caused by immune mediated changes in the ICC network. Funding Agencies CIHR

Striatal Acetylcholine Helps to Preserve Functional Outcomes in a Mouse Model of Stroke

Acetylcholine (ACh) has been suggested to facilitate plasticity and improve functional recovery after different types of brain lesions. Interestingly, numerous studies have shown that striatal cholinergic interneurons are relatively resistant to acute ischemic insults, but whether ACh released by these neurons enhances functional recovery after stroke is unknown. We investigated the role of endogenous striatal ACh in stroke lesion volume and functional outcomes following middle cerebral artery occlusion to induce focal ischemia in striatum-selective vesicular acetylcholine transporter-deficient mice (stVAChT-KO). As transporter expression is almost completely eliminated in the striatum of stVAChT-KO mice, ACh release is nearly abolished in this area. Conversely, in other brain areas, VAChT expression and ACh release are preserved. Our results demonstrate a larger infarct size after ischemic insult in stVAChT-KO mice, with more pronounced functional impairments and increased mortality than in littermate controls. These changes are associated with increased activation of GSK-3, decreased levels of β-catenin, and a higher permeability of the blood–brain barrier in mice with loss of VAChT in striatum neurons. These results support a framework in which endogenous ACh secretion originating from cholinergic interneurons in the striatum helps to protect brain tissue against ischemia-induced damage and facilitates brain recovery by supporting blood–brain barrier function.

An optimized acetylcholine sensor for monitoring in vivo cholinergic activity

The ability to directly measure acetylcholine (ACh) release is an essential first step towards understanding its physiological function. Here we optimized the GRABACh (GPCR-Activation–Based-ACh) sensor with significantly improved sensitivity and minimal downstream coupling. Using this sensor, we measured in-vivo cholinergic activity in both Drosophila and mice, revealing compartmental ACh signals in fly olfactory center and single-trial ACh dynamics in multiple regions of the mice brain under a variety of different behaviors