Respiratory modulation of afferent terminal excitability in the nucleus tractus solitarius

The excitability of vagal and carotid sinus nerve afferent terminals within the nucleus of the tractus solitarius has been studied in cats and rabbits using the technique of antidromic activation. Although the excitability of superior laryngeal nerve afferent terminals was observed to fluctuate in phase with the central respiratory cycle, no equivalent variations in sinus nerve terminal excitability were observed. Whilst recording from single identified vagal afferents, no respiratory-related changes in the excitability of baroreceptor afferent terminals could be demonstrated, though such modifications in terminal excitability were readily seen in lung stretch afferent terminals.     

Nitric oxide inhibits excitatory vagal afferent input to nucleus tractus solitarius neurons in anaesthetized rats

Objective

Endogenous nitric oxide (NO) has been implicated in the regulation of neuronal activity which mediates cardiovascular reflexes. However, there is controversy concerning the role of NO in the nucleus tractus solitarius (NTS). The present study aims to elucidate the possible physiological role of endogenous NO in modulating the excitatory vagal afferent input to NTS neurons.

Methods

All the experiments in the rat were conducted under anaesthetic conditions. Ionophoresis method was used for the application of NO donor or nitric oxide synthase (NOS) inhibitor, and single unit recording method was employed to detect the effects of these applications on vagal afferent- or cardio-pulmonary C-fibre reflex-evoked neuronal excitation in NTS.

Results

Ionophoresis applications of L-arginine (L-Arg), a substrate of NOS, and sodium nitroprusside (SNP), a NO donor, both attenuated the vagal afferent-evoked discharge by (51.5±7.6)% (n = 17) and (68.3±7.1)% (n = 9), respectively. In contrast, application of D-Arg at the same current exerted no overall effect on this input. Also, both L-Arg and SNP inhibited spontaneous firing of most of the recorded neurons. In contrast, ionophoresis application of NG-nitro-Larginine methyl ester (L-NAME) enhanced vagal afferent-evoked excitation by (66.3±11.4)% (n = 7). In addition, ionophoresis application of L-Arg and SNP significantly attenuated cardio-pulmonary C-fibre reflex-induced excitation in the tested NTS neurons.

Conclusion

Activation of local NO pathway in the NTS could suppress vagal afferent-evoked excitation, suggesting that NO is an important neuromodulator of visceral sensory input in the NTS.     

Firing pattern of neurons in the nucleus tractus solitarius: modulation by membrane hyperpolarization

Neurons in the ventral region of the nucleus tractus solitarius (NTS) of guinea pigs were studied using an in vitro brainstem slice preparation. One group of neurons was characterized electrophysiologically by a delay between the onset of a depolarizing stimulus and the first spike. This delay could be as large as 760 ms and was modulated by the membrane potential level preceding the stimulus. The firing rate during the depolarizing stimulus was also modulated by the preceding membrane potential level. A fast transient outward current, similar to A-current in molluscan neurons, appeared to be responsible for the delay in firing while a slower calcium-activated potassium current affected the firing rate. These data suggest that intrinsic membrane properties may play an important role in determining the firing pattern of NTS neurons. In vivo, inhibitory synaptic inputs could modulate the expression of these intrinsic properties during subsequent excitation.     

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.     

Collateral axonal projections from hypothalamic hypocretin neurons to cardiovascular sites in nucleus ambiguus and nucleus tractus solitarius

Hypocretin-1 (hcrt-1)-containing axons have been shown to have an extensive distribution within the central nervous system, although the total number of hypothalamic hcrt-1 neurons has been shown to be small. This suggests that hcrt-1 neurons may innervate central structures with similar function through collateral axonal projections. Retrograde tract-tracing techniques combined with immunohistochemistry were used in this study to investigate whether hypothalamic hcrt-1-containing neurons send collateral axonal projections to cardiovascular sites in the nucleus of the solitary tract (NTS) and in the nucleus ambiguus (Amb) in the rat. Fluorogold- (FG) and/or rhodamine (Rd)-labeled latex microspheres were microinjected into either the NTS or Amb at sites that elicited bardycardia responses (L-glutamate; 0.25 M; 10 nl). After a survival period of 10-15 days, the rats were sacrificed and tissue sections of the hypothalamus were processed immunohistochemically for the identification of hcrt-1-containing cell bodies. After injection of the tract-tracers into the NTS or Amb, retrogradely labeled neurons were observed within several hypothalamic regions; the paraventricular hypothalamic nucleus, lateral hypothalamic area, perifornical hypothalamic area, and posterior hypothalamus, bilaterally, but with an ipsilateral predominance. In addition, after NTS injections, retrogradely labeled neurons were found within the ipsilateral caudal arcuate nucleus. Of the total number (1107+/-97) of hcrt-1-immunoreactive neurons found bilaterally within the lateral and perifornical hypothalamic nuclei, 7.9+/-1.4% were found to be retrogradely labeled from the NTS, 16.4+/-1.8% from the Amb, and 3.1+/-0.5% from both medullary sites. Hcrt-1 neurons projecting to the NTS were found mainly in and around the perifornical hypothalamic region, with a smaller number in the caudal lateral hypothalamic area. On the other hand, those innervating the Amb were primarily observed within the caudal lateral hypothalamic area, with a smaller number in the perifornical hypothalamic area. Neurons with collateral axonal projections to NTS and Amb were observed within two specific hypothalamic areas: one group of neurons was found in the perifornical hypothalamic area, and the other was observed in the lateral hypothalamic region just dorsal to the retrochiasmatic component of the supraoptic nucleus. These data indicate that axons from hcrt-1 neurons bifurcate to innervate functionally similar cardiovascular-responsive sites in the NTS and Amb. Although the function of these hcrt-1-containing hypothalamic-medullary pathways is not known, they likely represent the anatomical substrate by which the lateral hypothalamic hcrt-1 neurons simultaneously coordinate autonomic-cardiovascular responses to different behaviors.     

Choline acetyltransferase activity in the nucleus tractus solitarius: Regulation by the afferent vagus nerve

The influence of nodose ganglionectomy or transection of the peripheral branches of the afferent vagus nerve on choline acetyltransferase (ChAT) activity in the nucleus tractus solitarius (NTS) was studied. ChAT activity was reduced in the microdissected caudal and intermediate portions of the NTS in vagotomized as well as ganglionectomized rats. However, only the ganglionectomy resulted in the degeneration of medullary nerve fibers. These results suggest that the changes in ChAT activity in the NTS are independent of neuronal degeneration and may be due to transynaptic modulation of ChAT activity by afferent vagal impulses. The presence of ChAT in the sensory nodose projection to the NTS, however, cannot be ruled out.     

Projection of nucleus tractus solitarius units influenced by hepatoportal afferent signal to parabrachial nucleus

In urethan-chloralose anesthetized rats, units in the nucleus tractus solitarius (NTS) which antidromically responded to electrical stimulation of the parabrachial nucleus (PB) were investigated for their responses to hepatic-vagal afferent signals. Among 63 NTS units examined, 25 (40%) were excited, 17 (27%) inhibited and the remaining 21 (33%) unaffected by single shock electrical stimulation of the hepatic branch of the vagus nerve. Topical application of Na+ produced an increase in discharge rate of 9 units and a decrease in 5 units. Portal infusion of hypertonic saline produced an increase in discharge rate of 3 units and a decrease in 5 units. Furthermore, 3 units responded to both topical application of Na+ and portal infusion of hypertonic saline.     

Characterization of neurons of the nucleus tractus solitarius pars centralis

Esophageal sensory afferent inputs terminate principally in the central subnucleus of the tractus solitarius (cNTS). Neurons of the cNTS comprise two major neurochemical subpopulations. One contains neurons that are nitric oxide synthase (NOS) immunoreactive (-IR) while the other comprises neurons that are tyrosine hydroxylase (TH)-IR. We have shown recently that TH-IR neurons are involved in esophageal-distention induced gastric relaxation. We used whole cell patch clamp techniques in rat brainstem slices combined with immunohistochemical and morphological reconstructions to characterize cNTS neurons. Postrecording reconstruction of cNTS neurons revealed two morphological neuronal subtypes; one group of cells (41 out of 131 neurons, i.e., 31%) had a multipolar soma, while the other group (87 out of 131 neurons, i.e., 66%) had a bipolar soma. Of the 43 cells in which we conducted a neurochemical examination, 15 displayed TH-IR (9 with bipolar morphology, 6 with multipolar morphology) while the remaining 28 neurons did not display TH-IR (18 with bipolar morphology, 10 with multipolar morphology). Even though the range of electrophysiological properties varied significantly, morphological or neurochemical distinctions did not reveal characteristics peculiar to the subgroups. Spontaneous excitatory postsynaptic currents (sEPSC) recorded in cNTS neurons had a frequency of 1.5 +/- 0.15 events s(-1) and an amplitude of 27 +/- 1.2 pA (Vh = -50 mV) and were abolished by pretreatment with 30 muM AP-5 and 10 muM CNQX, indicating the involvement of both NMDA and non-NMDA receptors. Some cNTS neurons also received a GABAergic input that was abolished by perfusion with 30-50 muM bicuculline. In conclusion, our data show that despite the heterogeneity of morphological and neurochemical membrane properties, the electrophysiological characteristics of cNTS neurons are not a distinguishing feature.     

Functional analysis of GABAA receptors in nucleus tractus solitarius neurons from neonatal rats

To gain insight into specific GABA_A receptor configurations functionally expressed in the nucleus tractus solitarius (NTS), we conducted several physiological and pharmacological assessments. NTS neurons were characterized in thin brain slices from 1–14 day old rats using whole-cell patch clamp recordings. GABA_A− receptor-mediated currents were detected in all neurons tested, with an average EC₅₀ of 22.2 μM. GABA currents were consistently stimulated by diazepam (EC₅₀=63 nM), zolpidem (EC₅₀=85 nM), loreclezole (EC₅₀=10.1 μM) and the neurosteroid 5α-pregnan-3α-hydroxy-20-one (3α-OH-DHP). In contrast, GABA-gated currents of the NTS were inhibited by the divalent cation Zn²⁺ (IC₅₀=33.6 μM) picrotoxin (IC₅₀=2.4 μM) and blockade of endogenous protein tyrosine kinase. GABA-activated currents were insensitive to furosemide (10–1000 μM) in all NTS neurons tested. Collectively, the data suggest that in neonatal rats, the predominant α subunit isoform present in GABA_A receptors of the NTS appears to be the α1 and/or α2 subunit. β2 and/or β3 subunits are the major β isoform, while the predominant γ subunit is likely γ2. Our data suggest the contribution to NTS GABA currents by α3–α6, β1, γ1 and δ subunits, if present, is minor by comparison.     

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.     

17beta-estradiol inhibits excitatory amino acid-induced activity of neurons of the nucleus tractus solitarius

The effects of 17beta-estradiol (17betaE2) on spontaneous and excitatory amino acid (EAA) induced nucleus tractus solitarius (NTS) neuronal activity were investigated by electrophysiological and immunohistochemical experiments in ovariectomized female Sprague-Dawley rats. Out of 62 NTS neurons tested, 42 were inhibited (68%) following iontophoretic application of 17betaE2 in a current-dependent manner. The averaged firing rate decreased from 3.06+/-0.40 to 0.78+/-0.17 Hz. The inhibitory responses were rapid in onset (within 1 min) and variable in duration (2-4 min). The inhibitory effects of 17betaE2 were blocked by simultaneously applied 17betaE2 antagonist ICI182,780, but not by GABA antagonist, bicuculline and phaclofen. L-Glutamate, AMPA or NMDA enhanced the activity of 71, 73 or 69% of NTS cells tested, respectively. The excitatory effects of EAA were significantly inhibited in the presence of 17betaE2. Fluorescent immunohistochemistry revealed that all subnuclei of the NTS contained high levels of estrogen receptors (ERs) immunoreactivity. These results suggest that 17betaE2 inhibits spontaneous and EAA-induced NTS neuronal activity through 17betaE2 activation of ERs.     

Sonic hedgehog signalling in neurons of adult ventrolateral nucleus tractus solitarius

The transmembrane receptor Patched (Ptc) mediates the action of the diffusing factor Sonic hedgehog (Shh), which is implicated in establishing morphogenetic gradients during embryonic development. Whereas alteration of Ptc function is associated with developmental abnormalities and brain tumors, its functional activity and roles in the adult brain have yet to be elucidated. Here we describe the complementary pattern of Shh and Ptc expression in the rat dorsal vagal motor nucleus and the ventrolateral nucleus tractus solitarius (vNTS), respectively. Those two interconnected structures regulate the cardiorespiratory function during hypoxia. Bath application of a subnanomolar concentration of aminoterminal Shh protein (ShhN) to a slice preparation of the vNTS induces a rapid decrease in neuronal firing followed by a bursting activity that propagates in the neuronal network. Intracellular current injections show that bursts result from an action on the neuronal membrane electro-responsiveness. Both inhibiting and bursting effects are blocked by the monoclonal Shh antibody 5E1 and may require the Ptc binding site of ShhN. Thus, ShhN acting on specific neuronal sites controls electrophysiological properties of differentiated neurons of the vNTS. We speculate on a retrocontrol of cardiorespiratory signals in the vNTS, by Shh generated in dorsal vagal motoneurons.     

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).     

A hepatoportal osmoreceptive afferent projection from nucleus tractus solitarius to caudal ventrolateral medulla

This study was performed to examine projection of nucleus tractus solitarii (NTS) neurons to the caudal ventrolateral medulla (cVLM) in rats. One hundred and seven neurons that responded antidromically to electrical stimulation of the cVLM were recorded within the NTS. Electrical stimulation of the hepatic branch of the vagus nerve (hepatic vagus) elicited facilitation on 62 neurons (facilitatory neurons) and suppression on 6 neurons (suppressed neurons). Effect of portal infusion of hypertonic saline was examined on 44 facilitatory and 4 suppressed neurons. Twelve facilitatory and 2 suppressed neurons showed a decrease in the discharge rate. One suppressed neuron showed an increase in the discharge rate. It is concluded that hepatoportal osmoreceptive signals are conveyed from the NTS to the cVLM. The responses are mostly characterized by the decrease in the discharge rate by portal infusion of hypertonic saline.     

Extended secondhand tobacco smoke exposure induces plasticity in nucleus tractus solitarius second-order lung afferent neurons in young guinea pigs

Infants and young children experiencing extended exposure to secondhand smoke (SHS) have an increased occurrence of asthma, as well as increased cough, wheeze, mucus production and airway hyper-reactivity. Plasticity in lung reflex pathways has been implicated in causing these symptoms, as have changes in substance P-related mechanisms. Using whole-cell voltage-clamp recordings and immunohistochemistry in brainstem slices containing anatomically identified second-order lung afferent nucleus tractus solitarius (NTS) neurons, we determined whether extended SHS exposure during the equivalent period of human childhood modified evoked or spontaneous excitatory synaptic transmission, and whether those modifications were altered by endogenous substance P. SHS exposure enhanced evoked synaptic transmission between sensory afferents and the NTS second-order neurons by eliminating synaptic depression of evoked excitatory postsynaptic currents (eEPSCs), an effect reversed by the neurokinin-1-receptor antagonist (SR140333). The recruitment of substance P in enhancing evoked synaptic transmission was further supported by an increased number of substance P-expressing lung afferent central terminals synapsing onto the second-order lung afferent neurons. SHS exposure did not change background spontaneous EPSCs. The data suggest that substance P in the NTS augments evoked synaptic transmission of lung sensory input following extended exposure to a pollutant. The mechanism may help to explain some of the exaggerated respiratory responses of children exposed to SHS.     

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.     

Inhibitory effect of cholecystokinin octapeptide on neurons in the nucleus tractus solitarius

We investigated the effect of the cholecystokinin octapeptide (CCK8) applied locally to neurons of the nucleus tractus solitarius (NTS). Results demonstrate an inhibitory effect of CCK8 on spike discharges including those related to respiration. It is suggested that CCK8 acts at this level through specific receptor mechanisms since CCK8-induced inhibitions were not reproduced by application of related peptides and were resistant to antagonists of different inhibitory transmitters.     

A direct hepatic osmoreceptive afferent projection from nucleus tractus solitarius to dorsal hypothalamus

Thirty units that responded antidromically to electrical stimulation of the zona incerta (ZI) or the dorsal portion of hypothalamus were recorded in the nucleus tractus solitarii (NTS). These units were analyzed in relation to hepatoportal afferent inputs. Electrical stimulation of the hepatic branch of the vagus nerve facilitated six units (facilitatory units) and suppressed 10 units (suppressed units). Effect of the portal infusion of hypertonic saline was examined on six facilitatory and eight suppressed units. One facilitatory unit and one suppressed unit increased their discharge rates in response to portal infusion. Four facilitatory units and one suppressed unit decreased their discharge rates in response to the same stimulation. Increased or decreased discharge rates in response to portal infusion of hypertonic saline were observed in units that responded antidromically to electrical stimulation of the paraventricular hypothalamic nucleus, the lateral hypothalamic area, or the ZI. It is concluded that hepatoportal osmoreceptive signals are conveyed to the hypothalamus or the ZI directly from the NTS.     

Absence of respiration modulation of carotid sinus nerve inputs to nucleus tractus solitarius neurons receiving arterial chemoreceptor inputs

The reflex responses to activation of the arterial chemoreceptors are dependent upon when in the respiratory cycle the chemoreceptor stimulus is given. To determine if the respiratory modulation of the chemoreflex occurs within the nucleus tractus solitarius (NTS), intracellular recordings were obtained in pentobarbital-anesthetized, paralyzed and mechanically ventilated cats, from 22 non-respiratory NTS cells which were depolarized following activation of the ipsilateral carotid body chemoreceptors (by close arterial injection of <100 μ1 CO₂ saturated bicarbonate). Activation of the ipsilateral carotid body chemoreceptors evoked depolarizations with amplitudes of 2.9–4.6 mV and durations of 2.1–5.9 s. Three of these cells also received a convergent excitatory input from the carotid sinus baroreceptors. Carotid sinus nerve (CSN) stimulation evoked either an excitatory post-synaptic potential (EPSPs) (n = 14, 8 monosynaptic) or an excitatory/inhibitory sequence (EPSP/IPSPs) (n = 8, 1 monosynaptic). CSN evoked PSPs were separately averaged (25–50 sweeps) during periods of phrenic nerve activity and phrenic nerve silence and during periods when the lungs were inflated and when the lungs were deflated. No parameter of the CSN evoked PSPs (latency, peak amplitude, duration) was altered during periods of phrenic nerve activity or lung inilation (all P values > 0.12, Wilxocon signed-rank test). The results suggest that there is no respiratory modulation of arterial chemoreceptor inputs by either central respiratory drive or lung stretch receptor afferent inputs at this early stage of the reflex arc.     

The excitation by neurotensin of nucleus tractus solitarius neurons induces apneustic breathing

The possible involvement of neurotensin in the regulation of respiratory drive has been tested on single brainstem respiratory related neurons and on the global respiratory output. The neuropeptide was locally applied either by microiontophoresis or by pressure injection in the dorsal and ventral respiratory areas of the anesthetized bivagotomized cat. Effects of neurotensin applications were studied, on the one hand on the firing discharge of respiratory related neurons and on the other hand on the phrenic nerve activity and on arterial blood pressure. An increase of the firing frequency of respiratory related neurons was induced by neurotensin applied by iontophoresis or by pressure injection (0.005-33.5 fmol/s) on single neurons. In the latter case, neurotensin was active at concentration 10(3) times lower than glutamate. A bilateral apneustic pattern was induced on the phrenic nerve activities by microinjection of neurotensin (0.23-0.54 pmol/s) in one ventrolateral nucleus tractus solitarius without alteration of arterial blood pressure. These results suggest that the release of neurotensin in the nucleus tractus solitarius regulates respiratory rhythmogenesis by increasing the inspiratory duration.