P.D Potts

Central mechanisms underlying short- and long-term regulation of the cardiovascular system.

1. Sympathetic vasomotor nerves play a major role in determining the level of arterial blood pressure and the distribution of cardiac output. The present review will discuss briefly the central regulatory mechanisms that control the sympathetic outflow to the cardiovascular system in the short and long term.

Functional organization of brain pathways subserving the baroreceptor reflex: studies in conscious animals using immediate early gene expression.

Abstract1. This paper reviews studies carried out in our laboratory in which we have used the c-fos functional mapping method, in combination with other methods, to determine the functional organization of central baroreceptor pathways as they operate in the conscious rabbit.2. First, we showed that periods of induced hypertension or hypotension each result in a specific and reproducible pattern of activation of neurons in the brainstem and forebrain. In particular, hypotension (but not hypertension) results in the activation of catecholamine neurons in the medulla and pons and vasopressin-synthesizing neurons in the hypothalamus.3. The activation of medullary cell groups in response to induced hypertension or hypotension in the conscious rabbit is almost entirely dependent on inputs from arterial baroreceptors, while the activation of hypothalamic vasopressin-synthesising neurons in response to hypotension is largely dependent on baroreceptors, although an increase in circulating angiotensin also appears to contribute.4. Discrete groups of neurons in the rostral ventrolateral medulla (RVLM) and A5 area in the pons are the major groups of spinally projecting neurons activated by baroreceptor unloading. In contrast, spinally projecting neurons in the paraventricular nucleus in the hypothalamus appear to be largely unaffected by baroreceptor signals.5. Direct afferent inputs to RVLM neurons in response to increases or decreases in arterial pressure originate primarily from other medullary nuclei, particularly neurons located in the caudal and intermediate levels of the ventrolateral medulla (CVLM and IVLM), as well as in the nucleus tractus solitarius (NTS).6. There is also a direct projection from barosensory neurons in the NTS to the CVLM/IVLM region, which is activated by baroreceptor inputs.7. Collectively, the results of our studies in conscious animals indicate that baroreceptor signals reach all levels of the brain. With regard to the baroreceptor reflex control of sympathetic activity, our studies are consistent with previous studies in anesthetized animals, but in addition reveal other previously unrecognized pathways that also contribute to this reflex regulation.

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.

Role of angiotensin II receptor subtypes in mediating the sympathoexcitatory effects of exogenous and endogenous angiotensin peptides in the rostral ventrolateral medulla of the rabbit

The pressor region in the rostral part of the ventrolateral medulla (VLM) in the rabbit contains a high density of the AT1 subtype of angiotensin (Ang) II receptor. In this study in anaesthetized barodenervated rabbits, we determined the effect of microinjection into the rostral VLM of the AT1 receptor antagonist losartan and the AT2 receptor antagonist PD123319 on resting arterial pressure and renal sympathetic nerve activity, and on the cardiovascular responses normally evoked by exogenous Ang II or Ang III in this region. Losartan (1 nmol) abolished the pressor and sympathoexcitatory responses normally evoked by exogenous Ang II, but PD123319 (1 nmol) had little effect on these responses. Both losartan (0.1-10 nmol) and PD123319 (0.1-1 nmol) had little effect on the resting arterial pressure and renal sympathetic nerve activity, except for a transient sympathoexcitatory response at the higher doses. In confirmation of previous findings, however, microinjection of the non-selective Ang receptor antagonist [Sar1,Thr8]Ang II (80 pmol) significantly decreased resting arterial pressure and sympathetic nerve activity. These results suggest that the sympathoexcitatory effects evoked by exogenous Ang II and III in the rostral VLM are mediated by AT1 receptors, but that the tonic sympathoexcitation produced by endogenous Ang peptides in the rostral VLM of the rabbit are mediated by receptors other than AT1 or AT2 receptors.

Role of angiotensin II receptors in the regulation of vasomotor neurons in the ventrolateral medulla.

1. There is a high density of angiotensin type 1 (AT1) receptors in various brain regions involved in cardiovascular regulation. The present review will focus on the role of AT1 receptors in regulating the activity of sympathetic premotor neurons in the rostral part of the ventrolateral medulla (VLM), which are known to play a pivotal role in the tonic and phasic regulation of sympathetic vasomotor activity and arterial pressure.

Effects of sinoaortic denervation on Fos expression in the brain evoked by hypertension and hypotension in conscious rabbits.

We have previously shown [Li and Dampney (1994) Neuroscience 61, 613-634] that periods of sustained hypertension and hypotension each induces a distinctive and reproducible pattern of neuronal expression of Fos (a marker of neuronal activation) in specific regions of the brainstem and forebrain of conscious rabbits. The aim of this study was to determine the contribution of afferent inputs from arterial baroreceptors to the activation of neurons in these various brain regions that is caused by a sustained change in arterial pressure. Experiments were carried out on rabbits in which the carotid sinus and aortic depressor nerves were cut in a preliminary operation. Following a recovery period of seven to 10 days, a moderate hypertension or hypotension (increase or decrease in arterial pressure of 20-30 mmHg) was induced in conscious barodenervated rabbits for 60 min by the continuous infusion of phenylephrine or sodium nitroprusside, respectively. In control experiments, barodenervated rabbits were subjected to the identical procedures except that they were infused with the vehicle solution alone. Compared with the effects seen in barointact rabbits, [Li and Dampney (1994) Neuroscience 61, 613-634] the number of neurons that expressed Fos in response to hypertension was reduced by approximately 90% in the nucleus of the solitary tract and in the caudal and intermediate parts of the ventrolateral medulla. In supramedullary regions, baroreceptor denervation resulted in a reduction of approximately 60% in hypertension-induced Fos expression in the central nucleus of the amygdala and in the bed nucleus of the stria terminalis, but no significant reduction in the parabrachial complex in the pons. Following hypotension, the number of neurons that expressed Fos in barodenervated rabbits, compared with barointact rabbits, [Li and Dampney (1994) Neuroscience 61, 613-634] was reduced by approximately 90% in the nucleus of the solitary tract, area postrema, and caudal, intermediate and rostral parts of the ventrolateral medulla. Baroreceptor denervation also resulted in a similar large reduction in hypotension-induced Fos expression in many supramedullary regions (locus coeruleus, midbrain periaqueductal grey, hypothalamic paraventricular nucleus, and in the central nucleus of the amygdala and the bed nucleus of the stria terminalis in the basal forebrain). In the supraoptic nucleus, hypotension-induced Fos expression in barodenervated rabbits was reduced by 75% compared to barointact animals, but was still significantly greater than in control animals. There was also a high level of Fos expression, much greater than in control animals, in the circumventricular organs surrounding the third ventricle (subfornical organ and organum vasculosum lamina terminalis). The results indicate that in conscious rabbits the activation of neurons that occurs in several discrete regions at all levels of the brain following a sustained change in arterial pressure is largely dependent upon inputs from arterial baroreceptors, with the exception of neurons in the circumventricular organs surrounding the third ventricle that are activated by sustained hypotension. The latter group of neurons are known to project to vasopressin-secreting neurons in the supraoptic nucleus, and may therefore via this pathway trigger the hypotension-induced release of vasopressin that occurs in the absence of baroreceptor inputs.

Use of c-fos functional mapping to identify the central baroreceptor reflex pathway: advantages and limitations.

Prolonged stimulation of many neurons results in the expression of the immediate early gene c-fos, which in turn cause the production of the protein Fos, whose presence in a cell can be detected by immunocytochemistry. This method has been used in both conscious and anaesthetized animals to identify central neurons involved in the baroreceptor reflex. In this paper we review the factors that can influence c-fos expression, with particular emphasis on the effects of different anaesthetic agents. We conclude that the c-fos method of functional mapping, when applied carefully and critically, is a very useful method of identifying central neurons that are activated by cardiovascular stimuli in conscious animals. Anaesthetic agents can significantly alter c-fos expression, and this effect differs greatly according to the type of anaesthetic used.

Fos expression in neurons projecting to the pressor region in the rostral ventrolateral medulla after sustained hypertension in conscious rabbits.

Previous studies in anaesthetized animals have shown that the baroreflex control of sympathetic vasomotor activity is mediated to a large extent by inhibitory inputs to sympathoexcitatory pressor neurons in the rostral part of the ventrolateral medulla. The aim of this study was to determine, in conscious rabbits, the distribution of neurons within the brain that have two properties characteristic of interneurons conveying baroreceptor signals to the rostral ventrolateral medulla: (i) they are activated by an increase in arterial pressure; and (ii) they project specifically to the rostral ventrolateral medulla pressor region. In a preliminary operation, an injection of the retrogradely transported tracer, fluorescent-labelled microspheres, was made into the physiologically identified pressor region in the rostral ventrolateral medulla. After a waiting period of one to eight weeks, hypertension was produced in the conscious rabbit by continuous intravenous infusion of phenylephrine at a rate sufficient to increase arterial pressure by approximately 20 mmHg, maintained for a period of 60 min. A control group of animals was infused with the vehicle solution alone. In confirmation of our previous study, hypertension produced by phenylephrine resulted in the neuronal expression of Fos (a marker of neuronal activation) in the nucleus of the solitary tract, area postrema, the intermediate and caudal parts of the ventrolateral medulla parabrachial complex, and in the central nucleus of the amygdala. Approximately 50% of the Fos-immunoreactive neurons in both the caudal and intermediate parts of the ventrolateral medulla were also retrogradely labelled from the rostral ventrolateral medulla pressor region; such double-labelled neurons were confined to a discrete longitudinal column located just ventrolateral to the nucleus ambiguus. Significant numbers of double-labelled neurons were also found in the nucleus of the solitary tract and area postrema, although these represented a much lower proportion (13-16%) of the total number of Fos-immunoreactive neurons in these regions. In the parabrachial complex, Fos-immunoreactive and retrogradely labelled neurons were largely separate populations, while in the amygdala they were entirely separate populations. In the control group of rabbits, virtually no double-labelled neurons were found in any of these regions. The results indicate that putative baroreceptor interneurons that project to the pressor region of the rostral ventrolateral medulla are virtually confined to the lower brainstem. In particular, they support the results of previous studies in anaesthetized animals indicating that neurons in the intermediate and caudal ventrolateral medulla convey baroreceptor signals to the rostral ventrolateral medulla pressor region, and extend them by demonstrating the precise anatomical distribution of these neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation of brain neurons by circulating angiotensin II. Direct effects and baroreceptor-mediated secondary effects

Circulating angiotensin II acts on neurons in circumventricular organs, leading to activation of central pathways involved in blood pressure regulation and body fluid homeostasis. Apart from this primary effect, an increase in the level of circulating angiotensin II may also activate brain neurons as a secondary consequence of the associated increase in blood pressure, which will stimulate arterial baroreceptors and thus activate central neurons that are part of the central baroreceptor reflex pathway. The aim of this study was to identify the population of neurons that are activated as a consequence of the direct actions of circulating angiotensin II on the brain, independent of secondary baroreceptor-mediated effects. For this purpose, we have mapped the distribution of neurons in the brainstem and forebrain that are immunoreactive for Fos (a marker of neuronal activation) following intravenous infusion of angiotensin II in conscious rabbits with chronically denervated carotid sinus and aortic baroreceptors. The distribution was compared with that evoked by the same procedure in two separate groups of barointact rabbits, in which angiotensin II was infused either at a rate similar to that in the barodenervated group, or at a rate approximately five times greater. In barodenervated rabbits, angiotensin II infusion evoked a significant increase in Fos expression, compared to control animals infused with the vehicle solution alone, in several forebrain nuclei (organum vasculosum of the lamina terminalis, subfornical organ, median preoptic nucleus, supraoptic nucleus, paraventricular nucleus, bed nucleus of the stria terminalis and suprachiasmatic nucleus), but little or no increase in Fos expression in any lower brainstem region. In barointact rabbits infused with angiotensin II at a similar rate to that in barodenervated rabbits, a similar degree of Fos expression was evoked in all of the above forebrain regions, but in addition a significantly greater degree of Fos expression was evoked in several medullary regions (nucleus tractus solitarius, area postrema, and ventrolateral medulla), even though the angiotensin II-evoked increase in mean arterial pressure (17 +/- 3 mmHg) was less than that evoked in the barodenervated rabbits (26 +/- 2 mmHg). In barointact rabbits infused with angiotensin II at the higher rate, the increase in mean arterial pressure was 29 +/- 3 mmHg. In these animals, the pattern of Fos expression was similar to that evoked in barointact rabbits infused at the lower rate, but the degree of Fos expression in all medullary regions and in some forebrain regions was significantly greater. The results of the present study, together with those of previous studies from our laboratory in which we determined the effects of phenylephrine-induced hypertension on brain Fos expression [Li and Dampney (1994) Neuroscience 61, 613-634; Potts et al. (1997) Neuroscience 77, 503-520], indicate that in conscious rabbits circulating angiotensin II activates primarily circumventricular neurons within the organum vasculosum of the lamina terminalis and subfornical organ, but not the area postrema, and this in turn leads to activation of neurons in other forebrain regions, including the median preoptic, supraoptic, paraventricular and suprachiasmatic nucleus as well as the bed nucleus of the stria terminalis. In contrast, the activation of neurons in medullary regions evoked by an increase in the level of circulating angiotensin II is primarily a secondary effect resulting from stimulation of arterial baroreceptors.

Activation of brain neurons following central hypervolaemia and hypovolaemia: contribution of baroreceptor and non-baroreceptor inputs

In the present study we have used the detection of Fos, the protein product of c-fos, to determine the distribution of neurons in the medulla and hypothalamus that are activated by changes in central blood volume. Experiments were conducted in both barointact and barodenervated conscious rabbits, to determine the contribution of arterial baroreceptors to the pattern of Fos expression evoked by changes in central blood volume, induced either by intravenous infusion of an isotonic modified gelatin solution, or by partial occlusion of the vena cava. These procedures resulted in a significant increase and decrease, respectively, in right atrial pressure over a 60 min period. In control experiments, barointact and barodenervated rabbits were subjected to the identical procedures except that no changes in central blood volume were induced. In comparison with the control observations, central hypervolaemia produced a significant increase in the number of Fos-immunoreactive neurons in the nucleus tractus solitarius, area postrema, the caudal, intermediate and rostral parts of the ventrolateral medulla, supraoptic nucleus, paraventricular nucleus, arcuate nucleus, suprachiasmatic nucleus and median preoptic nucleus. The overall pattern of Fos expression induced by central hypervolaemia did not differ significantly between barointact and barodenervated animals. Similarly, the overall pattern of Fos expression induced by central hypovolaemia did not differ significantly between barointact and barodenervated animals, but did differ significantly from that produced by hypervolaemia. In particular, central hypovolaemia produced a significant increase in Fos expression in the same regions as above, but also in the subfornical organ and organum vasculosum lamina terminalis. In addition, compared with central hypervolaemia, hypovolaemia produced a significantly greater degree of Fos expression in the rostral ventrolateral medulla and supraoptic nucleus. Furthermore, double-labelling for tyrosine hydroxylase immunoreactivity demonstrated that neurons in the ventrolateral medulla that expressed Fos following hypovolaemia were predominantly catecholamine cells, whereas following hypervolaemia they were predominantly non-catecholamine cells. Finally, double-labelling for vasopressin immunoreactivity demonstrated that the number of Fos/vasopressin immunoreactive cells in the supraoptic nucleus was approximately 10 times greater following hypovolaemia compared with hypervolaemia, but there were very few such double-labelled neurons in the paraventricular nucleus in response to either stimulus. The results demonstrate that central hypervolaemia and hypovolaemia each induces reproducible and specific patterns of Fos expression in the medulla and hypothalamus. The degree and pattern of Fos expression was unaffected by arterial baroreceptor denervation, indicating that it is primarily a consequence of inputs from cardiac receptors, together with an increase in the level of circulating hormones such as atrial natriuretic peptide, angiotensin II or vasopressin. Furthermore, the pattern of Fos expression produced by central hypervolaemia and hypovolaemia is distinctly different from that evoked by hypertension and hypotension, respectively [Li and Dampney (1994) Neuroscience 61, 613-634], particularly in hypothalamic regions. These findings therefore indicate that the central pathways activated by changes in blood volume are, at least in part, separate from those activated by changes in arterial pressure.