br Materials and methods br Results Bilateral microinjection

Materials and methods
Results Bilateral microinjections (n = 6) of 10 mM ACh (300–500 pmol) and 5 mM physostigmine (150–250 pmol) at the two selected caudal NTS sites caused within 1 min significant increases in respiratory frequency (from 54.5 ± 1.7 to 70.9 ± 4.1 breaths/min; +30.8 ± 7.0%; P < 0.001) that faded out within 5 min. No significant changes in mean arterial pressure occurred (Table 1). These effects were accompanied by decreases or even complete suppression of expiratory activity that persisted for longer time period (10–15 min) and recovered within 30 min. Progressive depressant effects on the cough reflex reached a maximum within 10 min. At that time cough responses induced by mechanical stimulation of the tracheobronchial tree were completely abolished, while citric Peramivir synthesis inhalation-induced cough responses were only strongly reduced both in the cough number and in the peak abdominal amplitude (see Fig. 2 and Table 2). Cough-related variables resumed control values within 60 min. To disclose whether ACh exerts its effects through nAChRs or mAChRs, the corresponding specific antagonists were employed. We injected 10 mM mecamylamine (n = 3; 300–500 pmol) or 10 mM scopolamine (n = 4; 300–500 pmol) and after an interval of ∼5 min 10 mM ACh/5 mM physostigmine into the same sites. Neither mecamylamine nor scopolamine caused obvious and significant effects on baseline respiratory activity (paired t-tests). Indeed, in both cases respiratory frequency remained fairly constant with variations lower than ±1% in each preparation. ACh-induced changes in baseline respiratory activity as well as in the cough reflex were not counteracted by mecamylamine, but were prevented by scopolamine (not shown). In two additional preparations, bilateral control microinjections of 10 mM ACh/5 mM physostigmine were performed at different medullary locations (4 trials) sufficiently far (>0.8 mm) from the responsive sites (see e.g. Nicholson, 1985; Lipski et al., 1988; Mutolo et al., 2007, 2012, 2014; Sykova and Nicholson, 2008; Cinelli et al., 2013, 2016). With respect to the responsive sites, they were performed into the NTS region 1 mm rostral and 1 mm lateral to the midline (1 trial), into the adjacent reticular formation (2 trials) and into the reticular formation located 1 mm more caudal at the same depth (1 trial). Control microinjections (2 trials for each location) were also performed into the nucleus cuneatus medialis and the nucleus tractus spinalis nervi trigemini. All these microinjections failed to induce changes in the breathing pattern as well as suppressant effects on the cough reflex. Control injections of equal volumes of the vehicle solution at the responsive sites performed in 3 preparations before drug administration were ineffective. The localization of injection sites was confirmed by the histological control of pipette tracks and the location of fluorescent beads. The localization of injection sites on a dorsal view of the medulla oblongata along with the distribution of injection sites within the caudal NTS and some control regions is reported in Fig. 1. Only the distribution of sites where 10 mM ACh/5 mM physostigmine were injected has been reported. The same figure also illustrates an example of the location of fluorescent beads microinjected into the caudal NTS.
Discussion This study shows for the first time that ACh microinjected into the caudal NTS causes strong depressant effects on the cough reflex through the activation of mAChRs. Present findings not only confirm that the caudal NTS is one of the most important sites involved in the modulation of the cough reflex in the rabbit, but also extend our previous results showing that the central mechanisms subserving nociception and cough share similar features (for review see Mutolo, 2017). We have fully discussed in our previous reports the reliability of microinjection procedures, the spread of the injectate and the localization of injection sites that were selected by using stereotaxic coordinates according to the atlas of Meessen and Olszewski (1949) and confirmed by the histological control (Mutolo et al., 2007, 2008, 2009, 2012, 2013, 2014; Cinelli et al., 2013, 2016). The absence of changes in the ongoing respiratory activity and especially in cough reflex responses following drug microinjections at sites sufficiently far from the responsive sites (>0.8 mm) as well as following vehicle microinjections into the responsive sites of the caudal NTS supports the specificity of drug-induced effects. The distance of control microinjections from the responsive sites derives from previous observations on the spread of the injectate (see e.g. Nicholson, 1985; Lipski et al., 1988; Mutolo et al., 2002a, 2005; Bongianni et al., 2008, 2010; Sykova and Nicholson, 2008). Given the very short lasting effects of ACh due not only to diffusion, but especially to acetylcholinesterase-induced degradation, we injected in combination 10 mM ACh and 5 mM physostigmine.