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.

Dorsomedial hypothalamus mediates autonomic, neuroendocrine, and locomotor responses evoked from the medial preoptic area.

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.

Cardiovascular and thermal responses evoked from the periaqueductal grey require neuronal activity in the 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.

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.

Microinjection of muscimol into the dorsomedial hypothalamus suppresses MDMA-evoked sympathetic and behavioral responses.

When given systemically to rats and humans, the drug of abuse 3,4 methylenedioxymethamphetamine (ecstasy, MDMA) elicits hyperthermia, hyperactivity, tachycardia, and hypertension. Chemically stimulating the dorsomedial hypothalamus (DMH), a brain region known to be involved in thermoregulation and in stress responses, causes similar effects. We therefore tested the hypothesis that neuronal activity in the DMH plays a role in MDMA-evoked sympathetic and behavioral responses by microinjecting artificial CSF or muscimol, a neuronal inhibitor, into the DMH prior to intravenous infusion of saline or MDMA in conscious rats. Core temperature, heart rate, mean arterial pressure and locomotor activity were recorded by telemetry every minute for 120 min. In rats previously microinjected with CSF, MDMA elicited significant increases from baseline in core temperature (+1.3+/-0.3 degrees C), locomotion (+50+/-6 counts/min), heart rate (+142+/-16 beats/min), and mean arterial pressure (+26+/-3 mmHg). Microinjecting muscimol into the DMH prior to MDMA prevented increases in core temperature and locomotion and attenuated increases in heart rate and mean arterial pressure. These results indicate that neuronal activity in the DMH is necessary for the sympathetic and behavioral responses evoked by MDMA.

Angiotensin-II is a putative neurotransmitter in lactate-induced panic-like responses in rats with disruption of GABAergic inhibition in the dorsomedial hypothalamus.

Intravenous sodium lactate infusions or the noradrenergic agent yohimbine reliably induce panic attacks in humans with panic disorder but not in healthy controls. However, the exact mechanism of lactate eliciting a panic attack is still unknown. In rats with chronic disruption of GABA-mediated inhibition in the dorsomedial hypothalamus (DMH), achieved by chronic microinfusion of the glutamic acid decarboxylase inhibitor L-allylglycine, sodium lactate infusions or yohimbine elicits panic-like responses (i.e., anxiety, tachycardia, hypertension, and tachypnea). In the present study, previous injections of the angiotensin-II (A-II) type 1 receptor antagonist losartan and the nonspecific A-II receptor antagonist saralasin into the DMH of "panic-prone" rats blocked the anxiety-like and physiological components of lactate-induced panic-like responses. In addition, direct injections of A-II into the DMH of these panic-prone rats also elicited panic-like responses that were blocked by pretreatment with saralasin. Microinjections of saralasin into the DMH did not block the panic-like responses elicited by intravenous infusions of the noradrenergic agent yohimbine or by direct injections of NMDA into the DMH. The presence of the A-II type 1 receptors in the region of the DMH was demonstrated using immunohistochemistry. Thus, these results implicate A-II pathways and the A-II receptors in the hypothalamus as putative substrates for sodium lactate-induced panic-like responses in vulnerable subjects.

The non-peptide CRH1-antagonist CP-154,526 elicits a paradoxical route-dependent activation of the HPA axis.

The corticotropin-releasing hormone (CRH) plays an important role in mediating physiological response to stress and is thought to be involved in the development of various psychiatric disorders. In this paper, we compare the differences between the effect of intraperitoneal (i.p.) and intraarterial (i.a.) administration of the non-peptide CRH1 antagonist CP-154,526 (CP) (10 and 20mg/kg) on plasma adrenocorticotropic hormone levels (ACTH), heart rate, MAP, and c-Fos expression in the paraventricular nucleus of the hypothalamus. Intraperitoneal, but not i.a., injection of CP resulted in an increase in plasma ACTH (from 105±13 to 278±51pg/ml after 20mg/kg). This effect was accompanied by a dramatic increase in c-Fos expression in cells immunoreactive for CRH in the paraventricular nucleus of the hypothalamus. When the drug was administered i.p., CP-induced activation of the HPA appears to mask the inhibitory effect of CP on stress-induced ACTH secretion, an effect which was readily apparent when the drug was given i.a. Intraperitoneal administration of CP also increased the baseline MAP which may account for previous reports that treatment with this drug attenuated the increases associated with stress. CP given by either route had no effect on baseline heart rate or stress-induced tachycardia. Thus, in all studies in which CP 154,526 is given, the route of delivery must be given careful consideration.

Microinjection of muscimol into caudal periaqueductal gray lowers body temperature and attenuates increases in temperature and activity evoked from the dorsomedial hypothalamus.

Microinjection of the neuronal inhibitor muscimol into the midbrain lateral/dorsolateral periaqueductal gray (l/dlPAG) suppresses increases in heart rate (HR) and mean arterial pressure (MAP) evoked by microinjection of the GABA(A) receptor antagonist bicuculline methiodide (BMI) into the dorsomedial hypothalamus (DMH) in rats. Injection of BMI into the DMH also increases body temperature (Tco) and motor activity. Here, our goal was to extend previous findings by examining the effect of microinjection of muscimol into the PAG on these thermogenic and behavioral responses in conscious freely moving rats. Microinjection of muscimol (300 pmol and 1 nmol) alone into the l/dlPAG reduced baseline Tco without affecting activity, HR, or MAP. Similar injection of a dose that failed to alter baseline Tco (100 pmol) suppressed the increases in Tco evoked from the DMH and significantly attenuated DMH-induced increases in locomotor activity. Whereas microinjection of 1 nmol muscimol into the ldlPAG abolished the increases in Tco evoked from the DMH and in fact lowered body temperature to a degree similar to that seen after this dose of muscimol alone, 1 nmol muscimol at adjacent sites outside the targeted region of the PAG had no significant effect on DMH-induced increases in Tco or any other parameter. These results indicate a role for neuronal activity in the l/dlPAG in (1) the temperature and behavioral responses to disinhibition of neurons in the DMH, and (2) the maintenance of basal body temperature in conscious freely moving rats.

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-induced cardiac stimulation and fever: common hypothalamic origins and brainstem mechanisms.

Our past results provide considerable evidence that activation of neurons somewhere in the region of the dorsomedial hypothalamus (DMH) plays a key role in the generation of many of the effects typically seen in "emotional" stress in rats, including activation of the hypothalamic-pituitary-adrenal (HPA) axis, the neuroendocrine hallmark of the generalized response to stress, and sympathetically mediated tachycardia. More recently, we demonstrated that (1) the tachycardia resulting either from chemical stimulation of the DMH or from experimental stress is markedly attenuated by microinjection of the GABAA receptor agonist muscimol, a neuronal inhibitor, into the medullary raphe pallidus (RP); and (2) the specific subregion of the DMH mediating stimulation-induced tachycardia corresponds to the dorsal hypothalamic area (DHA), a site where neurons projecting to the RP are densely concentrated. Thus, the pathway from neurons in the DHA to sympathetic premotor neurons in the RP may constitute a key relay mediating the increases in heart rate seen in emotional stress--a role that had never been proposed previously for either of these regions. Instead, sympathetic premotor neurons were known to exist in the RP but had been most closely associated with sympathetic thermoregulatory mechanisms, including activation of brown fat, the principal means for nonshivering thermogenesis in rats, and cutaneous vasoconstriction in the tail, an important method of conserving body heat in this species. These sympathetic effects serve to maintain body temperature in a cold environment or to increase it in fever--and are typically accompanied by tachycardia. Interestingly, we and others have now shown that (1) disinhibition of neurons in the DMH also increases body temperature, at least in part through activation of brown fat, (2) microinjection of the neuronal inhibitor muscimol into the DMH reduces experimental fever and the associated tachycardia in rats. We hypothesize that activation of neurons in the DMH mediates both the increased body temperature and cardiac stimulation produced in rats by experimental "emotional" stress and fever, and that these effects are mediated in large part through direct projections to sympathetic premotor neurons in the RP. Thus, this pathway may constitute a common effector circuit upon which a variety of forebrain inputs converge in response to diverse environmental challenges.

Characterization of the relationship between spontaneous locomotor activity and cardiovascular parameters in conscious freely moving rats.

In freely behaving rats, variations in heart rate (HR) and blood pressure (BP) are coupled closely with changes in locomotor activity (Act). We have attempted to characterize this relationship mathematically. In 10- and 16-week-old rats, HR, BP and Act were recorded telemetrically every minute for 2 days under 12h:12h light-dark cycling. After examining data for individual rats, we found that the relationship between Act and HR could be approximated by the negative exponential function HR(Act)=HRmax-(HRmax-HRmin)∗exp(-Act/Acte), where HRmax, HRmin, and Acte are constants. These constants were calculated separately for light and dark periods by non-linear curve fitting. HR corresponding to maximal locomotion was similar during the light and dark phases, while HR at rest during the dark phase was higher than during the light phase. The range of HR variability associated with Act during the dark phase was similar in young and older animals, but minimal HR was significantly lower in older rats. The relationship between Act and BP was approximated with a similar function. We have found no differences between BP at rest and at maximal locomotion between light and dark and between 10-week and 16-week-old rats. Our results indicate that in rats, cardiovascular parameters are coupled to locomotion to a high degree; however both the HR and the BP reach maximal values when locomotor activity is relatively low. We also found that the phase of daily cycle affects HR in conscious rats independent of locomotor activity.

Yohimbine is a 5-HT1A agonist in rats in doses exceeding 1 mg/kg.

Yohimbine is a prototypical alpha2-adrenergic receptor antagonist. Due to its relatively high selectivity, yohimbine is often used in experiments whose purpose is to examine the role of these receptors. For example, yohimbine has been employed at doses of 1-5 mg/kg to reinstate drug-seeking behavior after extinction or to antagonize general anesthesia, an effects presumably being a consequence of blocking alpha2-adrenergic receptors. In this report we characterized dose-dependent autonomic and behavioral effects of yohimbine and its interaction with an antagonist of 5-HT1A receptors, WAY 100,635. In low doses (0.5-2 mg/kg i.p.) yohimbine induced locomotor activation which was accompanied by a tachycardia and mild hypertension. Increasing the dose to 3-4.5 mg/kg reversed the hypertension and locomotor activation and induced profound hypothermia. The hypothermia as well as the suppression of the locomotion and the hypertension could be reversed by the blockade of 5-HT1A receptors with WAY 100635. Our data confirm that yohimbine possesses 5-HT1A properties, and demonstrated that in doses above 1mg/kg significantly activate these receptors.

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.

Synaptic and membrane properties of neurons in the dorsomedial hypothalamus.

Studies in intact rats have shown that the dorsomedial hypothalamus (DMH) plays a key role in generating stress-induced physiologic changes, including activation of the hypothalamic-pituitary-adrenal axis through direct projections to paraventricular hypothalamic nucleus (PVN). However, little is known about the cellular properties of DMH neurons. We employed whole-cell patch-clamp recording techniques to characterize membrane properties and spontaneous post-synaptic currents (PSCs) in DMH neurons, including those projecting to PVN (identified by prior injection of DiI into PVN), in rat hypothalamic slices. DMH neurons (n=86 total) had uniform membrane properties. However, PVN-projecting neurons (n=32) had higher action potential (AP) thresholds, and fired fewer APs in response to current injection. Spontaneous PVN-projecting neurons (n=20) also fired APs at lower rates (4.8+/-0.6 Hz) than spontaneous neurons of unknown projection (n=38; 7.3+/-1.1 Hz). Spontaneous PSCs were observed in all neurons: One population expressed rapid decay characteristics (1.5-2.0 ms) and was blocked by non-NMDA ionotropic glutamate receptor antagonists NBQX or CNQX. Remaining PSCs reversed near E(Cl), were blocked by the GABA(A) receptor antagonists picrotoxin or bicuculline methiodide (BMI), and had longer decay time constants (4.5-6.0 ms) that were modulated by pentobarbital. Tetrodotoxin markedly reduced the frequency of PSCs sensitive to NBQX but not to BMI. Thus, DMH is made up of electrophysiologically similar neurons and PVN-projecting neurons are less excitable than neurons of unknown projection. Furthermore, as suggested by studies in intact rats, neurons in the DMH, including those projecting to the PVN, are regulated by tonic GABA(A) and non-NMDA glutamate receptor-mediated synaptic transmission.

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.

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.

Independent of 5-HT1A receptors, neurons in the paraventricular hypothalamus mediate ACTH responses from MDMA.

Acute and chronic complications from the substituted amphetamine 3,4-methylenedioxymethamphetamine (MDMA) are linked to activation of the hypothalamic-pituitary-adrenal (HPA) axis. How MDMA activates the HPA axis is not known. HPA responses to stress are known to be mediated through the paraventricular (PVH) hypothalamus and to involve serotonin-1a (5-HT1A) receptors. We sought to determine if the PVH and 5-HT1A receptors were also involved in mediating HPA responses to MDMA. Rats were pretreated with either saline or a 5-HT1A antagonist, WAY-100635 (WAY), followed by a systemic dose of MDMA (7.5mg/kg i.v.). Animals pretreated with WAY had significantly lower plasma ACTH concentrations after MDMA. To determine if neurons in the PVH were involved, and if their involvement was mediated by 5-HT1A receptors, rats implanted with guide cannulas targeting the PVH were microinjected with the GABAA receptor agonist muscimol, aCSF, or WAY followed by MDMA. Compared to aCSF, microinjections of muscimol significantly attenuated the MDMA-induced rise in plasma ACTH (126 vs. 588pg/ml, P=<0.01). WAY had no effect. Our data demonstrates that neurons in the PVH, independent of 5-HT1A receptors, mediate ACTH responses to MDMA.

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.

Stress-free microinjections in conscious rats.

Microinjections are a major tool in modern neuroscience. Microinjection techniques in conscious animals typically involve four steps: (1) animal adapts to experimental setup; (2) injection system is filled and the microinjector is carefully inserted; (3) a drug solution is injected; (4) 1-2 min later the microinjector is carefully removed. Steps 2 and 4 are difficult to perform in rodents without disturbing the animal. This disruption can cause stress and accompanying tachycardia and hyperthermia - unwanted artifacts in physiological research. To reduce these effects, we altered the traditional approach. Our procedure of microinjection consisted of the following steps: (1) we filled the injection setup and fixed the microinjector in its guide cannula; (2) allowed an animal to adapt to the setup; (3) performed an experiment including microinjection(s); (4) removed the microinjector after the experiment was complete. The key change we incorporated was a 1m long piece of tubing with a small internal diameter; it allowed us to inject nanoliter volumes through the injector which had been placed into the guide cannula in advance. This way we avoided the usual manipulations related to microinjection, and minimized extraneous disturbances to the rat. In this report we describe the details of this technique in conscious rats and provide examples of the effects and the reproducibility of a 100 nL drug injection on cardiovascular function.

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.