Medial Nucleus Tractus Solitarius Research Articles

Distribution of nitric oxide synthase in rat dorsal vagal complex and effects of microinjection of nitric oxide compounds upon gastric motor function.

Nitric oxide (NO) has received attention as a vagal nonadrenergic-noncholinergic (NANC) mediator of gastrointestinal relaxation. The dorsal vagal complex (DVC) is the primary hindbrain site of vagal control of the gastrointestinal tract, and yet the subnuclear distribution of NO and its physiological effects have not been analyzed in this nucleus. Therefore, this study estimates the relative number of NO synthase (NOS)-containing neurons in subnuclear regions of the DVC, identifies NOS-containing vagal abdominal preganglionic neurons in the dorsal motor nucleus of the vagus, and defines a role of NO in the DVC in control of gastric motor function. The location of NADPH-diaphorase-positive staining (a marker of NOS activity) and NOS immunoreactivity overlap in the DVC. In the dorsal motor nucleus of the vagus there are positively stained cells caudal to the obex and at its most rostral extent, but not at the intermediate level. Intraperitoneal fluorogold combined with NADPH-diaphorase activity labels approximately 5% and 15% of fluorogold-immunoreactive cells in the caudal and rostral dorsal motor nucleus of the vagus, respectively. Thus, a portion of NOS-containing neurons are preganglionic vagal neurons projecting to the abdominal viscera. In the nucleus tractus solitarius, the majority of NADPH-diaphorase-positive cells are within the centralis, medial, and ventral/ventrolateral subnuclei. Fiber/terminal staining is present in the subnucleus centralis, subnucleus gelatinosus, subpostremal zone, and the medial nucleus tractus solitarius. The presence of NOS terminal staining implicates NO in afferent control of gastric function in the DVC (e.g., vago-vagal circuits in subnucleus gelatinosus). To determine a role of NO in the DVC, NO-related agents were microinjected into the DVC in alpha-chloralose-anesthetized rats while recording indices of gastric motor function. L-Arginine, microinjected into the DVC, significantly decreases intragastric pressure (-2.2 +/- 0.4 cm2, N = 12), and this effect is abolished by vagotomy. Microinjection of an NOS inhibitor, NG-nitro-L-arginine methyl ester, increases intragastric pressure (1.9 +/- 0.7 cm2, N = 10), with the greatest effect in the DVC rostral to the obex. Overall, it was concluded that tonic release of NO in the DVC mediates gastric relaxation, at least in anesthetized animals, and NOS-containing preganglionic neurons in the dorsal motor nucleus of the vagus may be "command" NANC neurons which control a variety of gastrointestinal functions.

Functional GABAA receptors on rat vagal afferent neurones.

1. In the present study, in vitro electrophysiology and receptor autoradiography were used to determine whether rat vagal afferent neurones possess gamma-aminobutyric acid (GABA)A receptors. 2. GABA (1-100 microM) and isoguvacine (3-100 microM) caused a concentration-dependent depolarization of the rat isolated nodose ganglion preparation at room temperature. When applied to the tissue 20 min before the agonist, SR95531 (3 microM) and bicuculline (3 microM) caused a parallel shift to the right of the GABA and isoguvacine concentration-response curves, yielding shifts of 81 fold and 117 fold for SR95531 and 4 fold and 12 fold for bicuculline, respectively. 3. Baclofen (10 nM-100 microM) was unable to elicit a depolarization of the rat isolated nodose ganglion preparation at either room temperature or at 36 degrees C, whilst 5-aminovaleric acid (10 microM), a GABAB receptor antagonist, was unable to antagonize significantly the GABA-induced depolarization at either room temperature or at 36 degrees C. 4. [3H]-SR95531 (7.2 nM), a GABAA receptor-selective antagonist, bound topographically to sections of rat brainstem. Specific binding was highest in the medial nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus nerve (DMVN). Binding was also observed in certain medullary reticular nuclei, in particular the parvocellular reticular nucleus. 5. Unilateral nodose ganglionectomy caused a reduction in GABAA binding site density in the medial NTS from 93 +/- 7 to 68 +/- 6 d.p.m./mm2. This procedure also caused a reduction in GABAA binding site density in the side of the NTS contralateral to the lesion, from 151 +/- 12 to 93 +/- 7 d.p.m./mm2. Sham surgery had no effect on the binding of [3H]-SR95531 in rat brainstem. 6. The present data provide evidence for the presence of GABAA receptors located on the soma and central terminals of rat vagal afferent neurones. Additionally, a population of GABAA receptors is evidenced postsynaptically in the rat NTS with respect to vagal afferent terminals. These data are discussed in relation to the functional pharmacology of GABA in this region of the NTS.

Is L-Dopa a Neurotransmitter of the Primary Baroreceptor Afferents Terminating in the Nucleus Tractus Solitarii of Rats?

Transmitter-like Dihydroxyphenylalanine (DOPA) is released from rat striata and blood pressure regulation centers in the lower brain stem. Exogenous DOPA itself elicits in vitro presynaptic and in vivo postsynaptic responses. All are stereoselective, and most are antagonized by DOPA methyl ester, a competitive DOPA antagonist. DOPA does not displace selective binding of α 2 , β , D 1 , and D 2 ligands in brain membrane preparations. It is possible to cooperate for effectiveness in Parkinson's disease. Meanwhile, DOPA induces DOPA ester-sensitive neuronal glutamate release from slices, even under inhibition of aromatic L-amino acid decarboxylase (AADC). The release by baroreceptor stimulation with phenylephrine has been abolished by bilateral sinoaortic denervation without modification of hypertension. DOPA microinjected into depressor sites of the medial nucleus tractus solitarii (NTS) identified by prior glutamate leads to dose-dependent decreases in blood pressure and heart rate (HR) in untreated rats and in those treated with central AADC inhibitor. The discussion also includes interactions between DOPAergic and GABAergic systems. GABA is an inhibitory neuromodulator for baroreflex at the level of NTS. GABA functions tonically via GABA A receptors to elicit increases in blood pressure and HR and to inhibit decreases in those by electrical aortic nerve stimulation. The third topic under discussion in this chapter is altered functions of the DOPA system in the NTS of adult spontaneously hypertensive rats (SHR). Basal DOPA release is lower in SHR than age-matched Wistar-Kyoto (WKY) rats. This release has been reduced by tetrodotoxin (TTX) to the same absolute levels in the two strains.

Presynaptic or postsynaptic location of receptors for angiotensin II and substance P in the medial solitary tract nucleus.

1. Microinjection of angiotensin (Ang) II or substance P (SP) into the medial nucleus tractus solitarii (nTS) produces similar decreases in arterial pressure and heart rate. We previously reported that some medial nTS neurons responsive to SP were also excited by Ang II, and that Ang II increased the release of SP from medulla slices. Both electrophysiological and anatomic data suggest that the cardiovascular effects of these peptides may be mediated by a common neuronal pathway consisting of SP-containing vagal afferent fibers with presynaptic Ang II receptors that innervate medial nTS neurons with SP receptors. To evaluate the validity of this model, we established the presynaptic or postsynaptic location of the receptors for Ang II and SP that mediate excitation of medial nTS neurons by determining the capacity of each peptide to activate the cell before and after blocking synaptic transmission in rat dorsal medulla slices. 2. Extracellular recordings were obtained from 55 medial nTS neurons responsive to Ang II or SP in 400-microns horizontal slices of the dorsal medulla. Neuronal excitation by Ang II and SP was tested before, during, and after reversal of synaptic blockade with low-Ca2+ (0.2 mM), high Mg2+ (5 mM) artificial cerebrospinal fluid (aCSF). Elimination of synaptically evoked short latency responses of the neuron to current pulses applied to afferent fibers in the solitary tract (TS) documented blockade of synaptic transmission by low-Ca2+ aCSF. In most cases, the basal firing rate of the cell increased slowly during perfusion with low-Ca2+ aCSF and stabilized after approximately 30 min at a higher level of spontaneous activity. Responses to the peptides and TS stimulation were also documented after synaptic blockade had been reversed by adding aCSF containing 2-mM Ca2+. 3. Of the 55 medial nTS neurons, 41 were responsive to Ang II; whereas, 50 of the 55 cells were responsive to SP. The neurons were divided into three subgroups on the basis of their responsiveness to Ang II and SP. Although most neurons were responsive to both Ang II and SP (n = 36), five other cells were excited only by Ang II, and 14 neurons were activated only by SP. Of the 55 neurons, 26 were also responsive to L-glutamate: 14 of 17 cells responsive to both Ang II and SP, all 5 neurons excited by Ang II but not by SP, and 7 of 10 neurons responsive only to SP were also excited by L-glutamate. The latency of the action potentials evoked by TS stimulation was much shorter in those neurons responsive only to Ang II (3.6 ms) than in cells excited by both Ang II and SP (6.8 ms) or responsive only to SP (7.4 ms). 4. In 21 of the 36 medial nTS neurons responsive to both Ang II and SP, Ang II continued to excite the cell when synaptic responses to TS stimulation were prevented by low-Ca2+ aCSF, but had no effect on the firing rate of the other 15 neurons during synaptic blockade. Excitation induced by Ang II was also prevented in two of the five medial nTS neurons responsive only to Ang II when synaptic transmission in the slice was blocked. Low-Ca2+ aCSF failed to prevent excitation by SP or L-glutamate in all medial nTS cells responsive to these agonists (n = 50 and n = 26, respectively). In contrast to these observations in medial nTS neurons, Ang II-induced excitation was not altered during synaptic blockade in any of the six dmnX cells studied. No responses to SP or L-glutamate were blocked in dmnX neurons, as also seen in the medial nTS. 5. When all medial nTS neurons responsive to Ang II were examined, the latencies of the response to TS stimulation were significantly shorter in those neurons with presynaptic Ang II receptors than in the group of cells with postsynaptic receptors. In addition, neurons with presynaptic Ang II receptors were distributed differently within the medial nTS than cells with postsynaptic Ang II receptors.(ABSTRACT TRUNCATED)

Repetitively firing neurons in the rat medial nucleus tractus solitarius is involved in cholecystokinin type-A receptor-mediated functions

Repetitively firing neurons in the rat medial nucleus tractus solitarius is involved in cholecystokinin type-A receptor-mediated functions

Afferent synaptic drive of rat medial nucleus tractus solitarius neurons: dynamic simulation of graded vesicular mobilization, release, and non-NMDA receptor kinetics.

1. We have developed a comprehensive mathematical model of an afferent synaptic connection to the soma of a medial nucleus tractus solitarius (mNTS) neuron. Model development is based on numerical fits to quantitative data recorded in our laboratory. This work is part of a continuing collaborative effort aimed at identifying and characterizing the mechanisms responsible for the non-linear integrative properties of this first synapse in the baroreceptor reflex. 2. The complete model consists of three major parts: 1) a Hodgkin-Huxley (HH)-type membrane model of the prejunctional sensory terminal bouton; 2) a multistage model describing vesicular storage, adenosine 3',5'-cyclic monophosphate (cAMP)- and Ca(2+)-dependent mobilization, release and recycling; and 3) a HH-type membrane model of the postjunctional mNTS cell that includes descriptions for a desensitizing non-N-methyl-D-aspartate (NMDA) ionic current that is responsible for the fast excitatory postsynaptic potentials (EPSPs) observed in mNTS cells. The membrane models for both the terminal bouton and the mNTS neuron are coupled to separate lumped fluid compartment models describing intracellular Ca2+ ion concentration dynamics. 3. Our modeling strategy is twofold. The first is to validate model performance by reproducing a wide variety of experimental data both from our laboratory and from the literature. The second is to explore the functional aspects of the model in order to gain a greater appreciation for the balance between presynaptic mechanisms (e.g., terminal membrane properties and vesicular dynamics) and postsynaptic mechanisms (e.g., non-NMDA receptor kinetics and neuronal dynamics) that underlie the afferent synaptic drive of mNTS neurons. 4. The model accurately reproduces EPSP dynamics recorded with the use of a wide range of stimulus protocols. The model can also mirror the unique pattern of graded frequency- and use-dependent reduction in peak EPSP magnitude observed experimentally through 60 s of constant, suprathreshold synaptic activation. We demonstrate how vesicular mobilization, recycling, and receptor kinetics can function synergistically in establishing synaptic transfer. Furthermore, we show that by allowing the aggregate rate of vesicle mobilization to respond in a use-dependent manner, it is possible to compensate for the attenuating affects of desensitization at elevated rates of stimulation. 5. Our simulations indicate that the low-frequency characteristics of this synapse are dominated by vesicular dynamics, whereas the high-frequency properties arise from a combination of Ca(2+)-dependent vesicular mobilization and the kinetics of the non-NMDA receptor. Desensitization can influence the peak magnitude and decay time of the EPSP, thereby affecting synaptic throughput. However, we demonstrate that, as the time course of neurotransmitter in the synaptic cleft decreases, the influence of desensitization should be somewhat diminished. As a result, the effective bandwidth of the synapse increases and becomes limited by the gating characteristics of the non-NMDA channel. 6. The model also includes a neuromodulatory aspect in that the frequency response of the synapse can be modulated by an adenylate cyclase-mediated regulatory mechanism. Although our simulations indicate the behavior of a limited number of possible neuromodulatory agents, the results demonstrate the pivotal role such agents could play in modifying synaptic transfer characteristics presynaptically. 7. Both continuous and burst-mode tract stimulation evoke patterns of action potentials in spontaneously active mNTS neurons that are mimicked very well by our model. Our simulations demonstrate that, as the rate of stimulation increases beyond approximately 20-30 Hz, the inherent low-pass frequency-response characteristics of the synapse limit the overall dynamic range of the mNTS neuron, causing the postsynaptic cell to "entrain" at frequencies within its normal operating range.

Dynamics of sensory afferent synaptic transmission in aortic baroreceptor regions on nucleus tractus solitarius.

1. Synaptic responses of medial nucleus tractus solitarius (mNTS) neurons to solitary tract (ST) activation were studied in a horizontal brain slice preparation of the rat medulla. Slices included sections of ST sufficiently long that the ST could be electrically activated several millimeters from the recording site of cell bodies in mNTS. 2. Three types of synaptic events were evoked in response to ST stimulation: simple excitatory postsynaptic potentials (EPSPs), simple inhibitory postsynaptic potentials (IPSPs), and complex EPSP-IPSP sequences. Simple EPSPs had substantially shorter latencies than IPSPs (3.39 +/- 0.65 ms, mean +/- SE, n = 42, vs. 5.86 +/- 0.71 ms, n = 6, respectively). 3. EPSP amplitude increased linearly with increasing hyperpolarization, with an extrapolated reversal potential near 0 mV. 4. EPSPs were maximal at < 0.5 Hz of sustained, constant-frequency ST stimulation (n = 14). EPSP amplitude declined to an average of 57.5% of control at 10 Hz after 2 s of sustained stimulation. With 1 min of sustained, 100-Hz stimulation, EPSP amplitude declined to near zero. 5. With stimuli intermittently delivered as 100-ms bursts every 300 ms, generally comparable average EPSPs were evoked during constant and burst patterns of ST stimulation. The amplitude of the initial EPSP in each burst was very well maintained even at intraburst stimulation rates of 100 Hz. 6. At resting membrane potentials, low constant frequencies of ST stimulation (< 5 Hz) reliably elicited action potentials and suppressed spontaneous spiking, but higher frequencies led to spike failures (> 85% at 100 Hz). Between 5 and 10 Hz, this periodic stimulation-suppression cycle clearly entrained action potential activity to the ST stimuli. Similar patterns of current pulses (5 ms) reliably evoked action potentials with each pulse to higher frequencies (50 Hz) without failures, and entrainment was similar to ST stimulation. 7. In a subset of nucleus tractus solitarius (NTS) neurons (3 of 9 studied), bursts of ST stimuli were as much as 50% more effective at transmitting high frequencies (> 10 Hz) of ST stimulation than the equivalent constant frequencies (P < 0.0001). 8. The long-latency simple IPSPs with no preceding EPSPs reversed to become depolarizing at potentials more negative than -62.9 +/- 7.0 mV (n = 5) and were blocked by the non-N-methyl-D-aspartate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (n = 3). The ST stimulation frequency-response relation of these IPSPs was similar to that for the short-latency EPSP response excited by ST synapses. Thus these IPSPs appear to be activated polysynaptically via a glutamatergic-GABAergic sequence in response to ST activation. 9. The results suggest that sensory afferent synapses in mNTS have limited transmission of high-frequency inputs. Both synaptic transmission and the characteristics of the postsynaptic neuron importantly contribute to the action potential transmission from afferent to NTS neuron and beyond. This overall frequency response limitation may contribute to the accommodation of reflex responses from sensory afferent inputs such as arterial baroreceptors within their physiological discharge frequency range.

5-Hydroxytryptamine 3 receptor modulation of excitatory amino acid release in the rat nucleus tractus solitarius

In vivo microdialysis was employed to measure release of endogenous l-glutamate (GLU) and l-aspartate (ASP) in the medial nucleus tractus solitarius of urethane anaesthetised rats. Basal extracellular levels of these amino acids were stable following a 90 min equilibration period (6.3 ± 0.24 and 3.4 ± 0.6 pmol/20 μl sample of GGLU and ASP, respectively). Basal levels of endogenous extracellular GLU and ASP were increased over 2-fold and 3-fold, respectively, following local administration of the selective 5-hydroxytryptamine (5-HT 3) receptor agonist phenylbiguanide (300 μM). Intracerebral administration of the selective 5-HT 3 receptor antagonist ondansetron (30 μM) blocked the effect of phenylbiguanide on GLU release whilst the effect on ASP was variable and complex. These data suggest that 5-HT 3 receptor activation in the rat nucleus tractus solitarius can affect excitatory amino acid neurotransmission in this region of the medulla oblongata.

Cardiovascular effects of NMDA and MK-801 infusion at area postrema and mNTS in rat

In urethane-anesthetized male Sprague-Dawley rats, microinfusion of N- methyl- D -aspartate (NMDA) into the area postrema (AP) at the dose of 10 ng produced significant decreases in mean arterial pressure (MAP) (−26 ± 5 mmHg), heart rate (HR) (−34 ± 6 bpm), renal blood flow, mesenteric blood flow, and iliac vascular resistance. In addition, microinfusion of the same dosage of NMDA into the medial nucleus tractus solitarius (mNTS) produced significant decreases in MAP (−33 ± 4 mmHg), HR (−33 ± 6 bpm), renal blood flow, mesenteric blood flow and vascular resistance, and iliac blood flow and resistance. MK-801 (dizocilpine) microinfusion alone produced no significant changes in MAP or HR when microinfused either into the AP or unilaterally into the mNTS; however, bilateral microinfusion of MK-801 into mNTS produced sustained hypertension and tachycardia, lasting about 30 min. MK-801 pretreatment at both AP and mNTS effectively blocked NMDA-induced cardiovascular responses. MK-801 microinfusion at AP significantly attenuated baroreceptor reflex-mediated bradycardia elicited by intravenous injection of phenylephrine, but did not alter reflex tachycardia elicited by intravenous nitroprusside. In conclusion, NMDA receptor-mediated neurotransmission is involved in the cardiovascular functions of both AP and mNTS. Both loci appear to be sites of action for mK-801.

Mechanisms of angiotensin-induced hypotension and bradycardia in the medial solitary tract nucleus

The selective angiotensin (ANG) II antagonists losartan (AT1) and CGP-42112A (AT2) were used to determine the receptor subtype and neuronal pathways that mediate the hypotension and bradycardia produced by 200 fmol of ANG II microinjected into the dorsal medial nucleus tractus solitarii (NTS) or dorsal motor nucleus of the vagus (dmnX) in anesthetized rats. At dorsal medial NTS sites (0.3 mm below the surface) where L-glutamate microinjections produced maximal decreases in mean arterial pressure (MAP) and heart rate (HR), ANG II (200 fmol, 50 nl, n = 16) elicited hypotension (-22 +/- 1 mmHg) and bradycardia (-26 +/- 2 beats/min). Although L-glutamate also suppressed respiration, ANG II injections in the medial NTS did not alter respiration. Losartan injected at the medial NTS site caused a dose-dependent reduction of ANG II-induced decreases in MAP and HR. At 2 pmol, the AT1 antagonist attenuated the response to ANG II, whereas 100 pmol abolished the effects of ANG II microinjections. In contrast, the AT2 antagonist CGP-42112A (100 pmol) had no effect on the responses to ANG II. Neither ANG II antagonist altered the cardiovascular effects of L-glutamate injections. Losartan injected into the dmnX blocked hypotension and bradycardia produced by ANG II at that site but did not prevent responses to subsequent ANG II injections in the medial NTS.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of area postrema by vasopressin and angiotensin II modulates neuronal activity in the nucleus tractus solitarius

There is an abundance of evidence suggesting that the area postrema (AP) is involved in the central actions of argininevasopressin (AVP) and angiotensin II (Ang II) on cardiovascular regulation. Furthermore, recent studies have shown that activation of the AP facililates the response of nucleus tractus solitarius (NTS) neurons to tractus stimulation. In the present study, using the perfused rabbit brain slice preparation, we examined the response of NTS neurons when AVP and Ang II were microinjected onto the AP. Spontaneous or solitary tract stimulation-induced neuronal activity was recorded extracellularly from the medial NTS before, during and after AVP or Ang II application. An increase or decrease in activity by more than 30% of the baseline value was considered excitatory or inhibitory. The effects of AVP were studied in 57 NTS cells, 14 of which were spontaneously active and 43 were driven by tract stimulation. Of the cells with evoked activity, 49% were excited, 19% were inhibited, and 32% did not respond. The percentage of cells responding to AVP was similar in spontaneously active cells. The effects of Ang II were tested in 85 cells including 54 with evoked activity and 31 with spontaneous activity. In NTS cells with evoked activity, AP application of Ang II caused inhibition in 37%, excitation in 7%, while 56% did not respond. The proportion of cells responding to Ang II was similar in spontaneously active cells. These results suggest that AVP may act on the AP to increase the excitatory response of NTS neurons while the actions of Ang II result in an inhibitory influence.

A Model of Synaptic Coupling Between Sensory Afferent and Medial Nucleus Tractus Solitarius Neurons in the Rat

The electrophysiological properties of the monosynaptic junction between sensory neurons and cells within a restricted region of the medial nucleus tractus solitarius (mNTS) have been studied using a brainstem slice preparation. These investigations have enabled us to develop a mathematical model of a sensory afferent synaptic connection to a mNTS neuron. Model development is based upon numerical fits to quantitative data recorded in our laboratory. This work is part of a continuing integrative effort aimed at identifying and characterizing the fundamental electrophysiological properties of this first synapse in the baroreceptor reflex.

L-glutamate as a neurotransmitter at baroreceptor afferents: Evidence from in vivo microdialysis

In vivo microdialysis was employed to measure release of endogenous l-glutamate and l-aspartate in the region of the dorsomedial medulla oblongata including the medial nucleus tractus solitarius of the anaesthetized rat. Basal extracellular levels of these amino acids were stable and increased over two-fold when the perfusate was changed to a high KCl (80 mM) artificial cerebrospinal fluid. This high K +-evoked release was calcium-dependent, while basal levels were insensitive to removal of calcium ions from the perfusate. An intravenous infusion of phenylephrine, which elevated blood pressure, caused a marked increase of both spontaneous and evoked release of glutamate. In contrast aspartate efflux was not significantly altered. The present data provide evidence that the excitatory amino acids, glutamate and aspartate, serve a neurotransmitter function in the nucleus tractus solitarius of the rat. Furthermore, the increase in glutamate release following baroreceptor activation with phenylephrine suggests that glutamate may be a neurotransmitter at baroreceptor afferent nerve terminals within the nucleus tractus solitarius. On the other hand, aspartate appears to be possibly an inter-neuronal transmitter in this brain region.

Morphology and projections of neurobiotin-labeled nucleus tractus solitarii neurons recorded in vitro

The suitability of the anterograde tracer neurobiotin to provide information about the morphology and projections of extracellularly or intracellularly recorded medial nucleus tractus solitarii (nTS) neurons was evaluated in horizontally oriented rat dorsal medulla in vitro slices. After responsiveness to angiotensin (Ang) II, substance P (SP), and l-glutamate was evaluated, neurons were labeled by electrophoresis of neurobiotin at the recording site. Extracellular application (2 μA for 2 min) produced discrete injection sites (40–70 μm) with a small group of labeled neurons. Ejections into the solitary tract documented that the tracer was not taken up by axons traversing the injection site. Neuronal perikarya, primary and secondary dendrites, and axons exhibited a dense Golgi-like appearance, with well-defined dendritic spines and axonal varicosities. Dendritic or axonal processes could be followed for more than 1 mm from the cell soma in a 50 μm thick section, documenting the horizontal architecture of the medial nTS. Intracellular electrophoresis filled the soma, primary and secondary dendrites, and axon of neurons characterized for responsiveness to peptides, l-glutamate and solitary tract stimulation. The location within the nTS and axonal projections of neurons responsive to Ang II and SP appeared to differ from those of cells responsive to Ang II and l-glutamate. Thus, either extracellular or intracellular application of neurobiotin in the in vitro slice can reveal differences in axonal or dendritic targets of neuronal subgroups responsive to different neurotransmitters or peptides and provide evidence for the likely autonomie significance of the neurons.

An ionic current model for neurons in the rat medial nucleus tractus solitarii receiving sensory afferent input.

1. Neurons from a horizontal slice of adult rat brainstem were examined using intracellular recording techniques. Investigations were restricted to a region within the nucleus tractus solitarii, medial to the solitary tract and centred on the obex (mNTS). Previous work has shown this restricted area of the NTS to contain the greatest concentration of aortic afferent baroreceptor terminal fields. Electrical stimulation of the tract elicited short-latency excitatory postsynaptic potentials in all neurons. 2. mNTS neurons were spontaneously active with firing frequencies ranging between 1 and 10 Hz, at resting potentials of -65 to -45 mV. These neurons did not exhibit spontaneous bursting activity. 3. Depolarizing current injection immediately evoked a finite, high-frequency spike discharge which rapidly declined to a lower steady-state level (i.e. spike frequency adaptation, SFA). Increasing depolarizations produced a marked increase in the peak instantaneous frequency but a much smaller increase in the steady-state firing level. 4. Conditioning with a hyperpolarizing prepulse resulted in a prolonged delay of up to 600 ms before the first action potential (i.e. delayed excitation, DE) with an attendant decrease in peak discharge rates. DE was modulated by both the magnitude and duration of the prestimulus hyperpolarization, as well as the magnitude of the depolarizing stimulus. Tetrodotoxin (TTX) eliminated spike discharge but had little effect on the ramp-like membrane depolarization characteristic of DE. 5. We have developed a mathematical model for mNTS neurons to facilitate our understanding of the interplay between the underlying ionic currents. It consists of a comprehensive membrane model of the Hodgkin-Huxley type coupled with a fluid compartment model describing cytoplasmic [Ca2+]i homeostasis. 6. The model suggests that (a) SFA is caused by an increase in [Ca2+]i which activates the outward K+ current, IK,Ca, and (b) DE results from the competitive interaction between the injected depolarizing current and the hyperpolarization-activated transient outward K+ currents, IA and ID. 7. We conclude that our ionic current model is capable of providing biophysical explanations for a number of phenomena associated with brainstem neurons, either during spontaneous activity or in response to patterned injections of current. This model is a potentially useful adjunct for on-going research into the central mechanisms involved in the regulation of both blood pressure and ventilation.

CNS innervation of airway-related parasympathetic preganglionic neurons: a transneuronal labeling study using pseudorabies virus

The CNS cell groups that innervate the tracheal parasympathetic preganglionic neurons were identified by the viral retrograde transneuronal labeling method. Pseudorabies virus (PRV) was injected into the tracheal wall of C8 spinal rats and after 4 days survival, brain tissue sections from these animals were processed for immunohistochemical detection of PRV. Retrogradely labeled parasympathetic preganglionic neurons were seen in three sites in the medulla: the compact portion of the nucleus ambiguus, the area ventral to the nucleus ambiguus, and the rostralmost portion of the medial nucleus tractus solitarius (NTS); this labeling pattern correlated well with the retrograde cell body labeling seen following cholera toxin ß-subunit injections in the tracheal wall. PRV transneuronally labeled neurons were found throughout the CNS with the most abundant labeling concentrated in the ventral medulla oblongata. Labeled neurons were identified along the ventral medullary surface, and in nearby areas including the parapyramidal, retrotrapezoid, gigantocellular and lateral paragigantocellular reticular nuclei as well as the caudal raphe nuclei (raphe pallidus, obscurus, and magnus). Serotonin (5-HT) neurons of the caudal raphe complex (B1–B3 cell groups) and ventromedial medulla were labeled as well as a few C1 adrenergic neurons. The A5 cell group was the major noradrenergic area labeled although a small number of locus coeruleus neurons were also labeled. Several NTS regions contained labeled cells including the commissural, intermediate, medial, central, ventral, and ventrolateral subnuclei. PRV infected neurons were present in the Kölliker-Fuse and Barrington's nuclei. In the rostral mesencephalon, the precommissural nucleus of the dorsal periventricular gray matter was labeled. Labeling was present in the dorsal, lateral and paraventricular hypothalamic nuclei. In summary, the airway parasympathetic preganglionic neurons are innervated predominantly by a network of lower brainstem neurons that lie in the same regions known to be involved in respiratory and cardiovascular regulation. These findings are discussed in relationship to some of the potential CNS mechanisms that may be operative in airway disorders as well as potentially involved in certain fatal respiratory conditions such as Ondine's curse and sudden infant death syndrome (SIDS).

Excitatory amino acid neurotransmission in superior laryngeal nerve-evoked inspiratory termination.

Superior laryngeal nerve (SLN) stimulation elicits a transient inhibition of inspiration (single shocks) or inspiratory termination (stimulus trains). The neural pathways mediating these responses are unknown, but the medial nucleus tractus solitarius (mNTS) has been implicated in the termination reflex. This study tested the hypothesis that SLN-evoked inspiratory termination requires excitatory amino acid (EAA) neurotransmission in medial aspects of the NTS. Experiments were conducted in decerebrate, vagotomized, and paralyzed adult cats. Inspiratory motor output was recorded from the phrenic nerve. After control responses to SLN stimulation were recorded, a unilateral injection of the EAA antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 mM) was made into the mNTS. The transient inhibitions were not altered by DNQX. Inspiratory termination elicited by stimulation of the SLN contralateral to the injection persisted after DNQX (n = 4). Stimulation of the ipsilateral SLN no longer elicited termination (5 of 9 animals) or did so only at greatly elevated thresholds (4 of 9). We conclude that EAA neurotransmission in the mNTS is not required in the transient reflex but is necessary for the production of the SLN-evoked inspiratory termination.

Involvement of the Area Postrema in the Regulation of Sympathetic Outflow to the Cardiovascular System

Summary: The circumventricular organs of the brain have been implicated in the central regulation of the cardiovascular system. The area postrema, which is the only circumventricular organ in the hindbrain, has received less attention than the others, but recent studies suggest that it may play an important role in the regulation of the cardiovascular system. Studies in rats and rabbits indicate that angiotensin II (Ang II)-dependent hypertension is abolished by lesioning of the area postrema. Additional studies indicate that the hypertension associated with Ang II involves a resetting of the arterial baroreflex to a higher pressure. This upward resetting requires an interaction of neurons in the area postrema with barosensitive neurons terminating in the medial nucleus tractus solitarius (mNTS). Another peptide, arginine vasopressin (AVP), has been shown to enhance the sympathoinhibitory influence of the arterial baroreflex via an action at the area postrema. Studies in rabbits suggest that the sympathoinhibitory response is due to resetting of the baroreflex to a lower pressure. Electrophysiological studies, using an in vitro brain slice preparation, have shown that activation of area postrema neurons projecting to the mNTS alters the responsiveness of mNTS neurons to afferent inputs. It is postulated that α-adrenergic mechanisms are involved in these interactions.

Localization of tyrosine hydroxylase in neuronal targets and efferents of the area postrema in the nucleus tractus solitarii of the rat.

Catecholamines in the nucleus tractus solitarii (NTS) have been implicated in autonomic responses to circulating hormones that act on neurons in the area postrema, the most caudal circumventricular organ in brain. We combined immunoperoxidase labeling of the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHAL) with immunogold-silver labeling of tyrosine hydroxylase to determine whether this enzymatic marker for catecholamines was present in efferents from the area postrema or their targets in the rat NTS. At survival periods of 10-12 days after PHAL injections into the area postrema, light microscopy revealed numerous varicose processes containing peroxidase reaction product for PHAL in the dorsomedial, medial, and commissural NTS. Some of these labeled processes were located near neuronal perikarya and processes containing immunogold-silver intensified reaction product for tyrosine hydroxylase. Electron microscopy of the commissural and dorsomedial NTS established that the majority of the labeling for PHAL was in axon terminals, whereas immunogold labeling for tyrosine hydroxylase was mainly in soma and dendrites. Only 3 out of 579 PHAL-labeled terminals also contained detectable tyrosine hydroxylase immunoreactivity. Fifty-eight percent (335/579) of the PHAL-labeled terminals formed synapses with recognized symmetric junctions, whereas the remainder lacked synaptic specializations within the examined series of serial sections. Of those PHAL terminals forming recognized symmetric junctions, 22% were on tyrosine hydroxylase-immunoreactive dendrites, 74% on unlabeled dendrites and 4% on unlabeled axon terminals. From a total of 1,250 observed contacts on tyrosine hydroxylase labeled dendrites, 88 (7%) contained PHAL, 9 (< 1%) contained TH, and 1,180 (93%) lacked detectable immunoreactivity and formed primarily symmetric synapses. We conclude that a few catecholamine, but mainly noncatecholamine efferents from the area postrema provide a monosynaptic, and most likely inhibitory input to target neurons both with and without tyrosine hydroxylase immunoreactivity in the dorsomedial and commissural NTS. Synapses between the efferent terminals from the area postrema and tyrosine hydroxylase labeled and unlabeled dendrites as well as unlabeled axons in these specific subnuclei of the NTS suggest multiple sites for modulation of gastric and cardiovascular reflexes in response to circulating peptides.

Increases in cytosolic Ca 2+ in rat area postrema/mNTS neurons produced by angiotensin II and arginine-vasopressin

It is well established that neurons in the dorsal medulla, including the area postrema and the medial nucleus tractus solitarius (mNTS), are involved in the central actions of circulating peptides such as angiotensin II (ANG II) and arginine vasopressin (AVP). This report describes a reparation that permits the identification and maintenance of area postrema/mNTS neurons in culture in which the cellular and potentially subcellular responses to neurotransmitters and neuropeptides on area postrema/mNTS cells can be investigated. Following 15–21 days in culture, the effects of ANG II and AVP on changes in intracellular Ca 2+ concentration ([Ca 2+] i) were examined. Both ANG II and AVP resulted in a rapid and transient increase in [Ca 2+] i reaching maximum in 15 s and returning towards baseline values within 180 s. The ANG II-mediated increase in [Ca 2+] i was almost completely abolished by the selective angiotensin AT 1 receptor subtype antagonist, losartan (DuP 753). These results suggest that ANG II and AVP modulate area postrema/mNTS neuronal activity by increasing intracellular Ca 2+.