Multimodal responses of taste neurons in the frog nucleus tractus solitarius

The responses of 216 neurons in the nucleus tractus solitarius (NTS) of the American bullfrog were recorded following taste, temperature, and tactile stimulation. Cells were classified on the basis of their responses to 5 taste stimuli: 0.5 M NaCl, 0.0005 M quinine-HCl (QHCl), 0.01 M acetic acid, 0.5 M sucrose, and deionized water (water). Neurons showing excitatory responses to 1, 2, 3, or 4 of the 5 kinds of taste stimuli were named Type I, II, III, or IV, respectively. Cells whose spontaneous rate was inhibited by taste and/or tactile stimulation of the tongue were termed Type V. Type VI neurons were excited by tactile stimulation alone. Of the 216 cells, 115 were excited or inhibited by taste stimuli (Types I-V), with 35 being Type I, 34 Type II, 40 Type III, 2 Type IV and 4 Type V. The remaining 101 cells were responsive only to tactile stimulation (Type VI). Of those 111 cells excited by taste stimulation (Types I-IV), 106 (95%) responded to NaCl, 66 (59%) to acetic acid, 44 (40%) to QHCl, 10 (9%) to water, and 9 (8%) to warming. No cells responded to sucrose. Of the 111 cells of Types I-IV, 76 (68%) were also sensitive to mechanical stimulation of the tongue. There was some differential distribution of these neuron types within the NTS, with more narrowly tuned cells (Type I) being located more dorsally in the nucleus than the more broadly tuned (Type III) neurons. Cells responding exclusively to touch (Type VI) were also more dorsally situated than those responding to two or more taste stimuli (Types II and III).     

Taste responses in the nucleus tractus solitarius of the chronic decerebrate rat

The ingestive behavior of decerebrate rats has been studied for some time, yet little is known of its neural substrates. While taste fibers in rats proceed from hindbrain to thalamus and ventral forebrain, these regions return centrifugal fibers to the hindbrain by which lower-order taste activity may be influenced. We examined the functional characteristics of taste neurons in the nucleus tractus solitarii (NTS) of chronic decerebrate rats in which this reciprocal communication was disrupted and compared them with those of intact controls. Nine Wistar rats were decerebrated at the supracollicular level. After a minimum of one week recovery, they were immobilized with Flaxedil, anesthetized locally and prepared for recording. The responses of 50 taste cells were isolated bilaterally from the NTS of these animals, while the activity of 50 additional neurons was recorded from 12 intact rats under the same conditions. Taste stimuli included 7 Na-Li salts, 3 sugars, HCl and citric acids, quinine HCl and NaSaccharin. Mean spontaneous activity in decerebrates was 6.5 spikes/s, 36.0% lower than the level in intact animals. Mean evoked activity was reduced by 32.6%. Analyses of the effects of stimulus quality, intensity and time course of the responses all indicated that the decrease in activity was attributable to the inability of taste cells in decerebrate rats to respond to demands for high discharge rates. This deficit could be responsible for the failure of these animals to develop conditioned taste aversions. Neurons from decerebrate preparations did, however, retain the broad sensitivity across stimuli that characterized taste cells in intact preparations. It was also typical that most neuron response profiles from decerebrates could be grouped into 3 loose clusters with peak sensitivities to acid-salt, salt or sugar. An analysis of similarities among stimulus activity profiles indicated that Na-Li salts, sugars and an acid-quinine complex represented 3 groups of stimulus quality; in intact animals, the primary distinction was between sweet and non-sweet stimuli. Moreover, the response to sodium saccharin lost its bitter component in decerebrates. These findings were in general agreement with those derived from acute decerebrate rats.     

Synaptic responses of neurons of the nucleus tractus solitarius in vitro

Postsynaptic responses of neurons in the nucleus tractus solitarius (NTS) have been studied in an in vitro slice preparation using extra- and intracellular recording. Single or paired pulse stimulations were delivered to afferent fibers within the tractus solitarius (TS) to activate orthodromic responses in these neurons. Most NTS neurons displayed an initial synaptic excitation followed by inhibition of spontaneous or evoked firing lasting up to 150-200 ms after stimulation. Excitatory postsynaptic potentials (EPSPs), recorded intracellularly, were increased in amplitude by membrane hyperpolarization. Large afterhyperpolarizations followed action potentials triggered by the EPSPs or evoked by intracellular current injections. Intracellular evidence for synaptic inhibition within the NTS included: (1) the presence, after Cl-injection, of flurries of spontaneous PSPs likely to be inverted inhibitory postsynaptic potentials; (2) reduction of the size of a test EPSP by a previous subthreshold TS conditioning volley; and (3) hyperpolarizing PSPs recorded in some neurons. Other NTS neurons exhibited prolonged excitatory responses to TS stimulation and could be local inhibitory interneurons. These results may help specify synaptic mechanisms in the NTS that could play an integrative role in the relay of visceral sensory inputs to higher order effectors.     

An atlas of the rat subpostremal nucleus tractus solitarius.

The nucleus tractus solitarius (NTS) in the dorsal medulla is the principal visceral sensory relay nucleus in the brain. In the rat, numerous lines of evidence indicate that the caudal NTS at the level of the area postrema serves as a major integrating site for coordinating cardiorespiratory reflexes and viscerobehavioral responses. This region of the caudal NTS not only exhibits high densities of binding sites for an impressive array of transmitters and modulators but microinjections of many of these same neuroactive substances into the rat subpostremal NTS elicit pronounced cardiorespiratory and visceral response patterns. This report provides an abbreviated atlas of the rat subpostremal NTS consisting of a series of transverse, sagittal, and horizontal plates. Photomicrographs, together with their corresponding schematic drawings, are provided for the serial sections generated from each reference plane.     

Enhanced serotonin-mediated responses in the nucleus tractus solitarius of spontaneously hypertensive rats

Previous studies have demonstrated that injection of serotonin into the nucleus tractus solitarius (NTS) elicits hypotension and bradycardia in rats. The present study sought to further characterize this response and to examine the role of serotonergic mechanisms in the NTS in cardiovascular regulation in spontaneously hypertensive (SHR) rats. Injections of picomole amounts of serotonin into the NTS of chloralose-anesthetized normotensive Sprague-Dawley (S-D) or Wistar-Kyoto (WKY) rats produced hypotension and bradycardia that were eliminated by prior injection into the NTS of the selective 5HT(2) antagonist sarpogrelate. Bilateral injection of sarpogrelate did not alter blood pressure or reflex changes in heart rate in response to phenylephrine-induced increases in blood pressure or nitroprusside-induced decreases in blood pressure. In SHR rats, the depressor response produced by injection of serotonin into the NTS was markedly larger than in WKY rats, and was larger than depressor responses previously reported for other excitatory substances injected into the NTS. In SHR rats bilateral injection of sarpogrelate produced an increase in blood pressure, although it did not alter baroreceptor-evoked changes in heart rate. These results provide further support for the hypothesis that stimulation of 5HT(2) receptors in the NTS contributes to cardiovascular regulation independent of the baroreceptor reflex. Furthermore, this serotonergic system is altered in SHR rats, apparently acting tonically to reduce blood pressure.     

Cardiovascular effects of Leu-enkephalin in the nucleus tractus solitarius of the rat

Microinjections of Leu-enkephalin into the dorsal vagal complex induced hypotension and bradycardia. Both naloxone, given at a dose conferring selectivity for μ receptors, and the S antagonist ICI 154,129 prevented the cardiovascular effects of Leu-enkephalin. Naloxone was also found to decrease the gain of the baroreflex. These results suggest that Leu-enkephalin is involved in cardiovascular regulation through activation of δ-, and possibly μ-, opioid receptors in the dorsal vagal complex.     

Characterization of stimulation-produced analgesia from the nucleus tractus solitarius in the rat

Electrical stimulation of the commissural region of the nucleus tractus solitarius (NTS) inhibits the tail-flick reflex evoked by noxious heat. This antinociception can be measured in the awake or pentobarbital anesthetized rat at current intensities that do not induce overt behavioral side effects. Glutamate microinjections into the NTS, but not immediately surrounding the NTS, also inhibit the tail-flick reflex, demonstrating that activation of NTS cell bodies, and not fibers of passage, mediates antinociception from this region. In contrast, morphine microinjections into the NTS have no effect on the tail-flick reflex in anesthetized rats. These findings provide further evidence that the NTS is involved in the modulation of nociception.     

Catecholamine inputs to the nucleus tractus solitarius

The projections of brainstem catecholaminergic (CA) cell groups to the rat nucleus tractus solitarius (NTS) were examined using 6-hydroxydopamine (6-OHDA) injections and glass microknife cuts. 6-OHDA (4 micrograms) was injected into the intermediate NTS, and this resulted in depletion of CA fluorescent varicosities from the NTS at this rostrocaudal level, except for varicosities along the periventricular edge of the NTS. In addition, a band of swollen fluorescent axons extended between the CA A1 cell group of the ventrolateral medulla and the lateral NTS. Microknife cuts were used to interrupt the projections of the CA A1 and A2 cell groups (located in the caudal NTS) and tissues were examined for changes in CA varicosity density within the intermediate NTS. Following transverse knife cuts of the intermediate NTS, rostral to the A2 cell group, fluorescent varicosities rostral to the cut virtually disappeared, and the fluorescence intensity of the ipsilateral A2 neurons caudal to the cut was increased. These cuts also eliminated the 6-OHDA-resistant varicosities along the periventricular NTS. After microknife cuts lateral to the intermediate NTS, the fluorescent varicosity density in the NTS was unchanged. These results indicate that the major CA projection to the NTS arises from the ipsilateral A2 cell group. The 6-OHDA-resistant varicosities arising from neurons caudal to the knife cut probably arise from the adrenergic C2 cell group.     

Blood pressure modulation by substance P in the rat nucleus tractus solitarius

Substance P in a dose of 0.1-10 ng injected into the nucleus tractus solitarii (NTS) of the rat caused hypotension, bradycardia and apnea whereas a dose of 100 ng led to no response. A substance P antagonist injected into the NTS abolished the cardiovascular responses to substance P. The antagonist alone increased blood pressure and heart rate. The data suggest a role for substance P in the cardiovascular regulation by the NTS.     

Substance P innervation of neurons projecting to the paraventricular hypothalamic nucleus in the rat nucleus tractus solitarius

After injection of WGA-HRP-colloidal gold in the rat paraventricular nucleus (PVN), retrogradely labeled neurons were found mainly in the medial and commissural subnuclei of the nucleus tractus solitarius (NTS) around 0.5 mm caudal to the obex which is closely related to cardiovascular function. Electron microscopic immunohistochemistry in these areas demonstrated synaptic contacts between retrogradely labeled neurons and substance P-immunoreactive terminals. Innervation of NTS-PVN projection systems by substance P is suggested.     

Gustatory functions of the nucleus tractus solitarius in the rabbit

Multiunit analysis revealed a rostral region of NTS containing cells responsive to taste stimulation of rostral tongue. Using representative stimuli for the 4 basic types of taste, maximal incidence and magnitude of response was found to NaCl, followed by HCl, sucrose and QHC1. Further analysis of temporal patterns of response to the tastants revealed differences among stimuli in latency and time course. A principal components analysis indicated that time course, apart from magnitude of response, could contribute to neural differentiation of tastants. Information was also derived on neural intensity functions for these basic types of tastants. Additional observations with sodium saccharin revealed minimal neuronal responsivity despite reported evidence of behavioral preference for this tastant by rabbits.     

Projections from the nucleus and tractus solitarius in the cat

After lesions in the nucleus of the tractus solitarius (NTS) in cats and kittens, the termination of degenerating fibers was localized using the Nauta and the Fink-Heimer techniques. The distribution of degenerating fibers was compared with that seen after lesions of the dorsal nucleus of the vagus (DNV) and after section of the ninth and tenth cranial nerves. The projection from the NTS is to the nucleus ambiguus (A), the other divisions of the NTS including the medial NTS and the ventrolateral NTS, the DNV, the medial reticular formation ventral to the NTS (probably the paramedian reticular nucleus), the nucleus intercalatus (INC), and the intermediate nucleus (INT). The probable functional significance of projections from the NTS to these medullary nuclei is discussed in relation to pathways of cardiovascular reflexes. Of particular note is the projection to the INT which may be part of a descending pathway to spinal cardiovascular neurons in the intermediolateral horn.     

Stimulation of 5-HT2 receptors in the nucleus tractus solitarius enhances NMDA receptor-mediated reflex-evoked bradycardiac responses in the rat

The modulation by 5-HT2 receptors in the nucleus tractus solitarius of the reflex bradycardia evoked by stimulation of peripheral baroreceptors and cardiopulmonary chemoreceptors, and their possible functional interactions with local NMDA receptors, were investigated in pentobarbital- and urethane-anaesthetized rats, respectively. Microinjection of the 5-HT2 receptor agonist, 2,5-dimethoxy-4-iodoamphetamine (0.1-0.5 pmol), into the nucleus tractus solitarius elicited a dose-dependent hypotension and bradycardia. Bilateral microinjections at the same site of a subthreshold dose of 2,5-dimethoxy-4-iodoamphetamine (0.05 pmol) significantly enhanced the aforementioned reflex-evoked bradycardiac responses. In contrast, local bilateral microinjections of the NMDA receptor antagonist, 2-amino-5-phosphonopentanoic acid (500 and 1000 pmol), reduced, in a dose-dependent manner, both reflex-evoked responses. The facilitatory effect of 2,5-dimethoxy-4-iodoamphetamine upon these reflex-evoked bradycardiac responses was prevented by prior local microinjection of low doses of either the selective 5-HT2 receptor antagonist, ketanserin (10 pmol), or 2-amino-5-phosphonopentanoic acid (100 pmol), which, on their own, did not affect the reflex-associated bradycardia. These data suggest that 5-HT2 receptors within the nucleus tractus solitarius participate in a facilitatory modulation of the reflex control of heart rate, probably through functional interactions with local NMDA receptors.     

Afferent projections to cardiovascular portions of the nucleus of the tractus solitarius in the rat

Superfusion onto hippocampal slices of low concentrations of the tetradecapeptide somatostatin (SS), or an SS analogue having CNS activity, reversibly hyperpolarized pyramidal neurons, as revealed by intracellular recording. The hyperpolarizations were accompanied by reductions in spontaneous and evoked spike discharge and in input resistance; the magnitude of the hyperpolarizations was not influenced by 10 mM MgCl2 added to the perfusate to block synaptic transmission.     

Microinjection of orexin into the rat nucleus tractus solitarius causes increases in blood pressure

Orexin A (OX-A) and orexin B (OX-B), also known as hypocretin-1 and hypocretin-2, have been suggested to play a role cardiovascular control. The nucleus tractus solitarius (NTS), located in the dorsal medulla plays an essential role in neural control of the cardiovascular system. Orexin-immunoreactive axons have been demonstrated within this nucleus suggesting that NTS may be a site through which OX acts to influence cardiovascular control. We report here that microinjection of OX-A into the NTS of urethane anesthetized rats causes increases in blood pressure (10⁻⁹ M, mean AUC=607.1±65.65 mmHg s, n=5) and heart rate (10⁻⁹ M, mean AUC=16.15±3.3 beats, n=5) which returns to baseline within 90 s. We show that these effects are dose related and site specific. Microinjection of OX-B into NTS elicited similar increases in BP (mean AUC=680.8±128.5 mmHg s, n=4) to that of OX-A suggesting specific actions at the OX₂R receptor. These observations support the conclusion that orexins act as chemical messengers in the NTS likely influencing the excitability of cardiovascular neurons in this region and thus regulating global cardiovascular function.     

Synaptic interaction of vagal afferents and catecholaminergic neurons in the rat nucleus tractus solitarius

Combined radioautography and immunocytochemistry were used to define the ultrastructure and synaptic relations between vagal sensory afferents and catecholaminergic (CA) neurons of the A2 group located within the nucleus tractus solitarius (NTS) of rat brain. The vagal afferents were radioautographically labeled by tritiated amino acids anterogradely transported from the nodose ganglion. Immunocytochemical labeling for tyrosine hydroxylase (TH) served for the identification of catecholaminergic neurons. The radiographically labeled axons seen by light microscopy were widely distributed throughout the more caudal NTS. The reduced silver grains were more densely distributed within the NTS located homolateral to the injected nodose ganglion. The radioautographically labeled processes were localized in regions containing catecholaminergic neurons as indicated by immunoreactivity for TH. Electron microscopic analysis of the medial NTS at the level of the obex demonstrated that the reduced silver grains were localized within axon terminals. The radioautographically labeled terminals were 2-3 microns in diameter, contained numerous small, clear and a few large, dense vesicles, and formed predominately axodendritic synapses. Many of the recipient dendrites contained immunoreactivity for TH. In rare instances, vagal afferents formed synaptic appositions with both TH-labeled and unlabeled axon terminals and neuronal soma. This study provides the first ultrastructural evidence that the catecholaminergic neurons within the NTS receive direct synapses from sensory neurons in the nodose ganglion.     

Strategies for cellular identification in nucleus tractus solitarius slices

The indistinct regional anatomy and intermixing of second order neurons with projection and interneurons make cellular studies more difficult within the nucleus tractus solitarius (NTS). Here, we outline experimental strategies to join in vitro electrophysiological with neuroanatomical protocols to discriminate specific subpopulations of NTS neurons. Horizontally cutting the brain stem produces slices in which electrical activation of the solitary tract (ST) is free of local interneuron contamination. Such ST excitatory synaptic currents (EPSCs) functionally identify second order NTS neurons by their minimal variation of latency (jitter). Sapphire blades, cold cutting temperatures and a mechanically stable microtome were critical to consistently obtain viable slices that were optimized for infrared and fluorescence microscopy. Anterogradely transported carbocyanine dye implanted on the aortic depressor nerve anatomically identified second order NTS neurons and their ST synaptic performance conformed to the minimal jitter signature of second order neurons. Retrograde tracers and green fluorescent protein labeled neurons afford two additional promising approaches for discriminating NTS neuron phenotypes in broader system contexts. Detailed methods and troubleshooting are described. Coupling tracing techniques with electrophysiology adds important new dimensions to NTS studies and such strategies provide bridging information between cellular mechanisms, neuroanatomy and systems integration.     

Muscarinic M2 receptor inhibition of calcium current in rat nucleus tractus solitarius

The cholinergic system in the central nervous system plays an important role in higher brain functions, through muscarinic receptors. The nucleus tractus solitarius is known to play a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic and swallowing functions. Voltage-dependent Ca2+ channels (VDCCs) serve as crucial mediators of membrane excitability and Ca2+-dependent functions such as neurotransmitter release, enzyme activity and gene expression. The purpose of this study was to investigate the effects of acetylcholine (Ach) on VDCC currents (I(Ca)) in the nucleus tractus solitarius using patch-clamp recording methods. In 68 out of 99 neurons, an application of ACh caused inhibition of N-type and P/Q-type I(Ba) in a concentration-dependent manner. Pretreatments with AF-DX116 (muscarinic M2 receptor antagonist) attenuated the ACh-induced inhibition of I(Ba). Intracellular dialysis of the Galpha(i)-protein antibody also attenuated the ACh-induced inhibition of I(Ba). These results indicate that ACh inhibits N-type and P/Q-type VDCCs via Gi-protein betagamma subunits mediated by M2 receptors in nucleus tractus solitarius.     

The effect of amiloride on taste-evoked activity in the nucleus tractus solitarius of the rat

Amiloride is an inhibitor of passive sodium transport. Its application to taste receptors blocks inward sodium current, suppresses sodium-induced neural activity and reduces the perceived intensity of NaCl. We recorded taste-evoked responses of single neurons in the nucleus tractus solitarius (NTS) of the rat before and after the lingual application of amiloride to determine which neurons would be affected, the degree of the effect and the subsequent form of the neural code for sodium. Responses to all 7 stimuli that contained Na⁺ or Li⁺ were suppressed by amiloride. Activity evoked by the 8 other stimuli was unaltered. NTS neurons could be divided into 4 subsets according to their response profiles: Group 1 (salt-sugar), Group 2 (salt), Group 3 4 (acid-salt-bitter). The entire effect of amiloride was discharged on cells in Groups 1 and 2; those in Groups 3 and 4 were unaffected. Following amiloride application, the neural code for sodium and lithium salts was highly similar to those for acids, bitter salts and quinine. Thus the activity of neurons in Groups 1 and 2 may be responsible for the distinction between ‘saltiness’ and sour-bitter tastes. The results imply that specific receptors are responsible for the recognition and transduction of sodium salts and that this specificity in the peripheral taste nerves to be manisfested in the NTS.     

Behavioural responses to electrical stimulation of the nucleus of the tractus solitarius of the cat

Electrical stimulation of the region of the nucleus tractus solitarius (NTS) significantly reduced both the lever-pressing rate for substantia nigra self-stimulation and the food-consumption in food-deprived cats. In a shuttle box the cats showed no tendency toward shuttling in response to NTS stimulations with randomly varied stimulus intensities. In contrast with these effects, stimulation to nucleus reticularis paramedianus yielded an aversive effect.