Jean-Pierre Kessler

Central serotonergic projections to the nucleus tractus solitarii: Evidence from a double labeling study in the rat

Projections from several brainstem serotonergic nuclei to the nucleus tractus solitarii were investigated in the rat. Experiments were performed using a double labeling method combining retrograde radioautographic tracing and serotonin immunohistochemistry. After injection of the radioactive tracer ([3H] wheat germ agglutinin) into the lateral nucleus tractus solitarii, nerve cell bodies exhibiting both radioautographic labeling and immunostaining were detected in all the serotonergic nuclei investigated, namely the nucleus raphe magnus, the ventromedial paragigantocellular nucleus, the nuclei raphe pontis, medianus and dorsalis, the medial lemniscus and the reticulotegmental nucleus of the pons. Most of the double labeled perikarya observed were in the nucleus raphe magnus, the adjacent part of the paragigantocellular nucleus and the nucleus raphe dorsalis. Nerve cell bodies retrogradely labeled but devoid of immunostaining were also observed, together with the double labeled perikarya, within serotonergic nuclei. These results provide direct evidence that brainstem serotonergic neurons contribute to the innervation of the nucleus tractus solitarii. They indicate that the nucleus raphe magnus and the nucleus raphe dorsalis constitute two major sources of central serotonergic projections to the nucleus tractus solitarii.

Evidence that activation of N-methyl-D-aspartate (NMDA) and non-NMDA receptors within the nucleus tractus solitarii triggers swallowing

Swallowing is a patterned motor activity generated by neurons located within the nucleus tractus solitarii (NTS). Previous experiments have shown that administration of excitatory amino acids within the NTS induces swallowing. The present study was undertaken to identify the receptor subtypes involved in this effect. Pressure microinjections of L-glutamate (10-100 pmol), quisqualate (0.1-10 pmol) and N-methyl-D-aspartate (NMDA, 0.1-10 pmol) were performed into the NTS of decerebrate rats. Glutamate and quisqualate microinjections elicited short series of swallows while NMDA microinjections induced long-lasting, rhythmic swallowing. Pretreatment with the selective NMDA antagonist, DL-2-amino-5-phosphonovalerate (50 pmol), almost completely suppressed the response elicited by NMDA (10 pmol) but did not induce a significant modification of swallowing triggered by either glutamate (25 pmol) or quisqualate (10 pmol). Pretreatment with 6-cyano-7-nitroquinoxaline-2,3-dione (50 pmol), a selective blocker of non-NMDA receptors, suppressed the swallows elicited by glutamate and strongly inhibited the response elicited by quisqualate microinjections. The same pretreatment induced only a slight modification of the swallowing elicited by NMDA. These data demonstrate that deglutition can be triggered by activating either NMDA or non-NMDA receptors localized within the NTS, and therefore suggest that both receptor subtypes may be involved in swallowing elicited under physiological conditions.

Immunohistochemical detection of glutamate in rat vagal sensory neurons

Vagal primary afferent neurons have their cell bodies located in the nodose (inferior) and jugular (superior) vagal ganglia and send terminals into the nucleus tractus solitarii (NTS) which lies in the dorsomedial medulla. The presence of glutamate (Glu)-containing neurons in the rat nodose ganglion was investigated using immunohistochemistry. Glu-immunoreactivity on nodose sections was found in neuronal perikarya and nerve fibers, but not in non-neuronal elements such as Schwann cells and satellite cells. Both immunoreactive and non-immunoreactive ganglion cells were observed. The immunoreactive ganglion cells amounted to about 60% of the nodose population. No specific intraganglionic localization was observed for the non-immunoreactive cells. Immunoreactive perikarya were slightly smaller than the non-immunoreactive ones, but no relationship was found between size and staining intensities of immunoreactive neurons. The present data indicate that immunodetectable Glu is present in a large population of vagal afferent neurons. They therefore add to a growing body of evidence suggesting that Glu may be the main neurotransmitter released by vagal afferent terminals within the nucleus tractus solitarii.

Vesicular glutamate transporters type 1 and 2 expression in axon terminals of the rat nucleus of the solitary tract

The nucleus of the solitary tract is the site of termination of primary afferent fibers running in the facial, glossopharyngeal and vagus nerves. The present study was performed to map the distribution of glutamatergic axons terminals in the rat nucleus of the solitary tract using immunodetection of vesicular glutamate transporter 1 and vesicular glutamate transporter 2. The two vesicular glutamate transporters were differentially distributed among nucleus of the solitary tract subdivisions. Vesicular glutamate transporter 1 immunoreactivity was mostly found in the lateral part of the nucleus (ventrolateral, interstitial and intermediate subdivisions) whereas vesicular glutamate transporter 2 labeling was distributed throughout the nucleus of the solitary tract. Electron microscope examination indicated that vesicular glutamate transporter immunoreactivity was localized in axon terminals filled with round synaptic vesicles. After injection of cholera toxin B subunit in sensory ganglia, anterograde labeling was found in vesicular glutamate transporter 1, as well as vesicular glutamate transporter 2-immunoreactive boutons. Double immunolabeling experiments allowed distinctions between terminals expressing either vesicular glutamate transporter 1 or vesicular glutamate transporter 2 or both vesicular glutamate transporter 1 and vesicular glutamate transporter 2 immunoreactivities. The latter population, expressing both transporters immunolabeling, completely disappeared after deafferentation induced by removal of sensory ganglia. This study indicates that vesicular glutamate transporter content identifies three different subpopulations of glutamatergic boutons in the nucleus of the solitary tract and provides definitive evidence that primary afferent neurons contribute glutamatergic terminals to the nucleus of the solitary tract.

Glutamatergic neurotransmission in the nucleus tractus solitarii: structural and functional characteristics.

Glutamate is the main excitatory transmitter in the central nervous system. As such, it plays a major role in transmitting and processing visceral sensory information within the nucleus tractus solitarii (NTS). Here, we review current knowledge on NTS glutamatergic transmission. We describe the main organizational features of NTS glutamatergic synapses as determined by work performed during the last decade using antibodies against glutamate receptors and transporters proteins. In light of these recent neuronatomical findings, we discuss some functional properties of developing and adult NTS glutamatergic synapses.

Inhibitory influence of monoamines and brainstem monoaminergic regions on the medullary swallowing reflex.

Swallowing is a polysynaptic reflex organized by a neuronal network localized mainly within the lateral region of the nucleus tractus solitarius (NTS). Monoamine microinjections (serotonin, noradrenaline; 1 nmol, 50 nl) within the swallowing region of the NTS produced a significant decrease in the number of swallows elicited by repetitive stimulation of the superior laryngeal nerve. Stimulation, with concentric bipolar electrodes, of several brainstem structures overlapping monoaminergic regions such as the nucleus raphe magnus, the nucleus raphe pallidus, the lemniscus medialis, the ventrolateral reticular formation, the locus coeruleus and the nucleus commissuralis, induced an inhibition of the laryngeal-initiated swallowing. These results indicate the existence, within the NTS, of a monoaminergic inhibition of the swallowing reflex which might originate from monoaminergic brainstem regions.

Effect of catecholamines on the swallowing reflex after pressure microinjections into the lateral solitary complex of the medulla oblongata.

The present study was carried out to elucidate the influence of catecholamines on swallowing, a polysynaptic reflex organized by an interneuronal network localized mainly within the lateral solitary complex (LSC) of the medulla oblongata. The effects of catecholaminergic agents were investigated in the rat, on rhythmic swallowing elicited by repetitive stimulation of the superior laryngeal nerve (SLN). Catecholaminergic agents were microinjected by pressure application, through multibarrelled glass micropipettes, into the LSC including the tractus solitarius, the swallowing region of the nucleus of the solitary tract and the adjacent reticular formation. Microinjections of noradrenaline (NA, 0.1-5 nmol, 50 nl) induced a significant decrease of the number and the amplitude of the rhythmic swallows elicited by stimulation of the ipsilateral SLN. This inhibitory effect was dose-related. Microinjections of clonidine (2.5 nmol, 50 nl), dopamine (0.25-2.5 nmol, 50 nl) and apomorphine (0.5 nmol, 50 nl), also inhibited swallowing. No significant modification of swallowing was induced by control injections of the vehicle (50 nl) within the active sites. Moreover the NA-induced inhibition of swallowing, was significantly antagonized by pretreatment with the alpha-adrenergic blocker phentolamine applied locally in the LSC. Furthermore neither blood pressure, nor respiratory rhythm were consistently modified by the catecholaminergic microinjections, indicating that the catecholamine-induced inhibition of swallowing was not a secondary side effect originating from alteration of these functions. It can therefore be concluded that the present results demonstrate the existence within the LSC of a catecholaminergic inhibition of the swallowing reflex. This inhibitory effect likely arises from activation of specific catecholaminergic receptors and affects the swallowing structures localized within the LSC, i.e., the laryngeal swallowing afferents running in the solitary tract and/or the swallowing interneurons within the nucleus of the solitary tract.

Silent synapses in developing rat nucleus tractus solitarii have AMPA receptors.

NMDA-only synapses, called silent synapses, are thought to be the initial step in synapse formation in several systems. However, the underlying mechanism and the role in circuit construction are still a matter of dispute. Using combined morphological and electrophysiological approaches, we searched for silent synapses at the level of the nucleus tractus solitarii (NTS), a brainstem structure that is a gateway for many visceral sensory afferent fibers. Silent synapses were detected at birth by using electrophysiological recordings and minimal stimulation protocols. However, anatomical experiments indicated that nearly all, if not all, NTS synapses had AMPA receptors. Based on EPSC fluctuation measurements and differential blockade by low-affinity competitive and noncompetitive glutamate antagonists, we then demonstrated that NTS silent synapses were better explained by glutamate spillover from neighboring fibers and/or slow dynamic of fusion pore opening. Glutamate spillover at immature NTS synapses may favor crosstalk between active synapses during development when glutamate transporters are weakly expressed and contribute to synaptic processing as well as autonomic circuit formation.

Mixed GABA―glycine synapses delineate a specific topography in the nucleus tractus solitarii of adult rat

Using combined morphological and electrophysiological approaches, we have determined the composition of inhibitory synapses of the nucleus tractus solitarii (NTS), a brainstem structure that is a gateway for many visceral sensory afferent fibres. Immunohistochemical experiments demonstrate that, in adult rat, GABA axon terminals are present throughout the NTS while mixed GABA–glycine axon terminals are strictly located to the lateral part of the NTS within subnuclei surrounding the tractus solitarius. Purely glycine axon terminals are rare in the lateral part of the NTS and hardly detected in its medial part. Electrophysiological experiments confirm the predominance of GABA inhibition throughout the NTS and demonstrate the existence of a dual inhibition involving the co‐release of GABA and glycine restricted to the lateral part of NTS. Since GABAA and glycine receptors are co‐expressed postsynaptically in virtually all the inhibitory axon terminals throughout the NTS, it suggests that the inhibition phenotype relies on the characteristics of the axon terminals. Our results also demonstrate that glycine is mostly associated with GABA within axon terminals and raise the possibility of a dynamic regulation of GABA/glycine release at the presynaptic level. Our data provide new information for understanding the mechanisms involved in the processing of visceral information by the central nervous system in adult animals.

Involvement of excitatory amino acids in the activity of swallowing-related neurons of the ventro-lateral medulla

Previous studies have shown that swallowing-related (SR) neurons are present in the ventro-lateral medulla (VLM), within and around the nucleus ambiguus. During deglutition, these SR neurons receive an excitatory input from the swallowing network located within the nucleus tractus solitarii and exhibit a swallowing activity, i.e. a burst of spikes occurring in close temporal relationship with the swallowing motor contraction. The present experiments were carried out to evaluate the possible contribution of excitatory amino acids (EAA) receptors to the swallowing activity of VLM neurons. The effects of ionophoretic or pressure applications of EAA agonists and antagonists were investigated on the activity of SR neurons located in the VLM of decerebrate rats. All SR neurons were excited by ionophoretic applications of N-methyl-D-aspartate (NMDA) and kainate, a non-NMDA receptor agonist. Furthermore, the swallowing response of the neurons was depressed by ionophoretic applications of both the broad spectrum EAA antagonist, tau D-glutamyl-glycine, and the selective NMDA antagonist, DL-2-amino-5-phosphonovalerate, and by pressure applications of the preferential non-NMDA receptors blocker, 6-cyano-7-nitroquinoxaline-2,3-dione. These results indicate that the swallowing activity of SR neurons located in the VLM depends on the activation of EAA receptors. They moreover suggest that both NMDA and non-NMDA receptor subtypes are involved.