Effects of Acetylcholine and Cholinergic Antagonists on the Activity of Nucleus of the Solitary Tract Neurons ⇑ Werner I

Brain Research 1659 (2017) 136–141 Contents lists available at ScienceDirect Brain Research journal homepage: www.elsevier.com/locate/bres Research report Effects of acetylcholine and cholinergic antagonists on the activity of nucleus of the solitary tract neurons ⇑ Werner I. Furuya a, Eduardo Colombari a, Alastair V. Ferguson b,1, Débora S.A. Colombari a,1, a Department of Physiology and Pathology, School of Dentistry, São Paulo State University, Araraquara, SP, Brazil b Department of Biomedical and Molecular Sciences, School of Medicine, Queen’s University, Kingston, ON, Canada article info abstract Article history: Previously we have demonstrated that microinjection of acetylcholine (ACh) into the intermediate Received 3 August 2016 nucleus of the solitary tract (iNTS) induced sympatho-inhibition combined with a decrease in the phrenic Received in revised form 11 January 2017 nerve activity (PNA), whereas in the commissural NTS (cNTS), ACh did not change sympathetic nerve Accepted 21 January 2017 activity (SNA), but increased the PNA. In view of these demonstrated distinctive effects of ACh in different Available online 25 January 2017 subnuclei of the NTS the current studies were undertaken to examine, using patch clamp techniques, the specific effects of ACh on the excitability of individual neurons in the NTS, as well as the neuropharma- Keywords: cology of these actions. Coronal slices of the brainstem containing either cNTS or iNTS subnuclei were Nicotinic receptors used, and whole cell patch clamp recordings obtained from individual neurons in these two subnuclei. Muscarinic receptors Acetylcholine In cNTS, 58% of recorded neurons (n = 12) demonstrated rapid reversible depolarizations in response to Patch clamp ACh (10 mM), effects which were inhibited by the nicotinic antagonist mecamylamine (10 lM), but unaffected by the muscarinic antagonist atropine (10 lM). Similarly, bath application of ACh depolarized 76% of iNTS neurons (n = 17), although in this case both atropine and mecamylamine reduced the ACh- induced depolarization. These data demonstrate that ACh depolarizes cNTS neurons through actions on nicotinic receptors, while depolarizing effects in iNTS are apparently mediated by both receptors. Ó 2017 Elsevier B.V. All rights reserved. 1. Introduction a number of other neurotransmitters, such as dopamine, GABA, substance P, angiotensin II and acetylcholine (ACh), have been sug- The nucleus of the solitary tract (NTS) is an important medullary gested to contribute to the neurotransmission of cardiorespiratory area that receives peripheral afferent information from cardiovas- signals in the NTS (Abdala et al., 2006; Andresen and Kunze, 1994; cular and respiratory systems (Ciriello et al., 1994; Cottle, 1964; Criscione et al., 1983; da Silva et al., 2008; Furuya et al., 2014; Palkovits and Zaborsky, 1977). Baroreceptors make their first Sapru, 1996; Tsukamoto et al., 1994). Choline acetyltransferase synapse mainly at the level of the intermediate subnucleus of the positive neurons, high choline acetyltransferase and acetyl- NTS (iNTS; Ciriello et al., 1994), while peripheral chemoreceptors cholinesterase activity, muscarinic and nicotinic receptors are project primarily to the commissural subnucleus of the NTS (cNTS; found in the NTS (Cheng et al., 2011; Ernsberger et al., 1988; Ciriello et al., 1994). Functional data have demonstrated that lesions Gotts et al., 2015; Helke et al., 1983; Simon et al., 1981; Wada of the iNTS abolish the baroreflex, whereas cNTS lesions impair the et al., 1989). In fact, microinjection of ACh or the muscarinic ago- pressor and bradycardia induced by chemoreceptor activation, nist carbachol into the iNTS of anaesthetized or awake rats induces leaving the baroreflex unchanged (Blanch et al., 2013; Colombari dose-dependent baroreflex-like hypotensive and bradycardic et al., 1996; Miura and Reis, 1972; Schreihofer et al., 1999). responses (Criscione et al., 1983; da Silva et al., 2008; Tsukamoto Although L-glutamate has been proposed as the major excita- et al., 1994). Similar responses are also produced by nicotinic tory neurotransmitter released by the baro- and chemoreceptors receptor activation in the iNTS (Dhar et al., 2000). More recently, afferent inputs in the NTS (Machado, 2001; Talman et al., 1980), we have shown that the microinjection of ACh in the iNTS produced sympatho-inhibitory responses and reduced phrenic frequency, whereas in cNTS microinjections of ACh did not change ⇑ Corresponding author at: Department of Physiology & Pathology, School of the sympathetic nerve activity (SNA), but increased the PNA Dentistry of Araraquara, UNESP Rua Humaitá, 1680, Araraquara 14801-903, SP, (Furuya et al., 2014). In this study, we also demonstrated that pre- Brazil. E-mail address: [email protected] (D.S.A. Colombari). treatment with mecamylamine (nicotinic antagonist) abolished all 1 DSAC and AVF are co-seniors. the effects of ACh injected into the iNTS or the cNTS, whereas http://dx.doi.org/10.1016/j.brainres.2017.01.027 0006-8993/Ó 2017 Elsevier B.V. All rights reserved. W.I. Furuya et al. / Brain Research 1659 (2017) 136–141 137 atropine (muscarinic antagonist) reduced only the effects of ACh ATR vs. 7.8 ± 2.4 mV after ATR, p < 0.05, n = 8) as illustrated in injected into the cNTS (Furuya et al., 2014). Fig. 4A and C). Similar recordings from ACh responsive iNTS neu- Cell recording studies have shown that ACh changes the current rons showed that mecamylamine (10 lM, 5 min), administration of NTS neurons. For instance, in medullary horizontal slices, ACh almost completely abolished the effects of a second application increases the firing frequency of iNTS neurons, and such effects of ACh on these neurons (9.5 ± 2.0 mV before MEC vs. are reduced by both muscarinic and nicotinic receptors blockade 2.9 ± 1.0 mV after MEC, p < 0.05, n = 8), as illustrated in (Shihara et al., 1999). In the same study, it was demonstrated that Fig. 4B and C. The cholinergic antagonists alone did not change muscarinic receptor activation inhibits K+ channels, while nicotinic the resting membrane potential of iNTS neurons. activation promotes Na+/K+ channels opening (Shihara et al., 1999; Shiraki et al., 1997). In another study, using NTS cell culture from 3. Discussion neonatal rats, it was found that both ACh and nicotine induced depolarizing current (Ueno et al., 1993). While these data demon- In the present study, we demonstrated, using whole cell patch strate that ACh depolarizes iNTS cells, resulting in increased firing clamp techniques, depolarizing effects of ACh on the majority of frequency, to date studies have not compared the effects of ACh on neurons recorded from NTS slices. These effects were observed in distinct NTS subnuclei. Given the different physiological responses both iNTS and cNTS and were found to be rapid, reversible and con- elicited by ACh in iNTS and cNTS, the present study was under- centration dependent. A significant proportion of NTS neurons taken to examine both the cellular effects and neuropharmacology were also found to be non-responsive, and it should be noted that of ACh in these NTS subnuclei. no hyperpolarizations were seen. The nicotinic receptor antagonist mecamylamine, inhibited the responses to ACh in both iNTS and 2. Results cNTS, suggesting roles for these receptors in both regions. In con- trast, the muscarinic antagonist atropine inhibits the ACh actions 2.1. Acetylcholine depolarizes cNTS neurons in the iNTS, but was without effect in the cNTS, suggesting a region-specific functional role for the muscarinic receptor in iNTS. We first examined the effects of 10 mM ACh (1 min bath perfu- Nicotinic and muscarinic receptors have been found in the NTS sion) on the membrane potential of 12 cNTS neurons. The majority (Cheng et al., 2011; Ernsberger et al., 1988; Gotts et al., 2015; Wada of these cells (7/12–58%) showed rapid, reversible depolarizations et al., 1989), although the differential localization within the cNTS (mean 6.9 ± 1.5 mV) as illustrated in Fig. 1C–E, while the remaining and iNTS of both receptors were not systematically studied using 5 neurons tested were unaffected. (-0.0 ± 0.7 mV). The 1.0 mM con- either immunohistochemical or receptor autoradiography proto- centration of ACh depolarized 2 out of 4 cNTS neurons, and the cols. In the present study, it was observed that in the cNTS, the magnitude of the depolarizations observed was smaller ACh evoked response was blocked by nicotinic receptors, while (4.5 ± 1.2 mV), (Fig. 1B, D-E). Finally, at a concentration of atropine did not change the depolarizing effect of ACh. In a previ- 0.1 mM ACh, no significant effects were observed on the mem- ous study using the in situ preparation, we have observed that ACh brane potential of 4 neurons tested (Fig. 1A, D-E). in the cNTS does not alter SNA, but increase the PNA and changed Next, we examined the ability of cholinergic antagonists to the sympatho-respiratory coupling (Furuya et al., 2014). In that modulate these depolarizing effects of ACh on cNTS neurons. In a study, at equimolar doses, atropine reduced and mecamylamine group of 7 neurons which showed clear depolarizing effects of blocked the tachypnea induced by ACh in the cNTS, whereas only ACh before bath administration of atropine (ATR; 10 lM, 5 min), the nicotinic receptor blockade was able to revert the change in responses to a subsequent similar administration of ACh were sympatho-respiratory coupling induced by ACh in the cNTS unaffected (7.6 ± 2.6 mV before ATR vs. 6.7 ± 1.9 mV after ATR, (Furuya et al., 2014) and to reduce the peripheral p > 0.05, n = 7) as illustrated in Fig. 2A and C. In 5 ACh responsive chemoreceptor-induced tachypnea (Furuya et al., 2014). Therefore, cNTS neurons mecamylamine (MEC; 10 lM, 5 min) administration even though in the in situ studies muscarinic receptors in the cNTS completely abolished the effects of a second application of ACh might have a role in mediating ACh-induced responses, it seems (10.4 ± 2.7 mV before MEC vs.

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