Joseph A. DiMicco

Previous studies suggest that sympathetic responses evoked from the preoptic area in anesthetized rats require activation of neurons in the dorsomedial hypothalamus. Disinhibition of neurons in the dorsomedial hypothalamus in conscious rats produces physiological and behavioral changes resembling those evoked by microinjection of muscimol, a GABA_A receptor agonist and neuronal inhibitor, into the medial preoptic area. We tested the hypothesis that all of these effects evoked from the medial preoptic area are mediated through neurons in the dorsomedial hypothalamus by assessing the effect of bilateral microinjection of muscimol into the DMH on these changes. After injection of vehicle into the dorsomedial hypothalamus, injection of muscimol into the medial preoptic area elicited marked increases in heart rate, arterial pressure, body temperature, plasma ACTH, and locomotor activity and also increased c-Fos expression in the hypothalamic paraventricular nucleus, a region known to control the release of ACTH from the adenohypophysis. Prior bilateral microinjection of muscimol into the dorsomedial hypothalamus produced a modest depression of baseline heart rate and body temperature but completely abolished all changes evoked from the medial preoptic area. Microinjection of muscimol just anterior to the dorsomedial hypothalamus had no effect on autonomic and neuroendocrine changes evoked from the medial preoptic area. Thus, activity of neurons in the dorsomedial hypothalamus mediates a diverse array of physiological and behavioral responses elicited from the medial preoptic area, suggesting that the latter region represents an important source of inhibitory tone to key neurons in the dorsomedial hypothalamus.

Stimulation of neurons in the lateral/dorsolateral periaqueductal grey (l/dlPAG) produces increases in heart rate (HR) and mean arterial pressure (MAP) that are, according to traditional views, mediated through projections to medullary autonomic centres and independent of forebrain mechanisms. Recent studies in rats suggest that neurons in the l/dlPAG are downstream effectors responsible for responses evoked from the dorsomedial hypothalamus (DMH) from which similar cardiovascular changes and increase in core body temperature (T_co) can be elicited. We hypothesized that, instead, autonomic effects evoked from the l/dlPAG depend on neuronal activity in the DMH. Thus, we examined the effect of microinjection of the neuronal inhibitor muscimol into the DMH on increases in HR, MAP and T_co produced by microinjection of N‐methyl‐d‐aspartate (NMDA) into the l/dlPAG in conscious rats. Microinjection of muscimol alone modestly decreased baseline HR and MAP but failed to alter T_co. Microinjection of NMDA into the l/dlPAG caused marked increases in all three variables, and these were virtually abolished by prior injection of muscimol into the DMH. Similar microinjection of glutamate receptor antagonists into the DMH also suppressed increases in HR and abolished increases in T_co evoked from the PAG. In contrast, microinjection of muscimol into the hypothalamic paraventricular nucleus failed to reduce changes evoked from the PAG and actually enhanced the increase in T_co. Thus, our data suggest that increases in HR, MAP and T_co evoked from the l/dlPAG require neuronal activity in the DMH, challenging traditional views of the place of the PAG in central autonomic neural circuitry.

Microinjection of the neuronal inhibitor muscimol into the dorsomedial hypothalamus (DMH) suppresses increases in heart rate (HR), mean arterial pressure (MAP), and circulating levels of adrenocorticotropic hormone (ACTH) evoked in air jet stress in conscious rats. Similar injection of muscimol into the caudal region of the lateral/dorsolateral periaqueductal gray (l/dlPAG) reduces autonomic responses evoked from the DMH, leading to the suggestion that neurons in the l/dlPAG may represent a descending relay for DMH-induced increases in HR and MAP. Here, we examined the role of neuronal activity in the caudal l/dlPAG on the increases in MAP, HR, and plasma ACTH seen in air jet stress in rats. Microinjection of muscimol into the caudal l/dlPAG reduced stress-induced increases in HR and MAP, while identical injections into sites just dorsal or into the rostral l/dlPAG had no effect. Microinjection of a combination of the glutamate receptor antagonists 2-amino-5-phosphonopentanoate (AP5) and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) into the caudal l/dlPAG decreased stress-induced increases in HR alone only at the end of the 20-min stress period but significantly accelerated return to baseline. Surprisingly, microinjection of muscimol into the caudal l/dlPAG also reduced the stress-induced increase in plasma ACTH by 51%. Compared with unstressed control rats, rats exposed to air jet stress exhibited ∼3 times the number of Fos-positive neurons in the l/dlPAG. These findings suggest that neurons in the l/dlPAG are activated in air jet stress and that this activity contributes to increases in HR, MAP, and plasma ACTH.

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.

Neurons in the dorsomedial hypothalamus (DMH) play key roles in physiological responses to exteroceptive (“emotional”) stress in rats, including tachycardia. Tachycardia evoked from the DMH or seen in experimental stress in rats is blocked by microinjection of the GABA_Areceptor agonist muscimol into the rostral raphe pallidus (rRP), an important thermoregulatory site in the brain stem, where disinhibition elicits sympathetically mediated activation of brown adipose tissue (BAT) and cutaneous vasoconstriction in the tail. Disinhibition of neurons in the DMH also elevates core temperature in conscious rats and sympathetic activity to least significant difference interscapular BAT (IBAT) and IBAT temperature in anesthetized preparations. The latter effects are blocked by microinjection of muscimol into the rRP, while microinjection of muscimol into either the rRP or DMH suppresses increases in sympathetic nerve activity to IBAT, IBAT temperature, and core body temperature elicited either by microinjection of PGE₂into the preoptic area (an experimental model for fever), or central administration of fentanyl. Neurons concentrated in the dorsal region of the DMH project directly to the rRP, a location corresponding to that of neurons transsynaptically labeled from IBAT. Thus these neurons control nonshivering thermogenesis in rats, and their activation signals its recruitment in diverse experimental paradigms. Evidence also points to a role for neurons in the DMH in thermoregulatory cutaneous vasoconstriction, shivering, and endocrine adjustments. These directions provide intriguing avenues for future exploration that may expand our understanding of the DMH as an important hypothalamic site for the integration of autonomic, endocrine, and behavioral responses to diverse challenges.

The study by tanaka and mcallen published in this issue of American Journal of Physiology–Regulatory Integrative and Comparative Physiology ([32][1]) addresses a subject of considerable interest in the field of thermoregulation and one that for many years has been central to our understanding of fever, the regulated increase in body temperature evoked by immune challenge. The notion that prostaglandin E2 (PGE2) plays a key role in the signaling that translates a peripheral immune challenge into changes in the activity of specific thermoregulatory circuits is well established, but exactly where (and how) this signaling occurs is not entirely clear. The median preoptic area of the hypothalamus is certainly one such site (for review, see Ref. [2][2]). However, PGE2 may reach other regions of the brain after being synthesized either locally, e.g., in the vascular endothelium of blood vessels throughout the central nervous system ([35][3]), or peripherally ([9][4]) so that relatively high levels appear in the cerebrospinal fluid in fever ([8][5], [10][6]). This and the fact that receptors for PGE2 are found elsewhere in the brain ([16][7], [29][8], [39][9]) invites the possibility that the action of PGE2 at other sites may well contribute to the febrile response. In this regard, it is somewhat surprising that the specific hypothesis examined–that one such site might be the medullary raphé–had not been tested previously. The evidence in support of such a suggestion represents convergent findings from several investigative directions over the past decade and makes a compelling case. First, neurons in the region of the raphé pallidus (RP) have emerged as key regulators of sympathetic activity responsible for both metabolic activation of interscapular brown adipose tissue (IBAT), the major thermogenic organ in rats, and cutaneous vasoconstriction in the tail, a principal means of conservation (and, conversely, dissipation) of heat in this species ([13][10], [14][11], [22][12], [23][13], [31][14]). Second, central administration of PGE2 results in increased fos expression, a marker for neuronal activation, in the region of the RP ([17][15], [18][16]). Third, neurons in the RP that are polysynaptically connected to IBAT express EP3 receptors ([36][17]), a prostaglandin E receptor subtype shown to be important in fever ([20][18], [34][19]). Finally, microinjection of the neuronal inhibitor muscimol into the RP blocks the increases in IBAT sympathetic nerve activity and fever evoked by central administration of PGE2 in anesthetized rats ([15][20], [17][15]). Taken together, these data point to neurons in the RP as obvious additional potential sites of action for the central febrigenic actions of PGE2. Nevertheless, as is often the case for attractive ideas that are solidly rooted in previous experimental findings, the data of Tanaka and McAllen ([32][1]) that are relevant to this point are decidedly negative. Microinjection of PGE2 directly into the region of the medullary RP failed to increase either IBAT or tail vasoconstrictor sympathetic nerve activity, thus appearing to preclude the possibility of a contribution for this mechanism to the febrile response in rats. However, several caveats are in order before the results of this potentially important study can be put into proper perspective. One issue relates to the doses of PGE2 and the volumes of injectate tested and the inherent difficulty involved in proving a negative. The authors employed doses ranging from 150–500 ng (i.e., 0.43–1.4 nmol) injected in volumes ranging from 300 nl to 1 μl. These doses are higher, in some cases by orders of magnitude, than those employed in similar microinjection studies to evoke febrile responses elsewhere in the central nervous system ([1][21], [11][22], [12][23], [27][24], [37][25]). In fact, the minimal dose injected (150 ng) is 15 times the dose shown to increase body temperature when injected intraceroventricularly in unanesthetized rats ([30][26]). In addition, the doses used span a relatively narrow range in pharmacologic terms–less than a log. The inverted U-shaped dose response curve is not uncommon in the realm of pharmacology, and it is to basic principles of this discipline we must always attend when any substance, endogenous or not, is used as a drug as is PGE2 here. Simply put, if a little is sufficient to elicit an effect, more may not always be better (see Refs. [7][27], [28][28], [33][29]), and it is not inconceivable that the authors may have missed a response that would have been apparent at a lower dose. Among the potential mechanisms through which microinjection of large doses could fail to elicit an effect might be spread or diffusion of the injected agent to other sites of action in adjacent regions, a potential complication of any microinjection study. In this case, this possibility is enhanced not only by the high doses injected but also by the facts that PGE2 is highly lipid soluble and is not catabolized in the mammalian brain ([19][30]). These attributes, combined with the relatively large volumes of injectate used in the study, should translate into the ability of high doses of microinjected PGE2 to reach and act at relatively distant sites in the brain. For example, EP3 and perhaps other subtypes of PGE receptors are detectable on neurons in the nearby ventrolateral medulla ([16][7], [39][9]), a site where, according to a preliminary report by Cao and Morrison ([6][31]), chemical disinhibition can suppress any increases in sympathetic nerve activity to IBAT evoked through the RP. Thus, it is not unreasonable to propose that hyperthermic effects of PGE2 acting at the RP may have been obscured by its action at this or other brainstem sites and that a careful dose response analysis might unmask them. While these caveats leave room for refinement and confirmation, the core finding of Tanaka and McAllen ([32][1]) that PGE2...

Disinhibition of neurons in the region of the dorsomedial hypothalamus (DMH) elicits sympathetically mediated tachycardia in rats through activation of the brain stem raphe pallidus (RP), and this same mechanism appears to be largely responsible for the increases in heart rate (HR) seen in air jet stress in this species. Neurons projecting to the RP from the DMH are said to be concentrated in a specific subregion, the dorsal hypothalamic area (DA). Here, we examined the hypothesis that the location of RP-projecting neurons in the DA correspond to the sites at which microinjection of bicuculline methiodide (BMI) evokes the greatest increases in HR. To determine the distribution of RP-projecting neurons in the DA, cholera toxin B was injected in the RP in four rats. A consistent pattern of retrograde labeling was seen in every rat. In the hypothalamus, RP-projecting neurons were most heavily concentrated midway between the mammillothalamic tract and the dorsal tip of the third ventricle dorsal to the dorsomedial hypothalamic nucleus ∼3.30 mm caudal to bregma. In a second series of experiments, the HR response to microinjections of BMI (2 pmol/5 nl; n = 76) was mapped at sites in the DA and surrounding areas in 22 urethane-anesthetized rats. All injection sites were located from 2.56 to 4.16 mm posterior to bregma, and the microinjections that evoked the largest increase in HR (i.e., >100 beats/min in some instances) were located in a region where RP-projecting neurons were most densely concentrated. Thus RP-projecting neurons in the DA may mediate DMH-induced tachycardia and thus play a role in stress-induced cardiac stimulation.