Brainstem mechanisms controlling cardiovascular reflexes in channel catfish.

Microinjections of kynurenic acid and kainic acid into the general visceral nucleus (nGV), homologous to the mammalian nucleus tractus solitarius of the medulla, in anesthestized, spontaneously breathing catfish were used to identify central areas and mechanisms controlling resting normoxic heart rate and blood pressure and the cardiovascular responses to hypoxia. Kynurenic acid, an antagonist of ionotropic glutamate receptors, significantly reduced resting normoxic heart rate but did not block the bradycardia associated with aquatic hypoxia. Kainic acid (an excitotoxic glutamatergic receptor agonist) also significantly reduced normoxic heart rate, but blocked the hypoxia-induced bradycardia. Neither kynurenic acid nor kainic acid microinjections affected blood pressure in normoxia or hypoxia. The results of this study indicate that glutamatergic receptors in the nGV are involved in the maintenance of resting heart rate and the destruction of these neurons with kainic acid abolishes the bradycardia associated with aquatic hypoxia.

Involvement of non-NMDA receptors in central mediation of chemoreflexes in the shorthorn sculpin, Myoxocephalus scorpius.

NMDA receptors mediate hypoxia-induced ventilatory frequency and blood pressure increases in fish. Here we continue to resolve whether non-NMDA receptors participate in chemoreflexes. Shorthorn sculpins, instrumented for cardiorespiratory measurements, were kept unrestrained or positioned in a stereotaxic frame. Chemoreflexes were elicited (hypoxia/NaCN-induced) before/after administration of either the specific AMPA receptor antagonist, GYKI52466 (systemically), or the specific kainate receptor antagonist, UBP293 (microinjections into fourth ventricle). Immunohistochemistry was performed on medullary cross-sections to identify non-NMDA receptor subunits in the chemoreflex-pathway. Kainate receptors mediate the chemoreflex-mediated increase in ventilation amplitude, since the response was abolished by UBP293. GYKI52466 attenuated the ventilatory frequency increase, and induced more regular breathing patterns and higher heart rate in both normoxic and hypoxic conditions, suggesting that AMPA receptors also partake in cardiorespiratory control. This together with immunohistochemical findings of both AMPA and kainate receptor subunits in the chemoreflex-pathway, show that non-NMDA receptors play a role in both chemoreflex-activation and normoxic cardiorespiratory regulation in fish.

Late onset of NMDA receptor-mediated ventilatory control during early development in zebrafish (Danio rerio).

Increased ventilation frequency (fV) in response to hypoxia in adult fish depends on ionotropic N-methyl-D-aspartate (NMDA) receptors. Nonetheless, the ontogeny of central control mechanisms mediating hypoxic ventilatory chemoreflexes in lower vertebrates has not been studied. Therefore, the aim of this study was to determine when the hypoxic ventilatory response during zebrafish (Danio rerio) development is mediated via NMDA receptors, by performing physiological experiments and western blot analysis of NMDA receptor subunits. Zebrafish larvae at stages 4-16 days post-fertilisation (dpf) were exposed to an hypoxic pulse in control groups and in groups treated with MK801 (NMDA receptor antagonist). The hypoxic increase in fV was present at all larval stages, and it matured during development. The reflex became MK801 sensitive at 8 dpf, but did not completely rely on a glutamatergic transmission until 13 dpf. This, together with changing subunit composition during the different stages (increasing amounts of NMDAR1 subunits and appearance of NMDAR2A subunits in adults), suggests that the amount of functional NMDA receptors needed to achieve a fully developed reflex is not attained until later stages. Furthermore, our results suggest that other non-NMDA receptor mechanisms are responsible for the hypoxia-induced increase in fV during the earlier developmental stages.

Identification of central mechanisms vital for breathing in the channel catfish, Ictalurus punctatus.

To investigate central respiratory control mechanisms in channel catfish, microinjections of kainic acid (causing chemical lesion of neurons) or kynurenic acid (an antagonist of N-methyl-D-aspartate (NMDA), kainate and alpha-amino-3-OH-5-methyl-4-isooxazole-propionic-acid (AMPA) receptors) were made into the general visceral nucleus (nGV) of the medulla in anaesthetised spontaneously breathing animals. Kainic acid abolished the ventilatory movements, indicating that neurons in the nGV are crucial for maintaining normal breathing. Kynurenic acid did not affect normal breathing, but abolished the ventilatory responses to hypoxia, showing that ionotropic glutamate receptors in the nGV are vital for the production of oxygen chemoreceptor activated respiratory reflexes. In addition, immunohistochemistry of brain slices showed that interneurons and nerve fibres in the nGV display NMDA-immunoreactivity, which corroborates the physiological experiments. The results of this study suggest that neurons and glutamatergic pathways in the nGV are essential for ventilatory functions and hypoxic reflexes in channel catfish.

N-methyl-D-aspartate receptors mediate chemoreflexes in the shorthorn sculpin Myoxocephalus scorpius.

Glutamate microinjected into the vagal sensory area in the medulla produces cardiorespiratory responses mimicking oxygen chemoreflexes in fish. Here we directly investigate whether these reflexes are dependent on the ionotropic N-methyl-D-aspartate (NMDA) glutamate receptor. Fish were equipped with opercular, branchial and snout cannulae for measurements of cardiorespiratory parameters and drug injections. Oxygen chemoreceptor reflexes were evoked by rapid hypoxia, NaCN added into the blood (internal, 0.3 ml, 50 microg ml(-1)) and the mouth (external, 0.5 ml, 1 mg ml(-1)), before and after systemic administration of the NMDA receptor antagonist MK801 (3 mg kg(-1)). Hypoxia produced an MK801-sensitive increase in blood pressure and ventilation frequency, whereas the marked bradycardia and the increased ventilation amplitude were NMDA receptor-independent. The fish appeared more responsive to externally applied cyanide, but the injections and MK801 treatment did not distinguish whether external or internal oxygen receptors were differently involved in the hypoxic responses. In addition, using single-labelling immunohistochemistry on sections from the medulla and ganglion nodosum, the presence of glutamate and NMDA receptors in the vagal oxygen chemoreceptor pathway was established. In conclusion, these results suggest that NMDA receptors are putative central control mechanisms that process oxygen chemoreceptor information in fish.

Evidence for glutamatergic mechanisms in the vagal sensory pathway initiating cardiorespiratory reflexes in the shorthorn sculpin Myoxocephalus scorpius.

Glutamate is a major neurotransmitter of chemoreceptor and baroreceptor afferent pathways in mammals and therefore plays a central role in the development of cardiorespiratory reflexes. In fish, the gills are the major sites of these receptors, and, consequently, the terminal field (sensory area) of their afferents (glossopharyngus and vagus) in the medulla must be an important site for the integration of chemoreceptor and baroreceptor signals. This investigation explored whether fish have glutamatergic mechanisms in the vagal sensory area (Xs) that could be involved in the generation of cardiorespiratory reflexes. The locations of the vagal sensory and motor (Xm) areas in the medulla were established by the orthograde and retrograde axonal transport of the neural tract tracer Fast Blue following its injection into the ganglion nodosum. Glutamate was then microinjected into identified sites within the Xs in an attempt to mimic chemoreceptor- and baroreceptor-induced reflexes commonly observed in fish. By necessity, the brain injections were performed on anaesthetised animals that were fixed by 'eye bars' in a recirculating water system. Blood pressure and heart rate were measured using an arterial cannula positioned in the afferent branchial artery of the 3rd gill arch, and ventilation was measured by impedance probes sutured onto the operculum. Unilateral injection of glutamate (40-100 nl, 10 mmol l(-1)) into the Xs caused marked cardiorespiratory changes. Injection (0.1-0.3 mm deep) in different rostrocaudal, medial-lateral positions induced a bradycardia, either increased or decreased blood pressure, ventilation frequency and amplitude and, sometimes, an initial apnea. Often these responses occurred simultaneously in various different combinations but, occasionally, they appeared singly, suggesting specific projections into the Xs for each cardiorespiratory variable and local determination of the modality of the response. Response patterns related to chemoreceptor reflex activation were predominantly located rostral of obex, whereas patterns related to baroreceptor reflex activation were more caudal, around obex. The glutamate-induced bradycardia was N-methyl-D-aspartate (NMDA) receptor dependent and atropine sensitive. Taken together, our data provide evidence that glutamate is a putative player in the central integration of chemoreceptor and baroreceptor information in fish.