br Acknowledgements This work was supported

Acknowledgements This work was supported by the National Key Technology Research and Development Program (2012BAD19B01) and General Financial Grant from the China Postdoctoral Science Foundation (2015M581754).
Introduction Cough is one of the most important defensive reflex brought into action by actually or potentially damaging events applied to the airways (Korpáš and Tomori, 1979). Peripheral and central mechanisms underlying nociception and cough share similar features; neuroactive agents involved in the central control of pain sensation (Millan, 2002; Yan et al., 2017) and concomitant reflex responses play a role also in the downregulation of the cough reflex (see Mutolo, 2017 also for further Refs. Mutolo et al., 2008, 2012, 2014; Cinelli et al., 2013, 2016). Several brainstem areas appear to contribute to the generation and regulation of cough responses in mammals (e.g. Gestreau et al., 1997; Bongianni et al., 1998; Jakus et al., 2008; Mutolo et al., 2002b; Poliacek et al., 2004, 2005, 2014; Shannon et al., 2004; Simera et al., 2013; see also Mutolo, 2017). Recent findings obtained mainly in the rabbit (for review see Mutolo, 2017) have led to the proposal that two medullary structures play a prominent role in the control of cough reflex responses and are sites of action of antitussive or protussive drugs, i.e. the caudal nucleus tractus solitarii (NTS), the first relay medullary station of the cough reflex pathway, and the caudal ventral respiratory group (cVRG), where hydroxychloroquine sulfate responsible for the expiratory component of the reflex are located (for the role of the cVRG see also Poliacek et al., 2007, 2010, 2015). Of note, afferent inputs from peripheral chemoreceptors and pulmonary rapidly adapting receptors converge on neurons of the caudal NTS (Mifflin et al., 1988; Mifflin, 1992; Machado, 2001; Kubin et al., 2006). However, recently evidence has been provided of the existence in the cat of important control mechanisms within the rostral NTS (Poliacek et al., 2017a,b; for review see Mutolo, 2017) very similar to those described for the caudal NTS in the rabbit. This could be possibly related to marked differences in the animal species. Besides its modulatory quality, acetylcholine (ACh) also acts as one of the most prominent neurotransmitters in the peripheral and central nervous system. Interestingly, cholinergic transmission profoundly affects the perception of pain via both nicotinic (nAChRs) and muscarinic (mAChRs) receptors (for review see Naser and Kuner, 2017). There is considerable direct and indirect evidence that ACh is widely distributed in the region of NTS (Kobayashi et al., 1978; Criscione et al., 1983; Ernsberger et al., 1988; Ruggiero et al., 1990; Zoccal et al., 2014) where both mAChRs and nAChRs are present. Muscarinic receptors are located in more caudal regions, including the commissural subnucleus, while nicotinic receptors are predominantly located at more rostral levels, i.e. in the medial, ventrolateral and ventral subnuclei (Maley, 1996; see also Furuya et al., 2014). ACh contributes to autonomic regulation. In particular, it is involved in the regulation of both cardiovascular (e.g. Shihara et al., 1999; Furuya et al., 2014, 2017; Zoccal et al., 2014) and respiratory activity (e.g. Haxhiu et al., 1984; Bianchi et al., 1995; Shao and Feldman, 2000, 2001, 2002, 2009; Shao et al., 2008; Boutin et al., 2017) through both nAChRs and mAChRs. However, only scanty knowledge is available on its contribution to the modulation of the cough motor pattern. Recently, it has been shown that (−)-nicotine administered via brainstem circulation or directly applied to the cVRG causes mecamylamine-insensitive inhibitory effects on mechanically-induced cough (Poliacek et al., 2015). In the present research, the possible role of ACh in the modulation of the cough reflex at the level of the caudal NTS of pentobarbital sodium-anesthetized, spontaneously breathing rabbits was investigated making use of microinjection techniques.