The effect of air puff stress on c-Fos expression in rat hypothalamus and brainstem: central circuitry mediating sympathoexcitation and baroreflex resetting.

Psychological stress evokes increases in sympathetic activity and blood pressure, which are due at least in part to an upward resetting of the baroreceptor-sympathetic reflex. In this study we determined whether sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM), which have a critical role in the reflex control of sympathetic activity, are activated during air puff stress, a moderate psychological stressor. Secondly, we identified neurons that are activated by air puff stress and that also project to the nucleus tractus solitarius (NTS), a key site for modulation of the baroreceptor reflex. Air puff stress resulted in increased c-Fos expression in several hypothalamic and brainstem nuclei, including the paraventricular nucleus (PVN), dorsomedial hypothalamus, perifornical area (PeF), periaqueductal gray (PAG), NTS and rostral ventromedial medulla, but not in the RVLM region that contains sympathetic premotor neurons. In contrast, neurons in this RVLM region, including catecholamine-synthesizing neurons, did express c-Fos following induced hypotension, which reflexly activates RVLM sympathetic premotor neurons. The highest proportion of NTS-projecting neurons that were double-labelled with c-Fos after air puff stress was in the ventrolateral PAG (29.3 ± 5.5%), with smaller but still significant proportions of double-labelled NTS-projecting neurons in the PVN and PeF (6.5 ± 1.8 and 6.4 ± 1.7%, respectively). The results suggest that the increased sympathetic activity during psychological stress is not driven primarily by RVLM sympathetic premotor neurons, and that neurons in the PVN, PeF and ventrolateral PAG may contribute to the resetting of the baroreceptor-sympathetic reflex that is associated with psychological stress.

Blockade of orexin receptors with Almorexant reduces cardiorespiratory responses evoked from the hypothalamus but not baro- or chemoreceptor reflex responses.

Orexin neurons form a restricted group in the dorsal hypothalamus. The group is centered on the perifornical area within the classic hypothalamic defense area, an area which when activated produces marked cardiovascular and respiratory effects. Central administration of orexin can produce cardiorespiratory effects, but the extent to which orexin contributes to such responses evoked from the perifornical hypothalamus is not clear. To determine this, we used the dual orexin receptor antagonist Almorexant to challenge the cardiorespiratory effects evoked by disinhibition of the perifornical hypothalamus. Bicuculline (10 and 20 pmol) was microinjected in the perifornical area before and after administration of Almorexant (15 mg/kg iv) or vehicle in urethane-anesthetized rats. Almorexant significantly reduced the pressor, tachycardic, renal sympathoexcitatory, and tachypneic responses to bicuculline (10 pmol, by 55%, 53%, 28%, 77%; 20 pmol, by 54%, 27%, 51%, 72%, respectively). Reductions of similar magnitude were observed with bicuculline microinjections centered on more caudal sites just peripheral to the orexin neuron group, which would likely have activated fewer orexin neurons. In contrast, Almorexant had no effect on the cardiorespiratory response of the chemoreflex (sodium cyanide injection) or the sympathetic component of the baroreflex. Thus orexin makes a major contribution to the cardiorespiratory response evoked from the perifornical area even though orexin neurons represent only a fraction of the output of this area. Orexin neurons may also mediate cardiorespiratory responses from non-orexin neurons in the caudal hypothalamus. However, under resting conditions, blockade of orexin receptors does not affect the chemo- and baroreflexes.

Synchronized activation of sympathetic vasomotor, cardiac, and respiratory outputs by neurons in the midbrain colliculi.

The superior and inferior colliculi are believed to generate immediate and highly coordinated defensive behavioral responses to threatening visual and auditory stimuli. Activation of neurons in the superior and inferior colliculi have been shown to evoke increases in cardiovascular and respiratory activity, which may be components of more generalized stereotyped behavioral responses. In this study, we examined the possibility that there are "command neurons" within the colliculi that can simultaneously drive sympathetic and respiratory outputs. In anesthetized rats, microinjections of bicuculline (a GABA(A) receptor antagonist) into sites within a circumscribed region in the deep layers of the superior colliculus and in the central and external nuclei of the inferior colliculus evoked a response characterized by intense and highly synchronized bursts of renal sympathetic nerve activity (RSNA) and phrenic nerve activity (PNA). Each burst of RSNA had a duration of ∼300-400 ms and occurred slightly later (peak to peak latency of 41 ± 8 ms) than the corresponding burst of PNA. The bursts of RSNA and PNA were also accompanied by transient increases in arterial pressure and, in most cases, heart rate. Synchronized bursts of RSNA and PNA were also evoked after neuromuscular blockade, artificial ventilation, and vagotomy and so were not dependent on afferent feedback from the lungs. We propose that the synchronized sympathetic-respiratory responses are driven by a common population of neurons, which may normally be activated by an acute threatening stimulus.

Activation of 5-hydroxytryptamine-1A receptors suppresses cardiovascular responses evoked from the paraventricular nucleus.

Activation of central 5-hydroxytryptamine-1A (5-HT(1A)) receptors powerfully inhibits stress-evoked cardiovascular responses mediated by the dorsomedial hypothalamus (DMH), as well as responses evoked by direct activation of neurons within the DMH. The hypothalamic paraventricular nucleus (PVN) also has a crucial role in cardiovascular regulation and is believed to regulate heart rate and renal sympathetic activity via pathways that are independent of the DMH. In this study, we determined whether cardiovascular responses evoked from the PVN are also modulated by activation of central 5-HT(1A) receptors. In anesthetized rats, the increases in heart rate and renal sympathetic nerve activity evoked by bicuculline injection into the PVN were greatly reduced (by 54% and 61%, respectively) by intravenous administration of (±)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), an agonist of 5-HT(1A) receptors, but were then completely restored by subsequent administration of WAY-100635, a selective antagonist of 5-HT(1A) receptors. Microinjection of 8-OH-DPAT directly into the PVN did not significantly affect the responses to bicuculline injection into the PVN, nor did systemic administration of WAY-100635 alone. In control experiments, a large renal sympathoexcitatory response was evoked from both the PVN and DMH but not from the intermediate region in between; thus the evoked responses from the PVN were not due to activation of neurons in the DMH. The results indicate that activation of central 5-HT(1A) receptors located outside the PVN powerfully inhibits the tachycardia and renal sympathoexcitation evoked by stimulation of neurons in the PVN.

Topographical specificity of regulation of respiratory and renal sympathetic activity by the midbrain dorsolateral periaqueductal gray.

The midbrain periaqueductal gray (PAG) mediates the physiological responses to a wide range of stressors. It consists of four longitudinal columns that have different anatomical connections and functional properties. Previous anatomical and behavioral studies have led to the hypothesis that the dorsolateral PAG, but not the adjacent lateral and dorsomedial subregions, is a key center that integrates the behavioral response to acute psychological threatening stimuli. In this study, we tested whether, consistent with this hypothesis, activation of neurons in the dorsolateral PAG evokes a pattern of cardiovascular and respiratory responses that is distinct from that evoked from surrounding regions. Arterial pressure, heart rate, renal sympathetic nerve activity (RSNA), and phrenic nerve activity (PNA) were recorded simultaneously in urethane-anesthetized rats. Microinjections of very small amounts of d,l-homocysteic acid (750 pmol in 15 nl) were made in sites throughout the dorsomedial, dorsolateral, and lateral PAG subregions. Increases in RSNA of similar magnitude accompanied by small to moderate increases in arterial pressure and heart rate were evoked from all three PAG subregions. In contrast, large increases in both PNA burst rate (respiratory rate) and overall respiratory activity were evoked only from a highly circumscribed region that corresponded closely to the dorsolateral PAG subregion at an intermediate to caudal level. Within this region, the evoked increases in RSNA and respiratory activity were highly correlated (r = 0.914, P < 0.001), suggesting the possibility that a common population of "command neurons" within the dorsolateral PAG may generate both sympathetic and respiratory responses from this region.

Vasomotor and respiratory responses evoked from the dorsolateral periaqueductal grey are mediated by the dorsomedial hypothalamus.

Activation of neurons in the dorsomedial hypothalamus (DMH) evokes increases in mean arterial pressure (MAP), sympathetic activity, heart rate (HR) and respiratory activity. Results of previous studies suggest that the DMH-evoked increases in MAP and HR are mediated by neurons within the periaqueductal grey (PAG), but a recent study has proposed that the converse is also true, i.e. that increases in MAP and HR evoked from the PAG depend upon neuronal activity in the DMH. In this study in anaesthetized rats, we examined the functional relationship between the DMH and PAG in regulating renal sympathetic nerve activity (RSNA) and respiratory activity (determined by measuring phrenic nerve activity (PNA)). Bilateral microinjections of the neuronal inhibitor muscimol into the DMH virtually abolished the increases in MAP, RSNA and PNA burst rate and amplitude evoked from the dorsolateral (dl) PAG. In contrast, multiple bilateral injections of much larger (10 times) doses of muscimol or of the local anaesthetic lignocaine into sites in the dlPAG at three different rostrocaudal levels did not reduce the magnitude or duration of the sympathetic vasomotor and respiratory responses evoked by disinhibition of neurons in the DMH. Thus, sympathetic vasomotor and respiratory responses generated from the dlPAG are dependent upon neuronal activity in the DMH, but not the converse. The results of this study together with those of previous studies indicate that the PAG regulates cardiovascular and respiratory function via both ascending projections to the DMH and descending projections to the ventral medulla, that originate from different PAG subregions.

Role of 5-HT(1A) receptors in the lower brainstem on the cardiovascular response to dorsomedial hypothalamus activation.

The dorsomedial hypothalamus (DMH) is an essential brain region for the integration of the physiological response to psychological stressors. The cardiovascular components of the response include increases in blood pressure, heart rate and the activity of sympathetic nerves to the kidney, skin, brown adipose tissue, and heart. Neurons in the rostral ventrolateral medulla (RVLM) and in the region of the medullary raphe are important components of the descending pathways that mediate the cardiovascular response to the DMH activation. Activation of 5-hydroxytryptamine 1A (5-HT(1A)) receptors in the brain leads to a suppression of the cardiac and sympathetic vasomotor components of the DMH-evoked response and of the response to acute psychological stress. In this study we showed that intracisternal injection of a low dose (1 microg/kg) of the 5-HT(1A) receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), significantly reduced the increases in heart rate and renal sympathetic nerve activity evoked by disinhibition of the DMH, but had no effect on these responses when injected intravenously. Subsequent intracisternal administration of the 5-HT(1A) receptor antagonist WAY-100635 restored the DMH-evoked cardiovascular responses to levels observed before 8-OH-DPAT administration. Bilateral microinjections of 8-OH-DPAT (200 pmol on each side) into the RVLM, however, did not significantly affect the cardiovascular response to disinhibition of the DMH. These observations demonstrate that activation of 5-HT(1A) receptors within the lower brainstem, but not in the RVLM, can powerfully suppress the cardiovascular response evoked from the DMH.

Effects of disinhibition of neurons in the dorsomedial hypothalamus on central respiratory drive.

Neurons within the dorsomedial hypothalamus (DMH) play a critical role in subserving the cardiovascular and neuroendocrine response to psychological stress. An increase in respiratory activity is also a characteristic feature of the physiological response to psychological stress, but there have been few studies of the role of DMH neurons in regulating respiratory activity. In this study we determined the effects of activation of DMH neurons on respiratory activity (assessed by measuring phrenic nerve activity, PNA) and the relationship between evoked changes in respiratory activity and changes in sympathetic vasomotor activity in spontaneously breathing urethane-anesthetized rats. Microinjections of bicuculline (4-40 pmol in 20 nl) into the DMH evoked dose-dependent increases in PNA burst frequency and amplitude. These were accompanied by dose-dependent decreases in mean tracheal CO(2) levels, indicative of hyperventilation. In control experiments, microinjections of bicuculline into sites adjacent to the DMH evoked much smaller or no changes in PNA. In experiments where renal sympathetic nerve activity (RSNA) was also measured, cycle-triggered averaging revealed that RSNA under resting conditions was partly correlated with the PNA, but in response to DMH disinhibition there was no consistent change in the amplitude of the respiratory-related variations in RSNA. The results indicate that DMH neurons can exert a powerful stimulatory effect on respiratory activity, causing hyperventilation. This is not associated with an increase in the degree of coupling between PNA and RSNA, indicating that the DMH-evoked increase in RSNA is not a consequence of increased central respiratory drive.

Calculation of threshold and saturation points of sigmoidal baroreflex function curves.

The logistic sigmoid function curve provides an accurate description of the baroreflex input-output relationship and is the most commonly used equation for this purpose. The threshold (Thr) and saturation (Sat) values for the baroreflex are commonly defined as the values of mean arterial pressure (MAP) at which the reflexly controlled variable (e.g., heart rate or sympathetic nerve activity) is within 5% of the upper or lower plateau, respectively, of the sigmoid function. These values are referred to here as Thr(5%) and Sat(5%). In many studies, Thr and Sat are calculated with the equations Thr = A(3) - 2.0/A(2) and Sat = A(3) + 2.0/A(2), where A(3) is the value of MAP at the point where the reflexly controlled variable is at the midpoint of its range and A(2) is the gain coefficient. Although it is commonly stated that the values of Thr and Sat calculated with these equations represent Thr(5%) and Sat(5%), we show here that instead they are significantly greater and less than Thr(5%) and Sat(5%), respectively. Furthermore, the operating range (difference between Thr and Sat) calculated with these equations is 32% less than the difference between Thr(5%) and Sat(5%). We further show that the equations that provide correct values of Thr(5%) and Sat(5%) are Thr(5%) = A(3) - 2.944/A(2) and Sat(5%) = A(3) + 2.944/A(2). We propose that these be used as the standard equations for calculating threshold and saturation values when a logistic sigmoid function is used to model the open-loop baroreflex function curve.

Modulation of the baroreceptor reflex by the dorsomedial hypothalamic nucleus and perifornical area.

Neurons within the dorsomedial hypothalamic nucleus (DMH) and perifornical area (PeF), which lie within the classic hypothalamic defense area, subserve the cardiovascular response to psychological stress. Previous studies have shown that electrical stimulation of the hypothalamic defense area causes inhibition of the cardiac and (in some cases) sympathetic components of the baroreceptor reflex. In contrast, naturally evoked psychological stress does not appear to be associated with such inhibition. In this study, we tested the effect of specific activation of neurons within the DMH and PeF on the baroreflex control of renal sympathetic nerve activity and heart rate in urethane-anesthetized rats. Microinjection of bicuculline (a GABA(A) receptor antagonist) into the DMH caused dose-dependent increases in heart rate and renal sympathetic activity, shifted the baroreflex control of both variables to higher levels (i.e., increased the upper and lower plateaus of the baroreflex function curves, and increased the threshold, midpoint, and saturation levels of mean arterial pressure). The maximum gain of the sympathetic component of the baroreflex was also increased, while that of the cardiac component was not significantly changed. Increases in the midpoint were very similar in magnitude to the evoked increases in baseline mean arterial pressure. Microinjection of bicuculline into the PeF evoked very similar effects. The results indicate that disinhibition of neurons in the DMH/PeF region not only increases sympathetic vasomotor activity and heart rate but also resets the baroreceptor reflex such that it remains effective, without any decrease in sensitivity, over a higher operating range of arterial pressure.