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Ventilatory responses to acute and long-term hypoxia are classically triggered by carotid chemoreceptors. The chemosensory inputs are carried within the carotid sinus nerve to the nucleus tractus solitarius and the brainstem respiratory centres. To investigate whether hypoxia acts directly on brainstem neurons or secondarily via carotid body inputs, we tested the ventilatory responses to acute and long-term hypoxia in rats with bilaterally transected carotid sinus nerves and in sham-operated rats. Because brainstem catecholaminergic neurons are part of the chemoreflex pathway, the ventilatory response to hypoxia was studied in association with the expression of tyrosine hydroxylase (TH). TH mRNA levels were assessed in the brainstem by in situ hybridization and hypoxic ventilatory responses were measured in vivo by plethysmography. After long-term hypoxia, TH mRNA levels in the nucleus tractus solitarius and ventrolateral medulla increased similarly in chemodenervated and sham-operated rats. Ventilatory acclimatization to hypoxia developed in chemodenervated rats, but to a lesser extent than in sham-operated rats. Ventilatory response to acute hypoxia, which was initially low in chemodenervated rats, was fully restored within 21 days in long-term hypoxic rats, as well as in normoxic animals which do not overexpress TH. Therefore, activation of brainstem catecholaminergic neurons and ventilatory adjustments to hypoxia occurred independently of carotid chemosensory inputs. O2-sensing mechanisms unmasked by carotid chemodenervation triggered two ventilatory adjustments: (i) a partial acclimatization to long-term hypoxia associated with TH upregulation; (ii) a complete restoration of acute hypoxic responsivity independent of TH upregulation.

Our aim was to determine whether the cardiovascular neurones in the rostro-ventrolateral medulla (CV-RVLM neurones) were involved in the sympathoexcitation induced by stimulation of 5-HT3 receptors in the region of the nucleus tractus solitarii (NTS). Experiments were performed in pentobarbitone-anaesthetized rats, artificially ventilated and paralysed with pancuronium bromide. Using extracellular recordings, different types of RVLM neurones were characterized: cardiovascular (CV), ventilation-related and baroreflex-insensitive (unidentified) neurones. The CV-RVLM cells were further subdivided into three populations according to their axonal conduction velocities: A (1.2 ± 0.1 m s−1), B (2.5 ± 0.2 m s−1) and C (6.8 ± 1.1 m s−1). Only the CV-RVLM neurones of the A and B categories were partially inhibited (−30 %) by a hypotensive dose (2.5 μg kg−1 i.v.) of clonidine. Microinjections into the region of the commissural NTS of 1-(m-chlorophenyl)-biguanide (CPBG, 2 nmol), a selective 5-HT3 receptor agonist, elicited an increase in both lumbar sympathetic nerve discharge (SND) and arterial pressure. In addition, this treatment produced a marked excitation of CV-RVLM neurones of the A and B categories, without affecting those of the C type, as well as ventilation-related and unidentified RVLM cells. The activity of the CV neurones in the caudo-ventrolateral part of the medulla oblongata (CV-CVLM) was not modified by 5-HT3 receptor stimulation in the NTS. Prior intra-NTS microinjections of ondansetron (300 pmol, a selective 5-HT3 receptor antagonist) into the region of the commissural NTS prevented the excitation of A and B CV-RVLM neurones induced by CPBG. Intracarotid administration of saline saturated with CO2 (chemoreceptor activation) elicited both an increase in the SND and an excitation of the clonidine-insensitive CV-RVLM neurones of the C type, without affecting A and B neurones. In conclusion, the sympathoexcitation elicited following 5-HT3 receptor stimulation in the region of the commissural NTS of pentobarbitone-anaesthetized rats seems to result from the excitation of two different pools of clonidine-sensitive CV-RVLM neurones. These neurones are apparently not involved in the sympathetic component of the chemoreceptor reflex. The rostro-ventrolateral medulla (RVLM) and nucleus tractus solitarii (NTS) are critically involved in the reflex control of sympathetic activity (Guyenet, Filtz & Donaldson, 1987; Sun & Guyenet, 1987; Spyer, 1994). The RVLM contains neurones that receive a number of inputs both peripheral and central in origin that influence sympathetic nerve activity (Sun & Guyenet, 1987; Spyer, 1994). Previous reports have described two populations of cardiovascular (CV)-RVLM neurones that project to the thoracic spinal cord (Brown & Guyenet, 1985; Sun & Guyenet, 1985). The first population consists of clonidine-sensitive cells with slow-conducting axons. The second population corresponds to cells that do not respond to hypotensive doses of clonidine, and are characterized by a much higher conduction velocity (Sun & Guyenet, 1986). The NTS is the site of termination of afferent fibres arising from arterial baroreceptors (baroreflex), cardiopulmonary chemoreceptors (Bezold-Jarisch reflex) and carotid chemoreceptors (chemoreflex) (Palkovits & Zaborsky, 1977; Kalia & Mesulam, 1980; Jordan & Spyer, 1986). For baro- and Bezold-Jarisch reflexes, which seem to share the same integrating mechanisms and pathways in the brain (Verberne & Guyenet, 1992), second-order neurones project from the NTS to the caudal ventrolateral part of the medulla (CVLM) (Gordon 1987; Guyenet et al. 1987; Verberne & Guyenet, 1992). These neurones exert an excitatory action on the GABAergic CVLM neurones that project to the RVLM where they inhibit the CV-RVLM neurones of this pressor area (Brown & Guyenet, 1985; Sun & Guyenet, 1985; Jeske, Reis & Milner, 1995). Some of the CV-RVLM neurones additionally constitute an efferent link in the sympathetic component of the chemoreflex (Guyenet & Brown, 1986; Sun & Reis, 1995). Indeed, it has been demonstrated that some NTS chemosensitive neurones have axonal projections to the RVLM (Koshiya & Guyenet, 1996). Within the NTS, serotonin (5-hydroxytryptamine, 5-HT) seems to be involved in the reflex control of blood pressure. Studies in both anaesthetized and conscious rats have shown that 5-HT2 receptor stimulation in the NTS elicits the typical CV responses of baroreceptor activation (Merahi, Orer & Laguzzi, 1992a; Callera, Bonagamba, Sevoz, Laguzzi & Machado, 1997a). On the other hand, stimulation of NTS 5-HT3 receptors elicits a chemoreceptor-like increase in arterial pressure and lumbar sympathetic activity (Merahi, Orer, Laporte, Gozlan, Hamon & Laguzzi, 1992b; Callera, Sevoz, Laguzzi & Machado, 1997b). In experiments aimed at analysing the mechanism (s) responsible for this sympatho-excitatory effect, we observed that stimulation of 5-HT3 receptors in the NTS did not inhibit the sympathetic component of the baroreflex (Nosjean, Franc & Laguzzi, 1995). Accordingly, it can be inferred that 5-HT3 receptor-mediated sympathoexcitation is not the consequence of the disruption of tonic baroreceptor sympathoinhibitory messages. However, our finding is compatible with the idea that CV-RVLM neurones may be involved in the sympathoexcitatory effect of 5-HT3 receptor stimulation in the NTS. In other experiments, we also observed that prior microinjections of pressor (nanomolar) doses of 5-HT3 receptor agonists into the NTS did not increase the sympathetic chemoreflex response (Sevoz, Callera, Machado, Hamon & Laguzzi, 1997). However, this observation does not rule out the possibility that under some physiological conditions, 5-HT released in the NTS may excite the NTS-RVLM sympathetic chemoreflex pathway (Koshiya & Guyenet, 1996), and the CV-RVLM neurones involved in this reflex. Indeed, as previously observed with the pressor chemoreflex response (Sun & Reis, 1995), we recently found that the microinjection of kynurenic acid, a glutamate receptor antagonist, into the RVLM blocked the pressor effects elicited by 5-HT3 receptor stimulation in this area (Sevoz, Hamon & Laguzzi, 1996b). In order to elucidate the possible role of CV-RVLM neurones in the sympathetic response to 5-HT3 receptor stimulation in the NTS, we have analysed the effects of intra-NTS microinjections of a potent and selective 5-HT3 receptor agonist, 1-(m-chlorophenyl)-biguanide (CPBG), as well as the effects of chemoreflex activation, on the activity of the different categories of RVLM neurones. In addition, the possible effects of intra-NTS administration of CPBG on the CV-CVLM neurones were also investigated.