ACh induced changes in respiratory frequency could be of int
ACh-induced changes in respiratory frequency could be of interest. They may reveal an important mechanism of respiratory modulation at the caudal NTS level. ACh-induced increases in respiratory frequency within this region have been also reported by Furuya et al. (2014). Changes in respiratory timing are typically associated with the function of a rhythm-generating mechanism (Von Euler, 1986; Feldman and Del Negro, 2006). It seems plausible that neurons in the caudal NTS are embedded in a ponto-medullary circuit implicated in the control of the respiratory timing (Budzinska et al., 1985a; Von Euler, 1986; Bianchi et al., 1995). On the other hand, it has been recently proposed that the NTS region has an important role in the neural control of breathing (Bautista and Dutschmann, 2014; Jones et al., 2015). Accordingly, it has also been reported that respiratory neurons of the caudal NTS project to the inspiratory neurons located in the preBötzinger complex, the recognized central pattern generator of inspiratory activity, as well as to the rostral respiratory portion of the ventral respiratory group (Alheid et al., 2011) and to the pons (Takakura et al., 2006; Song et al., 2011). All these medullary regions are involved in the regulation of respiratory frequency and probably in the respiratory responses to the peripheral chemoreceptor stimulation (see also Zoccal et al., 2014). ACh microinjections into the caudal NTS could directly or indirectly activate this circuitry and promote tachypnea. In addition, increases in respiratory frequency were accompanied by strong reductions or even complete inhibition of abdominal muscle activity, in agreement with previous studies in the cat showing that the caudal NTS has a clear role in the modulation of the expiratory cfse synthesis (Budzinska et al., 1985b). In fact, functional ablation of this area by focal cooling causes obvious increases in rhythmic expiratory activity. In our study, changes in respiratory frequency were relatively transient and disappeared within 5 min and were absent at the time when cough-suppressant effects were observed, thus suggesting that these two types of ACh-induced responses were subserved by different neural mechanisms. However, the observed depression in expiratory activity usually persisted and could be related to cough-suppressant effects. It is worth mentioning that ACh microinjections into the caudal NTS were less effective in reducing citric acid-induced cough. This outcome could possibly be related to a higher number of recruited acid-sensing receptors and to their deeper localization in the airways, as well as to their well-known association with the generation of tachypneic responses following cough bouts (Sant'Ambrogio and Widdicombe, 2001; Mutolo et al., 2009). As already mentioned, present results are in keeping with the proposal of similarities between neural mechanisms underlying nociception and cough. For instance, there is a large body of evidence pointing to the importance of muscarinic signaling in pain control at the level of the spinal dorsal horns. In particular, ACh receptors profoundly regulate nociceptive transmission in the spinal cord via pre- and postsynaptic mechanisms and the direct activation of mAChRs reduces pain in rodents and humans, while their inhibition induces hypersensitivity (for review see Naser and Kuner, 2017). An important issue is the source of cholinergic inputs to the caudal NTS. A functional cholinergic system has been described in the NTS region as well as a possible role of local cholinergic interneurons modulated by vagal afferent signals. While primary afferent fibers of the solitary tract use glutamate as the primary neurotransmitter, they could activate cholinergic interneurons (Kobayashi et al., 1978; Helke et al., 1983; Ruggiero et al., 1990; Maley, 1996; Shihara et al., 1999; Furuya et al., 2014; Zoccal et al., 2014). Furthermore, it has also been suggested that in addition to glutamate other neurotransmitters, including ACh, may contribute to the neurotransmission of cardiorespiratory signals in the NTS (Criscione et al., 1983; Andresen and Kunze, 1994; Tsukamoto et al., 1994; Machado, 2001; Machado and Bonagamba, 2005; Braga and Machado, 2006; Abdala et al., 2006; da Silva et al., 2008; Furuya et al., 2014; Zoccal et al., 2014). Cholinergic axons may also derive from the dorsal motor vagal nucleus and the nucleus ambiguus (Farkas et al., 1997). In addition, there are cholinergic projections to the brainstem from the ponto-mesencephalic tegmental cholinergic complex. However, their role in the modulation of arterial blood pressure and respiration within the NTS is not clear (Woolf and Butcher, 1989; Woolf, 1991). Further studies are needed to assess the source of ACh for the NTS and to disclose details on synaptic cholinergic mechanisms. In this context, it can be recalled that ACh microinjected into the caudal NTS of the rat causes increases in respiratory frequency through both mAChRs and nAChRs (Furuya et al., 2014). These results are in partial agreement with present findings and the discrepancy may be related to differences in the animal species employed.