Subpostremal Nucleus Tractus Solitarius Research Articles

Sympathetic and parasympathetic component of bradycardia triggered by stimulation of NTS P2X receptors

We have previously shown that activation of P2X purinoceptors in the subpostremal nucleus tractus solitarius (NTS) produces a rapid bradycardia and hypotension. This bradycardia could occur via sympathetic withdrawal, parasympathetic activation, or a combination of both mechanisms. Thus we investigated the relative roles of parasympathetic activation and sympathetic withdrawal in mediating this bradycardia in chloralose-urethane anesthetized male Sprague-Dawley rats. Microinjections of the selective P2X purinoceptor agonist alpha,beta-methylene ATP (25 pmol/50 nl and 100 pmol/50 nl) were made into the subpostremal NTS in control animals, after atenolol (2 mg/kg i.v.), a beta1-selective antagonist, and after atropine methyl bromide (2 mg/kg i.v.), a muscarinic receptor antagonist. The bradycardia observed with activation of P2X receptors at the low dose of the agonist is mediated almost entirely by sympathetic withdrawal. After beta1-adrenergic blockade, the bradycardia was reduced to just -5.1 +/- 0.5 versus -28.8 +/- 5.1 beats/min in intact animals. Muscarinic blockade did not produce any significant change in the bradycardic response at the low dose. At the high dose, both beta1-adrenergic blockade and muscarinic blockade attenuated the bradycardia similarly, -37.4 +/- 6.4 and -40.6 +/- 3.7 beats/min, respectively, compared with -88.0 +/- 11 beats/min in control animals. Double blockade of both beta1-adrenergic and muscarinic receptors virtually abolished the response (-2.5 +/- 0.8 beats/min). We conclude that the relative contributions of parasympathetic activation and sympathetic withdrawal are dependent on the extent of P2X receptor activation.

Electrophysiological evidence for the presence of NR2C subunits of N-methyl-D-aspartate receptors in rat neurons of the nucleus tractus solitarius

The nucleus tractus solitarius (NTS) plays an important role in the control of autonomic reflex functions. Glutamate, acting on N-methyl-D-aspartate (NMDA) and non-NMDA ionotropic receptors, is the major neurotransmitter in this nucleus, and the relative contribution of each receptor to signal transmission is unclear. We have examined NMDA excitatory postsynaptic currents (NMDA-EPSCs) in the subpostremal NTS using the whole cell patch clamp technique on a transverse brainstem slice preparation. The NMDA-EPSCs were evoked by stimulation of the solitary tract over a range of membrane potentials. The NMDA-EPSCs, isolated pharmacologically, presented the characteristic outward rectification and were completely blocked by 50 microM DL-2-amino-5-phosphonopentanoic acid. The I-V relationship of the NMDA response shows that current, with a mean (+/- SEM) amplitude of -41.2 +/- 5.5 pA, is present even at a holding potential of -60 mV, suggesting that the NMDA receptors are weakly blocked by extracellular Mg2+ at near resting membrane potentials. This weak block can also be inferred from the value of 0.67 +/- 0.17 for parameter delta obtained from a fit of the Woodhull equation to the I-V relationship. The maximal inward current measured on the I-V relationship was at -38.7 +/- 4.2 mV. The decay phase of the NMDA currents was fitted with one exponential function with a decay time constant of 239 +/- 51 and 418 +/- 80 ms at a holding potential of -60 and +50 mV, respectively, which became slower with depolarization (e-fold per 145 mV). The biophysical properties of the NMDA receptors observed in the present study suggest that these receptors in the NTS contain NR2C subunits and may contribute to the synaptic signal integration.

Neurotensin modulates synaptic transmission in the nucleus of the solitary tract of the rat

Whole-cell patch clamp recordings were made from neurons of the rat subpostremal region of the nucleus tractus solitarius (NTS) in transverse brainstem slices. Neurotensin (NT) enhanced the firing rate of action potentials from 0.8±0.4 Hz in control to 1.9±1.3 Hz ( n=9) and increased their decay time. The peak amplitude of the after-hyperpolarization was decreased by 34±5% ( n=9). These effects were associated with a depolarization of 4±1 mV ( n=10) in the resting membrane potential and an increase in the input resistance (from 768±220 MΩ to 986±220 MΩ; n=5) and were compensated by manually hyperpolarizing the cell to control values. In voltage clamp experiments NT decreased an outward current (from 488±161 to 340±96 pA at +40 mV; n=5) which reversed near the potassium equilibrium potential. In addition, NT increased the frequency of both excitatory and inhibitory spontaneous synaptic currents, an effect blocked by tetrodotoxin, and did not change the evoked excitatory or inhibitory postsynaptic currents. The selective NTR1 receptor antagonist SR48692 reversibly blocked the effects of NT on both action potentials and spontaneous synaptic currents. Our results suggest that NTR1 receptors can modulate post-synaptic responses in neurons of the subpostremal NTS by increasing cell excitability as a result of blockade of a potassium conductance.

Mechanisms mediating regional sympathoactivatory responses to stimulation of NTS A(1) adenosine receptors.

Selective activation of adenosine A(1) and A(2a) receptors in the subpostremal nucleus tractus solitarius (NTS) increases and decreases mean arterial pressure (MAP), respectively, and decreases heart rate (HR). We have previously shown that the decreases in MAP evoked by NTS A(2a) receptor stimulation were accompanied with differential sympathetic responses in renal (RSNA), lumbar (LSNA), and preganglionic adrenal sympathetic nerve activity (pre-ASNA). Therefore, now we investigated whether stimulation of NTS A(1) receptors via unilateral microinjection of N(6)-cyclopentyladenosine (CPA) elicits differential activation of the same sympathetic outputs in alpha-chloralose-urethane-anesthetized male Sprague-Dawley rats. CPA (0.33-330.0 pmol in 50 nl) evoked dose-dependent increases in MAP, variable decreases in HR, and differential increases in all recorded sympathetic outputs: upward arrow pre-ASNA >> upward arrow RSNA > or = upward arrow LSNA. Sinoaortic denervation + vagotomy abolished the MAP and LSNA responses, reversed the normal increases in RSNA into decreases, and significantly attenuated increases in pre-ASNA. NTS ionotropic glutamatergic receptor blockade with kynurenate sodium (4.4 nmol/100 nl) reversed the responses in MAP, LSNA, and RSNA and attenuated the responses in pre-ASNA. We conclude that afferent inputs and intact glutamatergic transmission in the NTS are necessary to mediate the pressor and differential sympathoactivatory responses to stimulation of NTS A(1) receptors.

On the immunohistochemical distribution of ionotropic P2X receptors in the nucleus tractus solitarius of the rat

ATP has been shown to excite neurones of the nucleus tractus solitarius (NTS) via the activation of P2X receptors. In the present study, the distribution of six P2X receptors (P2X 1–P2X 6) within the rat NTS was investigated by peroxidase immunohistochemistry. Immunopositive neurones for P2X receptor subtypes were detected in all divisions of the NTS, although the staining densities differed according to receptor subtype and sub-nuclei. P2X 1-immunopositive cells were distributed throughout the rostro-caudal extent of the NTS, while varicose fibres were mainly located along the postremal border. P2X 2 immunoreactivity was present in neurones and fibres located throughout the NTS. In the commissural NTS intense staining was observed medial of the solitary tract while in the sub-postremal NTS neurones were observed along the postremal border. A high density of P2X 3-positive neurones and fibres was observed in the sub-postremal NTS along the border of the area postrema and in the rostral NTS in the medial subdivision. In comparison to the staining observed with the other receptor antibodies, there was considerably reduced P2X 4 receptor immunoreactivity. P2X 4-positive neurones tended to be more sparsely distributed, and found mainly in the intermediate portion of the commissural NTS, and along the postremal border. In contrast, we observed dense staining for the P2X 5 receptor subtype in a majority of regions within the NTS. The most striking staining was observed in the intermediate subdivision at the level of the sub-postremal NTS and the medial portion of the rostral NTS. P2X 6 immunoreactive neurones were observed in the medial commissural NTS, along the postremal border and in the dorso-medial and medial subdivisions of the rostral NTS. Taken together, our findings confirm the presence of six P2X receptor subtypes within the NTS of the rat, consistent with a neurotransmitter role for ATP in the rat NTS. These results indicate the need for more extensive functional studies to elucidate the roles of the individual and heterodimeric assemblies of P2X receptor subtypes within the NTS.

Mechanisms mediating NTS P2x receptor-evoked hypotension: cardiac output vs. total peripheral resistance.

We have previously shown that P2x purinoceptor activation in the subpostremal nucleus tractus solitarius (NTS) produces dose-dependent decreases in mean arterial pressure (MAP), heart rate, efferent sympathetic nerve activity, and significant peripheral vasodilation. However, the relative roles of cardiac output (CO) and total peripheral resistance (TPR) in mediating this depressor response are unknown. Bradycardia does not necessarily result in decreased CO, because, with the greater filling time, stroke volume may increase such that CO may be unchanged. We measured changes in CO (via a chronically implanted flow probe on the ascending aorta) and MAP in alpha-chloralose- and urethane-anesthetized male Sprague-Dawley rats in response to microinjection of the selective P2x purinoceptor agonist alpha,beta-methylene ATP (25 and 100 pmol/50 nl) into the subpostremal NTS. TPR was calculated as MAP/CO. At the low dose of NTS P2x purinoceptor agonist, the reduction in MAP was primarily mediated by reductions in TPR (-31.3 +/- 3.3%), not CO (-8.7 +/- 1.7%). At the high dose, both CO (-34.4 +/- 6.6%) and TPR (-40.2 +/- 2.5%) contribute to the reduction in MAP. We conclude that the relative contribution of CO and TPR to the reduction in MAP evoked by NTS P2x purinoceptor activation is dependent on the extent of P2x purinoceptor activation.

Differential patterns of sympathetic responses to selective stimulation of nucleus tractus solitarius purinergic receptor subtypes.

1. Studies are described that indicate that stimulation of different purinergic receptor subtypes (A1, A2A and P2X) located in the sub-postremal nucleus tractus solitarius (NTS) evokes qualitatively and quantitatively different regional haemodynamic and efferent sympathetic responses. 2. Stimulation of A2A receptors evoked the most diverse pattern of regional sympathetic responses: preganglionic adrenal nerve activity (pre-ASNA) was increased, lumbar sympathetic nerve activity (LSNA) did not change, while renal (RSNA) and post-ganglionic adrenal (post-ASNA) sympathetic nerve activity was decreased. Stimulation of A1 receptors evoked qualitatively uniform, although quantitatively different, sympathoactivation: pre-ASNA > RSNA > LSNA. Stimulation of P2X receptors evoked qualitatively uniform, although quantitatively different, sympathoinhibition: RSNA=post-ASNA > LSNA = pre-ASNA. 3. These qualitatively and quantitatively different patterns of regional sympathetic responses strongly suggest that purinergic receptor subtypes may be specifically located and differentially expressed on NTS neurons/neural terminals that control different sympathetic outputs. Different NTS purinoceptors may contribute to patterned autonomic responses observed in specific physiological or pathological situations.

Characterisation of central adenosine A 1 receptors and adenosine transporters in mice lacking the adenosine A 2a receptor

The present study was designed to assess whether adenosine A 2a receptor knockout mice exhibit altered purine utilisation in brain nuclei. Specifically, the properties of adenosine transporters and adenosine A 1 receptors were characterised in brain membranes and on slide-mounted sections. The B MAX for [ 3H]nitrobenzylthioinosine ([ 3H]NBTI) binding (adenosine transporter density) was significantly reduced in brainstem membranes of homozygotes (560±52 fmol/mg protein, n=5, P<0.05, Kruskal–Wallis ANOVA) compared to wildtype (1239±213 fmol/mg protein) and heterozygous mice (1300±558 fmol/mg protein). Quantitative autoradiography data indicated that [ 3H]NBTI binding in the medulla oblongata of heterozygous mice was seen to decrease significantly ( P<0.05) in the subpostremal nucleus tractus solitarius (NTS), medial NTS, inferior olive and area postrema (AP). On the other hand, in the homozygous mice a decrease was seen in the medial NTS and AP. In the pons, [ 3H]1,3-dipropyl-8-cyclopentylxanthine ([ 3H]DPCPX) (adenosine A 1 receptor density) binding increased significantly ( P<0.05, Kruskal–Wallis ANOVA) in the lateral parabrachial nucleus, caudal pontine reticular nucleus and locus coeruleus of homozygotes compared to wildtype. In higher brain centres, [ 3H]NBTI binding was reduced in the paraventricular thalamic nucleus of both heterozygous and homozygous mice, whereas [ 3H]DPCPX binding was reduced in the hippocampus and lateral hypothalamus of heterozygotes. In homozygotes, [ 3H]DPCPX binding in the hippocampus increased compared to wildtype mice. The present study indicates that deletion of the A 2a receptor may have contributed to region-specific compensatory changes in purine utilisation in brain nuclei associated with autonomic, neuroendocrine and behavioural regulation.

Impaired arterial baroreflex regulation of heart rate after blockade of P 2-purinoceptors in the nucleus tractus solitarius

Activation of P 2x-purinoceptors in the nucleus tractus solitarius (NTS) via microinjection of ATP mimics baroreflex responses (bradycardia, hypotension); however, the physiological role of these receptors in cardiovascular control remains unclear. We tested whether blockade of these receptors attenuates arterial baroreflex control of heart rate (HR). Baroreflex-induced changes in HR (via graded i.v. infusion of phenylephrine and nitroprusside) were observed in seven α-chloralose/urethane anesthetized male Sprague-Dawley rats before and after microinjection of the purinergic P 2 receptor antagonist suramin (0.5 nmol in 50 nL) into the subpostremal NTS. Before suramin, typical baroreflex changes in HR were observed (maximum gain, G max = 2.94 ± 0.54 bpm/mmHg). Suramin markedly impaired baroreflex-induced changes in HR (gain = 0.02 ± 0.08 and 0.18 ± 0.09 bpm/mmHg for increases and decreases in mean arterial blood pressure, respectively); however, after 90–130 min, HR and baroreflex reactivity returned to control levels. Microinjections of vehicle into the same area did not alter baroreflex function. In addition, suramin did not alter the depressor responses to microinjections of glutamate into the same site of the NTS. We conclude that normal P 2x-purinoceptor function in subpostremal NTS may be necessary for baroreflex regulation of HR.

Activation of A 2a adenosine receptors in the nucleus tractus solitarius inhibits renal but not lumbar sympathetic nerve activity

The activation of adenosine A 2a receptors in the nucleus tractus solitarius (NTS) via microinjection of the selective agonist CGS 21680 elicits long-lasting, dose-dependent decreases in mean arterial pressure (MAP) and heart rate (HR) and preferential dilation of the iliac vascular bed in comparison to the renal and mesenteric vascular beds. We investigated whether differential changes in regional sympathetic output occur with A 2a receptor activation. In 24 chloralose/urethane anesthetized male Sprague–Dawley rats MAP, HR, renal (RSNA) and lumbar sympathetic nerve activity (LSNA) were recorded simultaneously. Data were analyzed as both the maximum decrease and the integral of the decrease over the duration of the depressor response. Microinjection of CGS 21680 (2 and 20 pmol in 50 nl volume) into the subpostremal NTS caused significant and dose-dependent decreases in MAP, HR and RSNA, however, did not significantly decrease LSNA in comparison to the effect of vehicle. Maximum responses of RSNA vs. LSNA in Δ% of control values were: −32±4 vs. −9±2, and −59±4 vs. −19±5 for low ( n=9) and high ( n=8) doses of CGS 21680 respectively; integral responses of RSNA vs. LSNA in Δ%×min were: −487±112 vs. −19±35 and −1258±164 vs. −175±126 for low and high doses of CGS 21680 respectively. Microinjections of vehicle ( n=7) did not alter integral hemodynamic or neural parameters. We conclude that activation of A 2a adenosine receptors in the NTS evokes differential changes in visceral vs. somatic sympathetic nerve activity which cannot explain differential vascular responses in terms of simple sympathetic withdrawal. Lack of significant inhibition of LSNA combined with preferential vasodilation in hindquarter vascular bed suggests that active vasodilation may be triggered by activation of A 2a adenosine receptors in the subpostremal NTS.

Purines and the nucleus tractus solitarius: effects on cardiovascular and respiratory function.

1. The roles of adenosine and adenosine 5'-triphosphate in cardiorespiratory regulation by the nucleus tractus solitarius (NTS) have been evaluated in a range of experiments, using micro-injections of selective agonists and antagonists of purinoceptors. 2. Adenosine injected into the caudal NTS decreases heart rate (HR), arterial blood pressure (BP) and respiratory frequency by an action at A2a receptors on glutamatergic nerve terminals. Microinjections of the A2a agonist CGS21680 caused falls in arterial BP and HR which were selectively antagonized by the A2a antagonist CGS15943. Injection of the A1 agonist CPA in the caudal NTS elevated BP and HR and its actions were selectively antagonized by the A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine. Adenosine enhanced glutamate release in the NTS. 3. In the rostral NTS, adenosine administration resulted in an elevation of BP. 4. ATP microinjection into the subpostremal NTS depressed HR and BP by an action on P2x purinoceptors, which are blocked by saramin. ATP microinjections differentially affected vascular conductance in regional peripheral vascular beds, with the most marked increases in conductance in the iliac bed and lesser effects in the superior mesenteric and renal vascular beds. 5. Recordings from renal and lumbar sympathetic nerves have demonstrated marked dose-dependent decreases in efferent activity following alpha, beta-methylene ATP injections into the NTS; however, only renal but not lumbar sympathetic nerve activity was inhibited following CGS21680 injections into the same site of the NTS. 6. ATP may function as a fast-acting neurotransmitter in the baroreceptor afferent pathway or in interneurons in the NTS. Adenosine is likely to play a role as a modulator of activity in baroreceptor and chemoreceptor pathways, fine tuning the functional output of both systems.

Activation of P 2x-purinoceptors in the nucleus tractus solitarius elicits differential inhibition of lumbar and renal sympathetic nerve activity

Activation of P 2x-purinoceptors in the nucleus tractus solitarius (NTS) via microinjection of α, β-methylene ATP ( α, β-MeATP) elicits large dose-dependent decreases in mean arterial pressure (MAP) and heart rate (HR) and preferential dilation of the iliac vascular bed in comparison to renal and mesenteric vascular beds. We investigated whether sympathoinhibition contributes to the depressor responses and whether differential changes in regional sympathetic output occur. In 43 chloralose/urethane anesthetized male Sprague–Dawley rats, MAP, HR, renal (RSNA) and lumbar sympathetic nerve activity (LSNA) were recorded. Data were analyzed as both the maximum decrease and the integral of the decrease over the duration of the depressor response. Microinjection of α, β-MeATP (25 and 100 pmol in 50 nl volume) into the subpostremal NTS caused significant and dose-dependent decreases in MAP, HR, RSNA and LSNA. However, the changes in RSNA were significantly greater than those observed in LSNA for both doses and both methods of analysis of data (maximum responses in Δ%: 84±3 vs 62±4, and 93±3 vs 74±4 for low and high dose of α, β-MeATP, respectively; integral responses in Δ%×min: 32±4 vs 18±3 and 179±7 vs 134±14 for low and high dose of α, β-MeATP, respectively). Blockade of P 2-purinoceptors in the NTS by the specific P 2-receptor antagonist suramin abolished responses to 100 pmol α, β-MeATP and microinjections of vehicle did not alter neural nor hemodynamic parameters. We conclude that activation of P 2x-purinoceptors in the NTS inhibits sympathetic nerve activity and evokes differential regional sympathetic responses. However, differential sympathoinhibition does not explain differential vascular responses to the activation of P 2x-purinoceptors in the NTS.

Activation of P 2-purinoceptors in the nucleus tractus solitarius mediate depressor responses

The purpose of the study was to examine the role of P 2 purinergic receptors in mechanisms of cardiovascular control mediated by the nucleus tractus solitarius (NTS), a major integrative site in the brainstem involved in the reflex coordination of cardiorespiratory and visceral response patterns. Microinjections of ATP and its analogues were made into the subpostremal NTS of anesthetized, spontaneously breathing rats. ATP, α,β-methylene ATP (αβ-meATP) and 2-methylthio-ATP (2-meSATP) produced significant dose-related reductions in arterial blood pressure. αβ-meATP was slightly more potent than ATP and 2-meSATP. Pretreatment with the P 2 receptor antagonist, suramin (0.5 nmol/rat), into the same NTS site 10 min prior to agonist administration completely blocked pronounced depressor response pattern elicited by the highest dose of αβ-meATP (0.1 nmol/rat). The present findings suggest that endogenous ATP may serve as a fast transmitter substance in NTS-mediated mechanisms of cardiovascular control.

Visceroendocrine responses elicited by neuropeptide Y in the nucleus tractus solitarius

Neuropeptide Y (NPY) has been shown to be localized in a number of CNS regions, including the nucleus tractus solitarius (NTS). In this meeting report, a brief overview is presented of recent studies from our laboratory examining the role of NPY in NTS-mediated mechanisms of cardiorespiratory and Visceroendocrine regulation. Microinjections of NPY, NPY analogs, or C-terminal NPY fragments were made into the subpostremal NTS of anesthetized spontaneously breathing rats. NPY elicited pronounced dose-related depressor responses, bradycardia, and reductions in respiratory minute volume. The overall cardiorespiratory response pattern elicited by NPY was mimicked by NPY 13/23;6, a fragment of NPY exhibiting selective agonist properties at presynaptic Y 2 receptors, whereas the y 1 receptor-selective analog, [Leu 31,Pro 34]NPY, and the C-terminal inactive fragment, NPY 26–36, were found to be ineffective. In an effort to further characterize intrinsic NTS mechanisms mediating the NPY-evoked response pattern, NPY microinjections were similarly made in a group of rats with bilateral glossopharyngeovagotomy (G-vagotomy) and in a group of rats decerebrated at the supracollicular level. The results showed that whereas decerebration did not appreciably affect the NTS-mediated cardiorespiratory responses elicited by NPY. G-vagotomy enhanced the NPY-evoked hypotension while at the same time abolishing the NPY-evoked bradycardia and reductions in tidal volume. Taken together, these observations with G-vagotomized animals, along with the results from microinjection studies using selective ligands for NPY receptors, suggest that NPY may modulate primary visceral afferent information via activation of Y 2 receptors distributed at presynaptic sites in the subpostremal NTS. On the other hand, the influence of reciprocal NPY-containing neuronal connections with rostral brain regions on NTS-mediated cardiorespiratory responses elicited by NPY appeared not to be consequential, at least in normal rats. However, when similar microinjections of NPY were made into the same subpostremal NTS sites of intact rats which were diabetic, there was a dramatic attenuation in cardiorespiratory responsiveness to NPY. These results suggest that NPY-sensitive mechanisms in the NTS mediating cardiorespiratory responses may conceivably be influenced by the increased hypothalamic NPYergic activity that has been observed in diabetic rats. These observations, together with recent findings from our laboratory showing that microinjection of NPY into the same subpostremal NTS sites potently stimulates insulin secretion, provide support for the notion that NPY may play a crucial modulatory role in NTS-mediated Visceroendocrine response patterns involved in cardiorespiratory control and nutrient homeostasis.

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.