Maria V. Zaretskaia

The dorsomedial hypothalamus and the response to stress: part renaissance, part revolution.

Emotional stress provokes a stereotyped pattern of autonomic and endocrine changes that is highly conserved across diverse mammalian species. Nearly 50 years ago, a specific region of the hypothalamus, the hypothalamic defense area, was defined by the discovery that electrical stimulation in this area evoked changes that replicated this pattern. Attention later shifted to the hypothalamic paraventricular nucleus (PVN) owing to (1) elucidation of its role as the final common pathway mediating activation of the hypothalamic-pituitary-adrenal (HPA) axis, a defining feature of the stress response and (2) the finding that the PVN was the principal location of hypothalamic neurons that project directly to spinal autonomic regions. Consequently, a primary role for the PVN as the hypothalamic center integrating the autonomic and endocrine response to stress was inferred. However, our findings indicate that neurons in the nearby dorsomedial hypothalamus (DMH)--a region originally included in the hypothalamic defense area--and not in the PVN play a key role in the cardiovascular changes associated with emotional or exteroceptive stress. Indeed, excitation of neurons in the parvocellular PVN and consequent recruitment of the HPA axis that occurs in exteroceptive stress is also signaled from the DMH. Thus, the DMH may represent a higher order hypothalamic center responsible for integrating autonomic, endocrine and even behavioral responses to emotional stress.

Chemical stimulation of the dorsomedial hypothalamus evokes non-shivering thermogenesis in anesthetized rats.

Disinhibition of neurons in the dorsomedial hypothalamus (DMH) by microinjection of the GABA(A) receptor antagonist bicuculline methiodide (BMI) elicits a range of autonomic and endocrine changes resembling those seen in experimental paradigms for emotional stress. Stress in rats is also known to provoke increases in body temperature resulting in part from sympathetically mediated activation of brown fat. The purpose of the present study was to examine the effect of microinjection of BMI into the DMH on core body temperature and temperature of brown fat in rats. In anesthetized preparations, microinjection of BMI 10 pmol/50 nl into the DMH and adjoining posterior hypothalamus elicited marked and rapid increases in temperature in interscapular brown adipose tissue (IBAT) and lesser delayed elevations in rectal temperature. Similar injections into the paraventricular nucleus or ventromedial hypothalamus had no effect on either parameter. Peak increases in IBAT were significantly correlated with both peak increases in core temperature and maximal increases in heart rate that accompanied these changes, and all of these effects were abolished by systemic treatment with propranolol 1 mg/kg. In conscious rats instrumented for telemetry, microinjection of BMI 10 pmol/100 nl into the DMH evoked marked increases in core temperature as well as heart rate, locomotor activity, and plasma ACTH. The increase in core temperature occurred with a delayed time course similar to that seen in anesthetized preparations. These results indicate that activation of neurons in the DMH provokes increases in body temperature resulting in large part from sympathoadrenally-mediated activation of brown fat.

Role of the dorsomedial hypothalamus in thermogenesis and tachycardia caused by microinjection of prostaglandin E2 into the preoptic area in anesthetized rats

Prostaglandin E2 (PGE2) acts in the preoptic area (POA) of the mammalian hypothalamus to increase body temperature and heart rate. Chemical stimulation of the dorsomedial hypothalamus (DMH), a region richly innervated by neurons in the POA, evokes sympathetically-mediated increases in heart rate and body temperature. We tested the hypothesis that neurons in the DMH mediate hyperthermia and tachycardia resulting from the action of PGE2 in the POA. Microinjection of PGE2 150 pmol/15 nl into the POA in urethane-anesthetized rats caused increases in body temperature and heart rate that were sharply reversed after injection of muscimol 80 pmol/100 nl into the DMH but not after similar injection of saline vehicle. Therefore, thermogenic and tachycardic actions of PGE2 in the POA are at least in part a consequence of neuronal activity in the region of the DMH.

Microinjection of muscimol into raphe pallidus suppresses tachycardia associated with air stress in conscious rats

Sympathetically mediated tachycardia is a characteristic feature of the physiological response to emotional or psychological stress in mammals. Activation of neurons in the region of the dorsomedial hypothalamus appears to play a key role in the integration of this response. Tachycardia evoked by chemical stimulation of the dorsomedial hypothalamus can be suppressed by microinjection of the GABAA receptor agonist and neuronal inhibitor muscimol into the raphe pallidus (RP). Therefore, we tested the hypothesis that neuronal excitation in the RP mediates tachycardia seen in experimental air stress in rats. Microinjection of the GABAA receptor antagonist bicuculline methiodide (BMI) into the RP evoked increases in heart rate. At the same sites, microinjection of muscimol (80 pmol (100 nl)−1) had no effect on heart rate under baseline conditions but virtually abolished air stress‐induced tachycardia, while microinjection of lower doses (10 or 20 pmol) produced transient but clear suppression. Microinjection of muscimol at sites outside the RP had no effect on stress‐induced tachycardia, although modest suppression was apparent after injection at two sites within 500 μm of the RP. In another series of experiments, microinjection of muscimol (80 pmol (100 nl)−1) into the RP failed to influence the changes in heart rate produced by baroreceptor loading or unloading. These findings indicate that activity of neurons in the RP plays a previously unrecognized role in the generation of stress‐induced tachycardia.

Microinjection of prostaglandin E2 and muscimol into the preoptic area in conscious rats: Comparison of effects on plasma adrenocorticotrophic hormone (ACTH), body temperature, locomotor activity, and cardiovascular function

The preoptic area (POA) is thought to play an important role in thermoregulation and fever. Local application of prostaglandin E2 (PGE2) to this region elicits increases in core body temperature, heart rate, and plasma levels of adrenocorticotrophic hormone (ACTH). Similar effects on body temperature and heart rate have also been reported after local application of the GABAA receptor agonist muscimol to the preoptic area. The purpose of this study was to assess and compare the effects of microinjection of PGE2 and muscimol into the preoptic area in the same chronically instrumented conscious rats on plasma levels of ACTH. Injection of either PGE2 (150 pmol/100 nL) or muscimol (20 or 80 pmol/100 nL) into the same sites in the preoptic area evoked increases in body temperature, heart rate, blood pressure, and plasma levels of ACTH, while significant increases in locomotor activity were apparent only after muscimol. These data confirm and extend previous findings and support the notion that neurons in the region of the preoptic area exert tonic inhibition on downstream mechanisms capable of increasing the activity of the hypothalamic-pituitary-adrenal (HPA) axis as well as sympathetic thermogenic and cardiac activity.

Stress- and lipopolysaccharide-induced c-fos expression and nNOS in hypothalamic neurons projecting to medullary raphe in rats: a triple immunofluorescent labeling study

Neurons in the rostral raphe pallidus (rRP) have been proposed to mediate experimental stress‐induced tachycardia and fever in rats, and projections from the dorsomedial hypothalamus (DMH) may signal their activation in these settings. Thus, we examined c‐fos expression evoked by air jet/restraint stress and restraint stress or by systemic administration of lipopolysaccharide (10 µg/kg and 100 µg/kg) as well as the distribution of the neuronal nitric oxide synthase (nNOS) in neurons retrogradely labeled from the raphe with cholera toxin B in key hypothalamic regions. Many neurons in the medial preoptic area and the dorsal area of the DMH were retrogradely labeled, and approximately half of those in the medial preoptic area and moderate numbers in the dorsal DMH were also positive for nNOS. Either stress paradigm or dose of lipopolysaccharide increased the number of c‐fos‐positive neurons and nNOS/c‐fos double‐labeled neurons in all regions examined. However, retrogradely labeled neurons positive for c‐fos were increased only in the dorsal DMH and adjoining region in both stressed and lipopolysaccharide‐treated groups, and triple‐labeled neurons were found only in this area in rats subjected to either stress paradigm. Thus, hypothalamic neurons that project to the rRP and express c‐fos in response to either experimental stress or systemic inflammation are found only in the dorsal DMH, and many of those activated by stress contain nNOS, suggesting that nitric oxide may play a role in signaling in this pathway.

Induction of Fos-immunoreactivity in the rat brain following disinhibition of the dorsomedial hypothalamus.

Activation of neurons in the dorsomedial hypothalamus (DMH) appears to play an important role in signaling the excitation of brain regions responsible for experimental fever and for many of the physiological and behavioral changes seen in experimental stress or anxiety in rats. Here, we examined the effect of disinhibition of the DMH by unilateral microinjection of bicuculline methiodide (BMI) on Fos expression in selected regions of the brain that have been implicated in anxiety and responses to stress and fever in rats. Disinhibition of the DMH resulted in dramatic increases in local Fos expression and also increased the numbers of Fos-positive neurons in the lateral septal nucleus and in both the parvocellular and magnocellular subdivisions of the paraventricular nucleus, with greater increases ipsilateral to the injection site in the DMH. However, microinjection of BMI had no significant effect on Fos expression in the bed nucleus of the stria terminalis, another forebrain area implicated in stress and anxiety. In the brainstem, disinhibition of the DMH increased Fos expression in the nucleus tractus solitarius and the ventrolateral medulla bilaterally with greater increases again ipsilateral to the site of the microinjection, and also in the midline rostral raphe pallidus. Thus, disinhibition of neurons in the DMH in conscious rats results in increases in Fos expression in selected forebrain and brainstem regions that have been implicated in stress-induced physiological changes, anxiety, and experimental fever.

The Orexin-1 receptor antagonist SB-334867 decreases sympathetic responses to a moderate dose of methamphetamine and stress

We recently discovered that inhibiting neurons in the dorsomedial hypothalamus (DMH) attenuated hyperthermia, tachycardia, hypertension, and hyperactivity evoked by the substituted amphetamine 3, 4-methylenedioxymethamphetamine (MDMA). Neurons that synthesize orexin are also found in the region of the DMH. As orexin and its receptors are involved in the regulation of heart rate and temperature, they would seem to be logical candidates as mediators of the effects evoked by amphetamines. The goal of this study was to determine if blockade of orexin-1 receptors in conscious rats would suppress cardiovascular and thermogenic responses evoked by a range of methamphetamine (METH) doses. Male Sprague-Dawley rats (n=6 per group) were implanted with telemetric transmitters measuring body temperature, heart rate, and mean arterial pressure. Animals were randomized to receive pretreatment with either the orexin-1 receptor antagonist SB-334867 (10mg/kg) or an equal volume of vehicle. Thirty minutes later animals were given intraperitoneal (i.p.) injections of either saline, a low (1mg/kg), moderate (5mg/kg) or high (10mg/kg) dose of METH. Pretreatment with SB-334867 significantly attenuated increases in body temperature and mean arterial pressure evoked by the moderate but not the low or high dose of METH. Furthermore, animals treated with SB-334867, compared to vehicle, had lower temperature and heart rate increases after the stress of an i.p. injection. In conclusion, temperature and cardiovascular responses to a moderate dose of METH and to stress appear to involve orexin-1 receptors. The failure to affect a low and a high dose of METH suggests a complex pharmacology dependent on dose. A better understanding of this may lead to the knowledge of how monoamines influence the orexin system and vice versa.

The role of orexin-1 receptors in physiologic responses evoked by microinjection of PgE2 or muscimol into the medial preoptic area

The medial preoptic area (mPOA) of the hypothalamus has long been thought to play an important role in both fever production and thermoregulation. Microinjections of prostaglandin E2 (PgE2) or the GABA(A) agonist muscimol into the mPOA cause similar increases in body temperature, heart rate, and blood pressure. Microinjections of these compounds however evoke different behavioral responses with muscimol increasing and PgE2 having no effect on locomotion. The purpose of this study was to determine the role of orexin-1 receptors in mediating these dissimilar responses. Systemic injections of the orexin-1 receptor antagonist SB-334867 reduced temperature and cardiovascular responses produced by microinjections of muscimol, but had no effect on either response produced by PgE2. SB-334867 did not significantly decrease locomotion evoked by microinjections of muscimol into the mPOA. These data suggest that there are two central nervous system circuits involved in increasing body temperature, heart rate and blood pressure: one circuit activated by muscimol, involving orexin neurons, and a separate orexin-independent circuit activated by PgE2.

Contribution of infralimbic cortex in the cardiovascular response to acute stress

The infralimbic region of the medial prefrontal cortex (IL) modulates autonomic and neuroendocrine function via projections to subcortical structures involved in the response to stress. We evaluated the contribution of the IL to the cardiovascular response evoked by acute stress. Under anesthesia (80 mg/kg ketamine-11.5 mg/kg xylazine), rats were implanted with telemetry probes or arterial lines for recording heart rate and blood pressure. Guide cannulas were implanted to target the IL for microinjection of muscimol (100 pmol/100 nl), N-methyl-d-aspartate (NMDA) (6 pmol/100 nl), or vehicle (100 nl). Microinjection of muscimol, an agonist of GABA(A) receptors, into the IL had no effect on stress-evoked cardiovascular and thermogenic changes in any of the paradigms evaluated (cage switch, restraint plus air-jet noise, or air-jet stress). However, microinjection of the excitatory amino acid NMDA into the IL attenuated the pressor and tachycardic response to air-jet stress. Pretreatment with the selective NMDA antagonist dl-2-amino-5-phosphonopentanoic acid (AP-5, 100 pmol/100 nl) blocked the effect of NMDA on the cardiovascular response to air-jet stress. We conclude that 1) the IL region is not tonically involved in cardiovascular or thermogenic control during stress or under baseline conditions, and 2) activation of NMDA receptors in the IL can suppress the cardiovascular response to acute stress exposure.