Effects of tachykinins on identified dorsal vagal neurons: an electrophysiological study in vitro.

Intracellular current-clamp recordings were performed using in vitro brainstem slice preparations to compare the actions of substance P, neurokinin A, neurokinin B and their agonists on rat dorsal vagal nucleus neurons with or without antagonists of neurokinin 1 and 2 receptors. The agonists used were either [Sar9,Met(O2)11]substance P or septide for neurokinin 1 and [Nle10]neurokinin A(4-10) for neurokinin 2 receptors. The antagonists were spantide, SR 140333 or RP 67580 for neurokinin 1 receptors and SR 48968 for neurokinin 2 receptors. Identification of vagal neurons was achieved electrophysiologically by testing antidromic responses and confirmed morphologically by an intracellular injection of biocytin. Of the 70 neurons tested, substance P led to depolarization in 36, hyperpolarization in six and no effect in 28. Depolarization was concentration dependent and generally associated with an increase of the membrane input resistance. Addition of tetrodotoxin (1 microM) to the medium had no effect on depolarization. RP 67580 (1 microM) blocked depolarization, but spantide and SR 140333 (microM to 50 microM) did not. Hyperpolarization was never observed using agonists. Neurokinin A and neurokinin 2 agonist induced concentration-dependent depolarization associated with an increase in membrane input resistance in eight of 14 neurons and in four of nine neurons, respectively. Depolarization was only partially abolished by the neurokinin 2 antagonist SR 48968. Neurokinin B had no effect in any of the eight neurons tested. These data prove that vagal neurons have neurokinin 1 and 2 receptors and that tachykinin could produce either depolarization or hyperpolarization. Since membrane potential variations were associated with an increase (during depolarization) or decrease (during hyperpolarization) in the membrane input resistance and since the reversal potential was close to the potassium equilibrium potential, we speculate that these effects are mediated by modulation of potassium conductance.

Up-regulation of substance P binding sites in the vagus nerve projection area of the cat brainstem after nodosectomy. A quantitative autoradiographic study.

Substance P (SP) regulates visceral functions in the nucleus of the solitary tract (NST) area. High affinity SP binding sites labelled with [3H]SP or [125I]SP show a heterogeneous distribution in the cat medulla with high densities in the rostral and dorso-caudal parts of both the median subnucleus of NST and the dorsal motor nucleus (DMN). We previously observed a significant loss of SP immunoreactivity in the vagal area of the cat after an ipsilateral nodosectomy. It was thus important to study the correlated plasticity of SP binding in the context of the regulation of receptor function. Whichever labelled ligand was used, a unilateral nodose excision was followed by an ipsilateral increase in SP binding in the NST (200%) and the DMN (300%) after 30 days of survival. This increase was region-specific and did not match exactly the decrease in SP immunoreactivity following nodosectomy. This SP receptor density up-regulation could be due to long-term deprivation of SP afferent fibres in the NST and partly in the DMN. In the latter the increase of SP receptors occurred in both the cytoplasm of large neurons and the neuropile and did not affect the glia. The up-regulation phenomenon seems to be specific for SP receptors in the cat (at least in the DMN) and may constitute a reactive mechanism against the injury of axotomy of DMN neurons.

Fine distribution of substance P-like immunoreactivity in the dorsal nucleus of the vagus nerve in cats.

The ultrastructure of substance P (SP)-immunoreactive elements in the cat dorsal motor nucleus of the vagus nerve was examined using pre- and post-embedding immunocytochemical procedures. Substance P-like immunoreactivity was observed in axon terminals and axon fibres which were mostly unmyelinated. Quantitative data showed that at least 16% of axon terminals contained SP. Their mean diameter was larger than that of their non-immunoreactive counterparts. Most (83%) SP-containing terminals were seen to contact dendrites but some were observed adjoining soma or entirely embedded in the cytoplasm of vagal neurons (4.5%). Only 0.5% were observed to contact soma of internuerons. A few immunoreactive axon terminals (4%) were observed in contact with non-immunoreactive axon terminals. Round agranular vesicles and numerous dense core vesicles were visible in most SP-containing axon terminals (84.6%). The immunogold procedure showed the preferential subcellular location of SP to be dense core vesicles. In 32.4% of cases, SP-containing terminals were involved in synaptic contacts that were generally of the asymmetrical Gray type 1 and mainly apposed dendrites. The theoretical total of synaptic contacts was 74.5% and this suggests the existence of weak non-synaptic SP innervation involving approximately 25% of SP-containing axon terminals. No axo-axonic synapses were observed in the dorsal vagal nucleus. These results support the hypothesis that SP found in the dorsal vagal nucleus originates partly from vagal afferents and is involved in direct modulation of visceral functions mediated by vagal preganglionic neurons.

Substance P-immunoreactivity in the dorsal medial region of the medulla in the cat: effects of nodosectomy.

The distribution of substance P in the vagal system of the cat was studied by immunohistochemistry. Substance P-immunoreactive cell bodies and fibres were observed in the nodose ganglion. Numerous substance P-immunoreactive terminals and fibres were localized in their bulbar projection area, i.e. throughout the caudo-rostral extent of the nucleus of the solitary tract. Four subnuclei, among the nine forming the nucleus of the solitary tract, were strongly labelled: interstitial, gelatinosus, dorsal and commissural. The dorsal motor nucleus of the vagus nerve also exhibited numerous substance P-immunoreactive terminals, sometimes closely apposed on the somata of preganglionic neurons. To determine the substance P component of the vagal afferent system a nodose ganglion was removed on one side. The ablation triggered ipsilaterally a large decrease of substance P immunoreactivity in the four subnuclei strongly labelled on normal cats. These results suggest the involvement of substance P-containing vagal fibres in integrative processes of the central regulation of cardiovascular, digestive and respiratory systems, viscerotopically organized throughout these four subnuclei. The nodose ablation also resulted in a decrease of substance P immunoreactivity in the ipsilateral dorsal motor nucleus of the vagus nerve, suggesting monosynaptic vago-vagal interactions.

A new interface chamber for the study of mammalian nervous tissue slices.

We describe a new interface-type chamber for electrophysiological studies in mammalian brain slices. Thermoregulation of the inner recording chamber is achieved using the Peltier effect and a feedback control unit. Between 15 and 40 degrees C, and for perfusion rates from 1 to 5 ml/min, the temperature can be maintained within +/- 0.1 degrees C of the command value; it can also be rapidly and reliably changed. An external bath, heated by a coiled resistor, generates a humidified, oxygenated atmosphere diffusing above the slices. Survival of neuronal tissue is excellent and stable intracellular recordings can be obtained using either sharp or patch-clamp micropipettes. Perfusion solutions can be readily exchanged, rendering this chamber suitable for the study of bath-applied neuroactive compounds.

Induction of Fos-like protein in neurons of the medulla oblongata after electrical stimulation of the vagus nerve in anesthetized rabbit.

Antibodies against the c-fos protein product Fos were used to map the first- and higher-order neurons in the rabbit medulla oblongata after electrical stimulation of the vagus nerve. Fos immunoreactivity appeared bilaterally except in the nucleus tractus solitarii. Seven areas were labeled: the nucleus tractus solitarii, the area postrema, the subnucleus lateralis caudalis magnocellularis medullar oblongata, the lateral reticular nucleus, the ambiguus nucleus, the dorsal part of the spinal trigeminal nucleus, the nucleus reticularis lateralis, the lateral border of the external cuneatus nucleus, the medial part of the inferior olivary nucleus (subnucleus beta). The last two areas have never been visualized with conventional tracing techniques and may represent higher-order neurons connected to visceral vagal pathways. No labeling was observed in the nodose ganglion.

Quantitative autoradiographic studies of 5-HT-accumulating neurones in the nodose ganglia of the cat after perikaryal or terminal uptake.

The distributions of ganglionar cell bodies, specifically labeled with 3H-5-HT, were studied in light microscope autoradiographs of the cat nodose ganglion. The size, number and localization of labeled cells were examined under different experimental conditions: after in vitro incubation of the ganglion with 3H-5-HT and after retrograde transport of 3H-5-HT injected into the nucleus of the solitary tract (NST) which receives primary visceral sensory projections from nodose ganglia cells of the vagal nerve. Following incubation of the nodose ganglia with a low concentration of 3H-5-HT (10(-6) M), some ganglionar cell bodies took up and retained the tracer. In both the right and left ganglia, they were significantly smaller in size than the unreactive neurones. The mean diameter of their perikaryon was 36.97 +/- 0.52 microns, compared with 45.76 +/- 0.87 microns in unreactive neurones. About 600 labeled cell bodies were counted in each ganglion, corresponding to 2-3% of the total nodose ganglion cell population. These reactive neurones were not localized in one particular area of the ganglia, but scattered throughout both of them. Following bilateral or unilateral microinjections of 3H-5-HT in the NST, retrogradely labeled cell bodies were observed, 24 h later, in the nodose ganglia. Their mean diameter was estimated to be 36.14 +/- 0.69 microns and they represented approximately 2% of the total ganglion cell population. As in in vitro experiments, the labeled cells were not grouped in any particular region of the ganglion. These experiments show that the distribution of both populations of labeled cells, observed under these conditions, are comparable. On an anatomical and quantitative basis one may reasonably suppose that the perikaryal or terminal uptakes concern the same neuronal population.

Substance P post-synaptically potentiates glutamate-induced currents in dorsal vagal neurons.

We examined the post-synaptic actions of glutamate, N-methyl-D-aspartate (NMDA) and substance P on dorsal vagal neurons, using the patch-clamp technique on brainstem slices of young rats. The vagal neurons were identified electrically and histologically. All vagal neurons responded to glutamate and NDMA and about 30% to substance P, with dose-dependent inward currents. The glutamate-induced currents were blocked partially by either CPP (3((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid) or CNQX (6-cyano-7-nitro-quinoxaline-2,3-dione), indicating that these currents resulted from the activation of at least two types of glutamate receptors: NMDA receptors and AMPA/kainate receptors. The NK1 receptor-selective antagonist, RP67580, blocked substance P-induced currents, suggesting that NK1 receptors do coexist with NMDA receptors and AMPA/Kainate receptors. Substance P potentiated the effects of glutamate. This potentiation lasted 10-20 min and was blocked by CPP and by RP67580, but not by CNQX, demonstrating that the increase in glutamate-induced currents resulted from the interaction between NK1 receptors and NMDA channels. These results provided the first evidence that the receptors for substance P and glutamate coexist on dorsal vagal neurons and interact with each other to modulate visceral efferent functions.

Serotonin-like immunoreactivity in paraffin-sections of the nodose ganglia of the cat.

The presence of serotonin (5-HT)-like immunoreactivity has been detected in ganglionar cell bodies of the nodose ganglia of the cat, using the peroxidase-antiperoxidase immunohistochemical method, applied to sections of ganglia previously embedded in paraffin. The antibodies were raised in rabbits following injection of 5-HT conjugated to bovine serum albumin, with formaldehyde as the condensation reagent. Immunoreactive cell bodies are morphologically and topographically described. The ubiquitous distribution and non-uniform labelling suggest that these 5-HT-containing vagal neurones may give rise to several functionally different 5-HT types in vagal afferent fibres.

Serotonergic projections from the nodose ganglia to the nucleus tractus solitarius: an immunohistochemical and double labeling study in the rat.

Possible projections of serotonin (5-HT)-immunoreactive neurons in the nodose ganglia (NG) to the nucleus tractus solitarius (NTS) were investigated in the rat using a double labeling method combining retrograde transport and 5-HT immunohistochemistry. After injection of a complex of colloidal gold-apo-horseradish peroxidase into the medio-caudal and commissural parts of the NTS, most of the 5-HT-immunoreactive neurons were found to be labelled by the gold complex. The present study provides direct evidence for the existence, in the rat, of a serotonergic NG-NTS system. This system may be involved in the regulation of blood pressure and vigilance states.

Selective retrograde transport of 3H-serotonin in vagal afferents.

A new serotonergic afferent vagal component has been demonstrated in the cat by radioautography. Twenty-four hours after a bilateral injection of tritiated serotonin (3H-5-HT) into the area of the nucleus of the solitary tract (NST), heavily and lightly labelled cell bodies were observed in the nodose ganglia. After unilateral injections of 3H-5-HT into the same area, labelled ganglionar cell bodies were found in the ipsilateral nodose ganglion. Some were also found in the contralateral one, suggesting a serotonergic crossed fibers component. Dense clusters of silver grains, depicting typical labelling of neuronal varicosities, were observed in the NST. After destruction of the serotonergic terminals with 5,7-dihydroxytryptamine, followed by injection of 3H-5-HT, the number of labelled cell bodies decreased dramatically in the ipsilateral nodose ganglia and the clusters of silver grains disappeared in the NST. After ligature or section of the supranodose vagal nerve, following injection of 3H-5-HT into the NST, no radioautographic reaction was observed in the homolateral nodose ganglia. The present study demonstrates the existence of a peripheral serotonergic system in vagal afferents. The physiological implications of this new serotonergic visceral pathway remain to be studied.

[Nucleus tractus solitarius, serotonin and regulation of vigilance]. / Noyau du faisceau solitaire, sérotonine et régulation de la vigilance.

A brief historical survey of the vagal influences on sleep is lightly sketched first by analyzing the hypnogenic effects of the medullar area of the solitary tract nucleus (STN) where the vagal afferents terminate. Then the vagal effects and more extensively the sinusal or aortic effects are developed. It is shown that serotonin could play a crucial role in this so-called "vago-aortic" sleep mechanisms. Finally, these results are discussed and confronted with other experimental and human pathological data.

Electrophysiological and morphological properties of type C vagal neurons in the nodose ganglion of the cat.

Some passive and active electrical properties of type C neurons were studied intracellularly, in situ, in the nodose ganglia of adult cats. From the neuronal responses to hyperpolarizing and depolarizing rectangular current pulses it was possible to determine the input resistance (34.4 M omega) and specific membrane resistance (2373 omega.cm2). Significant changes in magnitude and duration of the action potential evoked by vagal stimulation result from changes in the resting potential caused by the passage of steady polarizing currents across the cell membrane. The action potentials evoked by infranodose vagal stimulation had a long duration, a long latency and comprised several components. The fast main spike was followed by a long post-hyperpolarization. The double shock technique showed that the fast main potential was composed of an initial segment spike ('A spike') and a somatic spike ('S spike'), and made it possible to determine the somatic refractory periods. After electrical identification of the cells, horseradish peroxidase was injected ionophoretically into the soma, and it was shown that the central processes were about four times smaller in diameter than the peripheral processes.

A-current modifies the spike of C-type neurones in the rabbit nodose ganglion.

1. In the rabbit nodose ganglion, C-type fibre neurones (C neurones) can be divided into two subtypes according to their after-hyperpolarizing potential (AHP) i.e. those with a fast AHP only and those with a fast AHP and a subsequent slow AHP produced by a slow calcium-dependent potassium current. In addition we have shown that some C neurones can be divided into two groups according to the effect of membrane hyperpolarization on their spikes i.e. type 1 in which duration and amplitude do not change and type 2 in which duration and amplitude decrease significantly. 2. In the present report we studied the effect of A-current (IA) on spike duration, amplitude and slow AHP using intracellular recording techniques. 3. To detect the presence of IA, we first applied a series of increasing rectangular hyperpolarizing pulses to remove IA inactivation and then a short depolarizing pulse to trigger a spike. In type 1 C neurones the lag time of the spike in relation to hyperpolarization remains constant whereas in type 2 C neurones the spike only appears after IA inactivation and lag time in relation to hyperpolarization is lengthened. Thus, type 2 C neurones have an IA while type 1 C neurones do not. The fact that addition of cadmium did not change the lag time in type 2 C neurones shows that the IA is not calcium dependent. 4. Nodose neurones can be orthodromically activated by stimulation of the vagal peripheral process. In this way, after a hyperpolarizing pulse, IA can be fully activated by the orthodromic spike itself. Under these conditions it is possible to analyse the effects of IA on the spike. This was done by increasing either the hyperpolarizing potential, pulse duration, or the delay of the spike after the end of the pulse. We observed that maximum IA inactivation removal was always associated with the lowest duration and amplitude of the spike. 5. When IA inhibitors, 4-aminopyridine (4-AP) or catechol, were applied to type 2 C neurones, the delay of the spike after the hyperpolarization-depolarization test was no longer observed. In addition 4-AP abolished the shortening of the duration of the spike induced by steady hyperpolarization. 6. In type 2 C neurones with slow AHP, the IA-related decrease in spike duration was associated with a disappearance of the slow AHP. This indicates that IA decreases the calcium influx during the spike.(ABSTRACT TRUNCATED AT 400 WORDS)

Characteristics of the experimental reflex sleep induced by vago-aortic nerve stimulation.

1. Electrical stimulation of the baroceptive fibres of the vago-aortic trunks (VA St) reflexly induces, in the Ecéphale isolé (EI) cat preparation, a complete sleep cycle characterized by a progression of all stages of sleep previously described in chronic preparations (slow wave sleep, phasic slow wave sleep, paradoxical or REM sleep). Some qualitative and quantitative characteristics of this "vago-aortic" (VA) sleep are analysed and compared with those obtained in EI cats previously deprived of REM sleep. 2. VA sleep is not simply the consequence of chance occurrences, but the consequence of VA St. It is stimulus-bound, even if the interval between successive stimuli is as short as 7.5 min. Thus it is possible to impose an artificially regular cycle much shorter than the normal one. one. 3. VA St. precipitates the onset of the differrent sleep cycle episodes in the non-REM-deprived EI cat as well as in the REM-deprived EI cat. 4. REM sleep begins rapidly after stimulus onset, so that an experimental reflex REM narcolepsy may be obtained. This effect is particularly noteworthy in REM-deprived cats. In this situation, REM sleep episodes with cataplexy (muscular atonia and areflexia) can immediately follow the onset of stimulation.