L-dopaergic components in the caudal ventrolateral medulla in baroreflex neurotransmission.

L-3,4-Dihydroxyphenylalanine (L-DOPA) is probably a transmitter of the primary baroreceptor afferents terminating in the nucleus tractus solitarii; L-DOPA functions tonically to activate depressor sites of the caudal ventrolateral medulla, which receives input from the nucleus tractus solitarii [Misu Y. et al. (1996) Prog. Neurobiol. 49, 415-454]. We have attempted to clarify whether or not L-DOPAergic components within the caudal ventrolateral medulla are involved in baroreflex neurotransmission in anesthetized rats. Electrolytic lesions of the right nucleus tractus solitarii (1 mA d.c. for 10 s, 10 days before measurement) selectively decreased by 45% the tissue content of L-DOPA in the dissected ipsilateral caudal ventrolateral medulla. Electrolytic lesions did not decrease dopamine, norepinephrine and epinephrine levels. During microdialysis of the right caudal ventrolateral medulla, extracellular levels of L-DOPA, norepinephrine, epinephrine and 3,4-dihydroxyphenylacetic acid were consistently detectable using high-performance liquid chromatography with electrochemical detection. However, extracellular dopamine levels were lower than the assay limit. Baroreceptor activation by i.v. phenylephrine selectively evoked L-DOPA without increasing the levels of norepinephrine, epinephrine and 3,4-dihydroxyphenylacetic acid. This L-DOPA release was suppressed by acute lesion in the ipsilateral nucleus tractus solitarii. Intermittent stimulation of the right aortic depressor nerve (20 Hz, 3 V, 0.3 ms duration, for 30 min) repetitively and constantly caused L-DOPA release, hypotension and bradycardia, without increases in levels of norepinephrine, epinephrine and 3,4-dihydroxyphenylacetic acid. Local inhibition of L-DOPA synthesis with alpha-methyl-p-tyrosine (30 microM) infused into the ipsilateral caudal ventrolateral medulla gradually decreased basal levels of L-DOPA and 3,4-dihydroxyphenylacetic acid without decreasing norepinephrine and epinephrine. The inhibition of L-DOPA synthesis interrupted L-DOPA release and decreased by 65% depressor responses elicited by aortic nerve stimulation; however, it produced no effect on bradycardic responses. CoCl2 (119 ng), a mainly presynaptic inhibitory transmission marker, and L-DOPA methyl ester (1 microg), a competitive L-DOPA antagonist, when microinjected into depressor sites of the right caudal ventrolateral medulla, reduced by 60% depressor responses to transient ipsilateral stimulation of the aortic nerve (20 Hz, 3 V, 0.1 ms duration, for 10 s). No changes in bradycardic responses were observed. There may exist an L-DOPAergic relay from the nucleus tractus solitarii to the caudal ventrolateral medulla. L-DOPAergic components in the caudal ventrolateral medulla are involved in baroreflex neurotransmission via a baroreceptor-aortic depressor nerve-nucleus tractus solitarii-caudal ventrolateral medulla relay in the rat.     

Inhibitory effects evoked from the rostral ventrolateral medulla are selective for the nociceptive responses of spinal dorsal horn neurons

The aim of the present study was to determine whether or not descending control of spinal dorsal horn neuronal responsiveness following neuronal activation at pressor sites in the rostral ventrolateral medulla is selective for nociceptive information. Extracellular single-unit activity was recorded from 49 dorsal horn neurons in the lower lumbar spinal cord of anaesthetized rats. The 30 Class 2 neurons selected for investigation responded to noxious (pinch and radiant heat) and non-noxious (prod, stroke and/or brush) stimulation within their cutaneous receptive fields on the ipsilateral hindpaw. The excitatory amino acid, DL-homocysteic acid, was microinjected into either the rostral or the caudal rostral ventrolateral medulla at sites that evoked increases in arterial blood pressure. Effects of neuronal activation at these sites were then tested on the responses of Class 2 neurons to noxious and non-noxious stimulation within their excitatory receptive fields. The noxious pinch and radiant heat responses of Class 2 neurons were depressed, respectively to 13+/-3.8% (n=23) and to 16+/-3.7% (n=18) of control, following stimulation at sites in the rostral rostral ventrolateral medulla. In contrast, the low-threshold (prod) responses of eight Class 2 neurons tested were not depressed following neuronal activation at the same sites. When tested, control injections of the inhibitory amino acid, GABA, at the same sites in the rostral rostral ventrolateral medulla had no significant effects on neuronal activity. Neither intravenous administration of noradrenaline (to mimic the pressor responses evoked by DL-homocysteic acid microinjections in the rostral ventrolateral medulla) nor activation at pressor sites in the caudal rostral ventrolateral medulla had any significant effect on neuronal responsiveness.With regard to sensory processing in the spinal cord, these data suggest that descending inhibitory control that originates from neurons in pressor regions of the rostral rostral ventrolateral medulla is highly selective for nociceptive inputs to Class 2 neurons. These data are discussed in relation to the role of the rostral ventrolateral medulla in executing the changes in autonomic and sensory functions that are co-ordinated by higher centres in the CNS.     

Excitatory projections from the anterior hypothalamus to periaqueductal gray neurons that project to the medulla: a functional anatomical study

The present study was designed to investigate the organization of excitatory projections from regions of the anterior hypothalamus that are known to co-ordinate autonomic and sensory functions to medullo-output neurons in the periaqueductal gray. The induction of Fos protein was used to identify neurons in the periaqueductal gray that were activated synaptically by chemical stimulation at sites in the anterior hypothalamus from which either increases or decreases in arterial blood pressure were evoked (pressor sites and depressor sites, respectively). This was combined with retrograde tracing using fluorescent latex microspheres from sites in the medulla. When compared to control animals, neuronal activation at pressor sites in the anterior hypothalamus evoked Fos-like immunoreactivity in significantly more neurons in all but one sub-division of the periaqueductal gray (P at least < 0.05). The majority of Fos-positive neurons following a pressor response were located in the caudal half of the periaqueductal gray where significantly more neurons contained Fos-like immunoreactivity in lateral than in any other sub-division (P < 0.01). In all but two of 14 subdivisions of the periaqueductal gray, the numbers of neurons that expressed Fos-like immunoreactivity following stimulation at depressor sites in the anterior hypothalamus were not significantly different from controls. When neuronal activation at pressor or depressor sites in the anterior hypothalamus was combined with retrograde tracing from the rostral ventrolateral medulla, nucleus raphe magnus and/or nucleus raphe obscurus the majority of double-labelled neurons were located in the caudal half of the periaqueductal gray. Comparisons between the numbers of double-labelled neurons that resulted from different combinations of hypothalamic and medullary injection sites revealed that neuronal activation at pressor sites in the anterior hypothalamus combined with retrograde tracing from the rostral ventrolateral medulla resulted in the greatest numbers of double-labelled neurons. The identification of double-labelled neurons indicates that medullo-output neurons in the periaqueductal gray receive excitatory inputs predominantly from pressor compared to depressor sites in the anterior hypothalamus. These results are discussed in relation to the roles of the different longitudinal columns of the periaqueductal gray, and the organisation of their projections to the medulla, in the co-ordination of autonomic and sensory functions.     

Electrical stimulation of the C1 region of the rostral ventrolateral medulla of the rat increases mean arterial pressure and adrenaline release in the posterior hypothalamus

By using intracerebral dialysis in combination with high performance liquid chromatography and electrochemical detection, extracellular posterior hypothalamic adrenaline, noradrenaline, 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid were measured in the anaesthetized rat and changes in their levels monitored following administration of tranylcypromine and electrical stimulation of the rostral ventrolateral medulla. Tranylcypromine (10 mg/kg i.p.) administration decreased basal extracellular 3,4-dihydroxyphenylacetic acid and 5-hydroxyindole acetic acid levels with a simultaneous increase in adrenaline and noradrenaline levels. Electrical stimulation of the C1 area of the rostral ventrolateral medulla increased (+56.6%) extracellular adrenaline levels in the posterior hypothalamus with a simultaneous increase in mean arterial pressure (+48 mm Hg) compared to prestimulation control values. No change was seen in posterior hypothalamic extracellular levels of noradrenaline, 3,4-dihydroxyphenylacetic acid and 5-hydroxyindole acetic acid during the stimulation period. Electrical stimulation of areas close to but outside the C1 region had no effect on either mean arterial pressure or posterior hypothalamic extracellular levels of the amines or the metabolites. The increase in adrenaline levels in the hypothalamus during stimulation of the C1 region supports the evidence for an adrenergic pathway from the rostral ventrolateral medulla to the hypothalamus and suggests that the increase in mean arterial pressure during electrical stimulation to the C1 region may relate to a specific increase in adrenaline levels.     

Brainstem loci for activation of electrodermal response in the cat.

Brainstem loci from which electrodermal responses could be elicited were systematically explored by direct-stimulation techniques in chloralose-anesthetized and decerebrate cats. Reactive sites of the greatest amplitude were found to extend from the rostral border of the posterior hypothalamus, through the ventrolateral reticular formation of the pons and the medulla, to the cervical cord. Stimulation of these sites elicited stable, reproducible electrodermal responses of 10-30 mV in amplitude. In addition, it was found that stimulation of the ventrolateral extent of the lower brainstem evoked similar responses in the decerebrate preparation. Electrodermal responses could not be elicited from the dorsal medulla, the posterior commissure, or the midline region. The electrodermal response could be elicited from an apparently hypothalamus through the ventrolateral brainstem.     

Hypoxia-induced Fos expression in neurons projecting to the pressor region in the rostral ventrolateral medulla

Previous studies in anaesthetized animals have shown that the hypoxia-induced increase in sympathetic vasomotor activity is largely dependent on synaptic excitation of sympathoexcitatory pressor neurons in the rostral part of the ventrolateral medulla. The primary aim of this study was to determine, in conscious rabbits, the distribution of neurons within the brain that have properties characteristic of interneurons conveying excitatory inputs to the rostral ventrolateral medullary pressor region in response to systemic hypoxia. In a preliminary operation, a retrogradely-transported tracer, fluorescent-labelled microspheres, was injected into the physiologically-identified pressor region in the rostral ventrolateral medulla. After a waiting period of one to two weeks, the conscious rabbits were subjected to moderate hypoxia (induced by breathing 10% O₂ in N₂) for a period of 60 min. Control groups of animals were exposed to room air or to mild hypoxia (12% O₂ in N₂). Moderate hypoxia resulted in a modest hypertension of approximately 15 mmHg, and in the expression of Fos (a marker of neuronal activation) in many neurons in the nucleus tractus solitarius, the rostral, intermediate and caudal parts of the ventrolateral medulla, the Kölliker–Fuse nucleus, locus coeruleus, subcoeruleus and A5 area in the pons as well as in several midbrain and forebrain regions, including the periaqueductal grey in the midbrain and the paraventricular, supraoptic and arcuate nuclei in the hypothalamus. Fos expression was also observed in these regions in rabbits subjected to mild hypoxia or normoxia, but it was much reduced compared to rabbits subjected to moderate hypoxia. Approximately half of the neurons in the ventrolateral medulla, 27% of neurons in the nucleus tractus solitarius, and 49–81% of neurons in the locus coeruleus, sub-coeruleus and A5 area that expressed Fos following moderate hypoxia were also immunoreactive for tyrosine hydroxylase, and were therefore catecholamine cells. Approximately half of the neurons in the nucleus tractus solitarius and two-thirds of neurons in the Kölliker–Fuse nucleus that expressed Fos following moderate hypoxia were retrogradely labelled from the rostral ventrolateral medullary pressor region. Similarly, approximately one quarter of Fos-positive cells in the caudal and intermediate ventrolateral medulla were retrogradely labelled, but very few Fos-positive/retrogradely-labelled cells were found in other pontomedullary or suprapontine brain regions.

The results indicate that systemic hypoxia results in activation of neurons in several discrete nuclei in the brainstem and forebrain, including neurons in all the major pontomedullary catecholamine cell groups. However, neurons that are activated by systemic hypoxia and that also project to the rostral ventrolateral medullary pressor region are virtually confined to the lower brainstem, primarily in the nucleus tractus solitarius and Kölliker–Fuse nucleus and to a lesser extent the caudal/intermediate ventrolateral medulla. In a previous study from our laboratory, we determined the distribution of neurons in the brainstem that are activated by hypertension and that also project to the rostral ventrolateral medullary pressor region [Polson et al. (1995) Neuroscience 67, 107–123]. Comparison of the present results with those from this previous study indicates that the hypoxia-activated neurons in the nucleus tractus solitarius and Kölliker–Fuse nucleus that project to the rostral ventrolateral medulla are likely to be interneurons conveying excitatory chemoreceptor signals, while those in the caudal/intermediate ventrolateral medulla are likely to be mainly interneurons conveying inhibitory baroreceptor signals, activated by the rise in arterial blood pressure associated with the hypoxia-induced hypertension.     

Localization and action of adenosine A2a receptors in regions of the brainstem important in cardiovascular control

In vitro autoradiography and central microinjections of a P1 adenosine A2a receptor antagonist have been employed to investigate a possible role for centrally located adenosine A2a receptors in modulation of the baroreceptor reflex. In vitro autoradiography using [125I]4-(2-[7-amino-2-[2-furyl][3,2,4]triazolol[2,3-a][1,3,5]tr iazin-5-yl-amino]ethyl)phenol ([125I]ZM241385), the high-affinity adenosine A2a receptor antagonist, revealed a heterogeneous distribution of adenosine A2a binding sites within the lower brainstem of the rat. Image analysis showed high levels of binding in rostral regions of both the nucleus tractus solitarius and the ventrolateral medulla. Intermediate levels of binding were observed in the commissural nucleus tractus solitarius and the dorsal vagal motor nucleus, with low levels of binding in caudal regions of the nucleus tractus solitarius and the ventrolateral medulla, and the hypoglossal nucleus. Unilateral microinjections of unlabelled ZM241385 into the nucleus tractus solitarius had no effect on baseline levels of arterial pressure, heart rate and phrenic nerve activity recorded in anaesthetized, artificially ventilated rats. However, microinjections of ZM241385 reduced the bradycardia evoked by stimulation of the ipsilateral aortic nerve. In contrast, ZM241385 had no effect on the depressor response or the reduction in phrenic nerve activity evoked by aortic nerve stimulation. Our results indicate that adenosine A2a binding sites are located in a number of brainstem regions involved in autonomic function, consistent with the idea that adenosine acts as a neuromodulator of a variety of cardiorespiratory reflexes. Specifically, the data support the hypothesis that adenosine A2a receptors located within the nucleus tractus solitarius are activated during baroreceptor stimulation and have an important modulatory role in the pattern of cardiovascular changes associated with this reflex.     

Cardiorespiratory responses to chemical stimulation of the caudal most ventrolateral medulla in the cat.

Chemical stimulation of caudal ventrolateral medulla evoked both pressor and depressor responses. The pressor sites were generally located caudal to depressor sites. Effects on heart rate were variable. Significant increases in minute ventilation were also observed, which were primarily due to changes in respiratory frequency.     

An enkephalin-containing pathway from nucleus tractus solitarius to the pressor area of the rostral ventrolateral medulla of the rabbit

A technique combining retrograde tracing of wheat germ-conjugated gold particles with immunocytochemical demonstration of enkephalin-containing neurons was used to study intramedullary enkephalin-containing pathways to the pressor area of the rostral ventrolateral medulla in rabbits. The rostral ventrolateral medulla represents a main source of bulbospinal sympathoexcitatory neurons, and is critical to the tonic and reflex control of blood pressure. Firstly, the distribution of enkephalin-positive neurons and terminal fibres in rabbit medulla were described, with special reference to a moderately dense terminal plexus in the rostral ventrolateral medulla. Then, retrograde tracing studies were conducted; the rostral ventrolateral medullary pressor region was first localized by injection of L-glutamate (25 nmol in 50 nl). Slow (30-min) injections of wheat germ-gold (1.00 microliter) were then made at the same coordinates, resulting in a restricted injection site corresponding to the C1 pressor area, verified by the presence of tyrosine hydroxylase- and neuropeptide Y-containing neurons. Transported gold was revealed by silver reduction, and enkephalin immunoreactive cells were revealed by immunocytochemistry. Enkephalin-positive gold-containing neurons were found primarily in the nucleus tractus solitarius, especially in the commissural and medial intermediate subnuclei. Cells in the nucleus tractus solitarius containing other transmitters (substance P. galanin, neuropeptide Y and catecholamines) did not show the same degree or pattern of double-labelling, suggesting that the transport was not due to non-specific silver reduction or spread from the pipette track. The potential importance of this endogenous intramedullary opiate system is discussed in terms of medullary control of the cardiovascular system. It is hypothesized that this opiate projection from the nucleus tractus solitarius to the rostral ventrolateral medulla could play an important modulatory function, influencing baroreceptor or other cardiopulmonary reflex pathways involved in the primary regulation of the cardiovascular system. Furthermore, this pathway could represent a central substrate underlying opiate effects on the cardiovascular system during such conditions as hemorrhagic shock, stress or opiate intoxication.     

Distribution of neurons projecting to the rostral ventrolateral medullary pressor region that are activated by sustained hypotension.

Hypotension produces a reflex increase in the activity of sympathetic vasomotor and cardiac nerves. It is believed that the reflex sympathoexcitation is due largely to disinhibition of sympathoexcitatory neurons in the rostral ventrolateral medulla, but it is possible that it may also be mediated by excitatory inputs from interneurons that are activated by a fall in blood pressure. The aim of this study in conscious rabbits was to identify and map neurons with properties that are characteristic of interneurons conveying excitatory inputs to the rostral ventrolateral medullary pressor region in response to hypotension. In a preliminary operation, a retrogradely-transported tracer, fluorescent-labelled microspheres, was injected into the functionally-identified pressor region in the rostral ventrolateral medulla. After a waiting period of at least one week, a moderate hypotension (decrease in arterial pressure of approximately 20 mmHg) was induced in conscious rabbits for 60 min by the continuous infusion of sodium nitroprusside. In confirmation of a previous study from our laboratory, [Li and Dampney (1994) Neuroscience 61, 613–634] hypotension resulted in the expression of Fos (the protein product of c-fos, a marker of neuronal activation) in many neurons in several distinct regions in the brainstem and hypothalamus. Some of these regions (nucleus tractus solitarius, area postrema, caudal and intermediate ventrolateral medulla, parabrachial complex in the pons, and paraventricular nucleus in the hypothalamus) also contained large numbers of retrogradely-labelled cells. Approximately 10% of the Fos-positive neurons in the nucleus tractus solitarius, and 15–20% of Fos-positive neurons in the caudal and intermediate ventrolateral medulla were also retrogradely-labelled from the rostral ventrolateral medullary pressor region. In other brain regions, very few double-labelled neurons were found.In previous studies from our laboratory, we have determined the distribution of neurons in the brainstem that project to the rostral ventrolateral medullary pressor region and that are also activated by hypertension [Polson et al. (1995) Neuroscience 67, 107–123] or by hypoxia. [Hirooka et al. (1997) Neuroscience 80, 1209–1224] Comparison of the present results with those from these previous studies indicate that although hypotension and hypoxia both elicit powerful reflex sympathoexcitatory responses, the central pathways subserving these effects in conscious animals are fundamentally different. Hypoxia activates rostral ventrolateral medullary sympathoexcitatory neurons mainly via a major direct excitatory projection from the nucleus tractus solitarius, as well as from the Kölliker–Fuse nucleus in the pons, while in contrast the activation of these neurons in response to hypotension appears to be due mainly to disinhibition, mediated via inhibitory interneurons. In addition, however, inputs originating from excitatory interneurons in the nucleus tractus solitarius and caudal and intermediate parts of the ventrolateral medulla appear to contribute to the hypotension-evoked activation of sympathoexcitatory neurons in the rostral ventrolateral medulla.     

Serotonin(3) receptor stimulation in the nucleus tractus solitarii activates non-catecholaminergic neurons in the rat ventrolateral medulla

The present study was performed to determine whether or not the increased arterial pressure triggered by 5-HT(3) receptor stimulation in the nucleus tractus solitarii and underlain by a sympathoexcitation is associated with the activation of ventromedullary cells known to be involved in vascular regulation, i.e. the C1 and A1 catecholaminergic cells. For this purpose, double immunohistochemical labeling for tyrosine hydroxylase and c-fos protein was performed all along the ventrolateral medulla after microinjection of 1-(m-chlorophenyl)-biguanide, a selective and potent 5-HT(3) receptor agonist, into the nucleus tractus solitarii of alpha-chloralose/urethane-anaesthetized rats. This treatment produced a significant elevation of arterial pressure ( approximately +35 mm Hg). Concomitantly, a significant increase in the number of c-fos expressing neurons was observed in the rostral ventrolateral medulla (+63%), in particular in its most anterior part (+78%), and in the medullary region surrounding the caudal part of the facial nucleus (+91%). Retrograde labeling with gold-horseradish peroxidase complex showed that at least some of these activated c-fos expressing cells project to the spinal cord. However, the number of double-stained neurons, i.e. c-fos and tyrosine hydroxylase positive neurons, did not increase at any level of the ventrolateral medulla. In contrast, under the same alpha-chloralose/urethane anesthesia, systemic infusion of sodium nitroprusside appeared to produce a hypotension and a marked increase in the density of such double c-fos and tyrosine hydroxylase expressing cells in the rostral ventrolateral medulla and the caudal medullary region surrounding the caudal part of the facial nucleus.These data indicate that medullary catecholaminergic C1 and A1 neurons are not involved in the pressor effect elicited by 5-HT(3) receptor stimulation in the nucleus tractus solitarii. However, this 5-HT(3) receptor-mediated effect is clearly associated with the excitation of (non-catecholaminergic) neurons within the pressor region of the ventral medulla.     

The pressor effect of angiotensin-(1-7) in the rat rostral ventrolateral medulla involves multiple peripheral mechanisms

OBJECTIVE:

In the present study, the peripheral mechanism that mediates the pressor effect of angiotensin-(1-7) in the rostral ventrolateral medulla was investigated.

METHOD:

Angiotensin-(1-7) (25 pmol) was bilaterally microinjected in the rostral ventrolateral medulla near the ventral surface in urethane-anesthetized male Wistar rats that were untreated or treated (intravenously) with effective doses of selective autonomic receptor antagonists (atenolol, prazosin, methyl-atropine, and hexamethonium) or a vasopressin V₁ receptor antagonist [d(CH₂)₅ -Tyr(Me)–AVP] given alone or in combination.

RESULTS:

Unexpectedly, the pressor response produced by angiotensin-(1-7) (16±2 mmHg, n = 12), which was not associated with significant changes in heart rate, was not significantly altered by peripheral treatment with prazosin, the vasopressin V₁ receptor antagonist, hexamethonium or methyl-atropine. Similar results were obtained in experiments that tested the association of prazosin and atenolol; methyl-atropine and the vasopressin V₁ antagonist or methyl-atropine and prazosin. Peripheral treatment with the combination of prazosin, atenolol and the vasopressin V₁ antagonist abolished the pressor effect of glutamate; however, this treatment produced only a small decrease in the pressor effect of angiotensin-(1-7) at the rostral ventrolateral medulla. The combination of hexamethonium with the vasopressin V₁ receptor antagonist or the combination of prazosin, atenolol, the vasopressin V₁ receptor antagonist and methyl-atropine was effective in blocking the effect of angiotensin-(1-7) at the rostral ventrolateral medulla.

CONCLUSION:

These results indicate that angiotensin-(1-7) triggers a complex pressor response at the rostral ventrolateral medulla that involves an increase in sympathetic tonus, release of vasopressin and possibly the inhibition of a vasodilatory mechanism.     

The mapped pattern of kainate on blood pressure responses is similar to that of l-proline in the ventrolateral medulla of the rat

Kainate is an excitatory amino acid receptor agonist with a structure similar to the amino acid l-proline. Our previous studies demonstrated that microinjections of l-proline into the ventrolateral medulla (VLM) of the rat induce a mapped pattern of blood pressure responses distinct from l-glutamate, and the depressor response to l-proline in the caudal VLM (CVLM) is abolished by the kainate/AMPA receptor antagonist CNQX. The present study investigated whether kainate produces the l-proline-mapped pattern of responses in the VLM, compared with the pattern by AMPA. Kainate is known to activate AMPA receptors at higher concentrations. Therefore, responses to kainate were investigated at a low concentration. Microinjections of AMPA or NMDA showed the pattern of the l-glutamate-type; a pressor response in the rostral VLM and caudal pressor area (CPA) and a depressor response in the CVLM. Microinjections of kainate showed depressor responses in the CVLM but minor pressor responses in the rostral VLM, suggesting the same responses to l-proline. However, the response sites in the CPA did not enable us to clearly determine the l-proline-type. Further trials at sites defined by a pressor response to l-glutamate in the CPA, successive injections of l-proline and kainate produced no response, indicating that l-glutamate responding neurons in the CPA are not sensitive to l-proline and kainate. These results suggest that kainate stimulation in the VLM produces a mapped pattern of ABP responses similar to the mapped pattern with l-proline. Kainate receptors could therefore be involved in the depressor response to l-proline in the medulla.     

Localization and action of adenosine A2a receptors in regions of the brainstem important in cardiovascular control

In vitro autoradiography and central microinjections of a P₁ adenosine A_2a receptor antagonist have been employed to investigate a possible role for centrally located adenosine A_2a receptors in modulation of the baroreceptor reflex. In vitro autoradiography using [¹²⁵I]4-(2-[7-amino-2-[2-furyl][1,2,4]triazolol[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol ([¹²⁵I]ZM241385), the high-affinity adenosine A_2a receptor antagonist, revealed a heterogeneous distribution of adenosine A_2a binding sites within the lower brainstem of the rat. Image analysis showed high levels of binding in rostral regions of both the nucleus tractus solitarius and the ventrolateral medulla. Intermediate levels of binding were observed in the commissural nucleus tractus solitarius and the dorsal vagal motor nucleus, with low levels of binding in caudal regions of the nucleus tractus solitarius and the ventrolateral medulla, and the hypoglossal nucleus. Unilateral microinjections of unlabelled ZM241385 into the nucleus tractus solitarius had no effect on baseline levels of arterial pressure, heart rate and phrenic nerve activity recorded in anaesthetized, artificially ventilated rats. However, microinjections of ZM241385 reduced the bradycardia evoked by stimulation of the ipsilateral aortic nerve. In contrast, ZM241385 had no effect on the depressor response or the reduction in phrenic nerve activity evoked by aortic nerve stimulation.Our results indicate that adenosine A_2a binding sites are located in a number of brainstem regions involved in autonomic function, consistent with the idea that adenosine acts as a neuromodulator of a variety of cardiorespiratory reflexes. Specifically, the data support the hypothesis that adenosine A_2a receptors located within the nucleus tractus solitarius are activated during baroreceptor stimulation and have an important modulatory role in the pattern of cardiovascular changes associated with this reflex.     

Vagal modulation of responses elicited by stimulation of the aortic depressor nerve in neurons of the rostral ventrolateral medulla oblongata in the rat.

Stimulation of cervical vagal afferents inhibits central sympathetic outflows in part by inhibiting the ongoing activity of putative baroreceptive neurons in the rostral ventrolateral medulla oblongata. The aim of the present study was to examine the electrophysiological characteristics of vagal responses and their interactions with responses elicited by stimulation of the aortic nerve in neurons there. The study focused on the role of the long-lasting, late-onset vagal inhibition, which is likely to play an important role in the tonic inhibitory effects of vagal afferent stimulation. In vivo intracellular recordings were obtained from 33 neurons that received convergent inputs from aortic and vagal afferents. Sixty-four percent of these neurons exhibited a late inhibition following electrical stimulation of myelinated vagal afferents (mean onset latency of 100+/-5 ms). The average duration of late inhibition (294+/-19 ms) exceeded the duration of the cardiac cycle. As a consequence of this, sustained vagal stimulation diminished the effect of rhythmic baroreceptor inputs in neurons that exhibited late vagal inhibition. Simultaneous activation of aortic and vagal afferents significantly increased the magnitude of late inhibition, even in those neurons where stimulation of the aortic nerve alone did not elicit a response (n = 15). This suggested that the convergence between vagal and aortic afferent inputs occurred in inhibitory inteneurons antecedent to the recorded rostral ventrolateral medulla oblongata neurons. Focal stimulation of the caudal part of the nucleus of the solitary tract also elicited a late-onset inhibition in 73% of the neurons that responded to stimulation of the aortic nerve. This inhibition appeared to be similar to the late vagal inhibition, except for its shorter average onset latency (64+/-7 ms). Based on this observation, it is proposed that inhibitory inteneurons that mediate late inhibition to rostral ventrolateral medulla oblongata neurons may lie within the caudal part of the nucleus of the solitary tract. The present study established that activation of myelinated vagal afferents exerts a complex modulation over the ongoing and evoked activity of neurons that respond to stimulation of the aortic nerve. The complex interaction that occurs between aortic and vagal inputs in neurons of the rostral ventrolateral medulla may be implicated in long-term modulation of sympathetic outflows in response to changes in the activation of visceral receptors supplied by vagus afferents. The modulation elicited by late vagal inhibition may help to adjust cardiovascular outflows according to requirements set by the thoraco-abdominal visceral environment.     

Hypothalamic, midbrain and bulbar areas involved in the defense reaction in rabbits

The present study mapped neuroanatomical sites in the hypothalamus and periaqueductal gray (PAG) of the rabbit which, when stimulated electrically, evoked the cardiorespiratory components of the defense reaction (CRDR). This included increases in heart rate, blood pressure, hindlimb blood flow and respiration rate. All of the components of the CRDR were elicited by electrical stimulation of the posterior hypothalamus, at sites dorsal and medial to the fornix. Although there were regions throughout the PAG in which electrical stimulation elicited concomitant increases in blood pressure, hindlimb blood flow and respiration rate, only stimulation of the dorsal PAG evoked tachycardia. Injection of horseradish peroxidase into the rostral ventrolateral medulla (RVLM) led to heavy retrograde and anterograde labeling in the region of the hypothalamus that yielded the CRDR when stimulated electrically. Heavy labeling was also observed in the dorsal and ventral PAG. The results of this study provide evidence that the posterior hypothalamus and the dorsal PAG are nodal structures in the mediation of the CRDR and that cells in posterior hypothalamus, dorsal PAG and ventral PAG make monosynaptic connections with the RVLM.     

Parabrachial pons mediates hypothalamically induced renal vasoconstriction

The role of the parabrachial region of the dorsal rostral pons (PB) in mediating control of renal blood flow and of systemic arterial blood pressure was investigated in nine cats anesthetized with chloralose-urethan. Electrical stimulation through electrodes placed stereotaxically in lateral and medial positions in the hypothalamus (LH and MH) in PB and in ventrolateral reticular formation (VLRF) of each cat elicited pronounced systemic arterial pressor responses and renal vasoconstrictions. Stimulation parameters were adjusted so that renal flow responses elicited from each site were equal. Following a unilateral lesion in the PB, responses of renal vasoconstriction induced by hypothalamic stimulation were attenuated, but responses of arterial pressure were not altered. Stimulation of the VLRF, posterior to the lesion, consistently produced undiminished systemic pressor responses and renal vasoconstriction throughout the durations of the experiments excluding decay of renal vascular responsiveness. Thus, the data suggest that pathways mediating renal vasoconstriction in response to hypothalamic stimulation was discrete and pass through the parabrachial region, whereas pathways mediating systemic vasoconstriction in response to hypothalamic stimulation are distinct or less compact.     

Involvement of the purinergic system in central cardiovascular modulation at the level of the nucleus ambiguus of anaesthetized rats

Anatomical studies have demonstrated the existence of purinergic P2 receptors in the nucleus ambiguus (NA), a site containing cardiac vagal motoneurons. However, very little is known about the functional role of these receptors in central cardiac vagal regulation. The aims of our study were to evaluate the following: (1) the blood pressure and heart rate responses following purinoceptor activation within the NA; (2) the role of purinoceptors and excitatory amino acid (EAA) receptors in mediating the cardiovascular responses evoked by ATP and L-glutamate stimulation of NA; and (3) the role of NA purinoceptors in mediating the cardiovascular responses of the Bezold-Jarisch reflex. In anaesthetized rats, microinjection of L-glutamate (5.0 nmol/50 nl) into the NA induced a marked and immediate onset bradycardia with minimal change in arterial pressure. Microinjection of ATP into the NA induced a dose-dependent (0.31-6.0 nmol/50 nl) bradycardia and pressor responses. It is noteworthy that the bradycardia occurred either before or simultaneously with a pressor response (when present), indicating that it was not a baroreceptor reflex mediated response due to the rise in arterial pressure. The pressor response was prevented by α(1)-adrenergic blockade with prazosin, whereas muscarinic blockade with methyl-atropine abolished the evoked bradycardia. Ipsilateral microinjection of PPADS (a P2 receptor antagonist; 500 pmol/100 nl) into the NA significantly attenuated the ATP-induced bradycardia but spared the pressor response. In contrast, PPADS in the NA had no effect on the L-glutamate-evoked bradycardic response. Ipsilateral injection of kynurenic acid (a non-selective EAA receptor antagonist; 10 nmol/50 nl) into the NA totally blocked the bradycardia induced by l-glutamate and partly attenuated the ATP induced bradycardia. Finally, both the depressor and the bradycardic responses of the Bezold-Jarisch reflex were attenuated significantly (P < 0.01 and P < 0.05, respectively) following bilateral microinjection of PPADS into the NA. These results identify ATP and purinergic P2 receptors within the ventrolateral medulla as excitatory to cardiovagal neurons. Additionally, our data show that P2 receptors within the ventrolateral medulla are integral to the cardiovascular responses of the Bezold-Jarisch reflex.     

Right atrial stretch activates neurons in autonomic brain regions that project to the rostral ventrolateral medulla in the rat

Activation of the cardiac mechanoreceptors results in changes in sympathetic nerve activity and plays an important role in the responses elicited by elevated blood volume. Stimulation of the reflex influences several key autonomic regions, namely the paraventricular nucleus (PVN), the nucleus of the tractus solitarius (NTS) and the caudal ventrolateral medulla (CVLM). Neurons in these regions project directly to the rostral ventrolateral medulla (RVLM), a critical region in the generation of sympathetic vasomotor tone. The aim of the present experiments was to determine whether neurons in the PVN, NTS and CVLM that are activated by cardiac mechanoreceptor stimulation also project to the RVLM. Animals were prepared, under general anesthesia, by microinjection of a retrogradely transported tracer into the pressor region of the RVLM, and the placement of a balloon-tipped cannula at the junction of the right atrium and the superior vena cava. On the experimental day, in conscious rats, the balloon was inflated to stimulate cardiac mechanoreceptors (n = 9), or left uninflated (control, n = 8). Compared with controls, there was a significantly increased number of Fos-immunoreactive neurons (a marker of activation) in both the PVN (2.5-fold) and NTS (two-fold), but this was not seen in the CVLM. Compared with controls, a significant number of the neurons in the PVN (8%) and NTS (4.0%) that projected to the RVLM were activated. The data suggest that subgroups of RVLM-projecting neurons located in the PVN and NTS are involved in the central reflex pathway activated by cardiac mechanoreceptor stimulation.