Orexinergic Neurotransmission in Temperature Responses to Amphetamines

poster abstractDerivatives of amphetamines are widely abused all over the world. After long-term use cognitive, neurophysiological, and neuroanatomical deficits have been reported. Neurophysiological deficits are enhanced by hyperthermia, which itself is major mortality factor in drug abusers. Temperature responses to injections of methamphetamine are multiphasic and include both hypothermic and hyperthermic phases, which are highly dependent on ambient temperature and previous exposure to the drug. Also, amphetamine derivatives differentially affect various neuromediator systems, such as dopaminergic, noradrenergic and serotonergic. Temperature responses to methamphetamine (Meth) at room temperature have non-trivial dose-dependence, which is far from being understood. Intermediate doses of Meth cause less hyperthermia than both low and high doses of the drug. Also, maxima of all responses have different latency responses to low and high doses are virtually immediate, while a response to an intermediate dose appears to be delayed. In our previous modeling study we demonstrated that such dose-dependence could be explained by interaction of inhibitory and excitatory drives induced by Meth [1]. Recently, we have published data on the involvement of orexinergic neurotransmission in Meth-induced temperature responses [2] where the low dose (10 mg/kg, i.p.) of SB-334867 (SB), an antagonist of the first type of orexin receptors (ORX1), was injected 30 min prior to various doses of Meth. While this dose of antagonist clearly suppressed the response to low (1 mg/kg) and intermediate (5 mg/kg) doses of Meth, the effect was statistically significant only at the late phase (t > 60 min) of the response to intermediate dose. At the early phase (t < 60 min) any drug-related changes were marred by stress-induced temperature fluctuations resulting from two intraperitoneal injections. In a separate set of experiments a high dose of the same antagonist (30 mg/kg, i.p.) suppressed the effect of low doses of Meth even more, but in contrast, it significantly amplified the responses to the higher doses (5 and 10 mg/kg) of Meth. Understanding the mechanism that differentially affect excitatory and inhibitory components of temperature responses can have profound importance for explaining cases of life-threatening hyperthermia after Meth administration. Therefore, we performed a mathematical modeling study to provide mechanistic interpretation of SB action. Our previous model [1] was created to describe Meth-sensitive compartments and dynamics of the neural populations defining temperature responses for various doses of Meth. We hypothesized that a specific distribution of orexin receptors over the structures involved in the neural control of temperature is responsible for the complex dependence of the Meth-induced responses on the dose of orexin antagonist. To test this hypothesis we extended the model by incorporating ORX receptors that mediated Meth- and stress-dependent inputs. We showed that the low dose of antagonist almost fully suppresses the responses to both stress and intermediate doses of Meth by disruption of the corresponding inputs to the control structures. This allows hypothesizing that the excitatory component in temperature response to both stress and low dose of Meth is mediated by ORX1 receptors. Amplification of the response to the high dose of Meth at high dose of the antagonist points out to the involvement of a mechanism different from ORX1 receptor blockade. We speculate that at high doses SB becomes non-specific to ORX1 receptors and starts affecting ORX2 receptors. Further, ORX2 activation disinhibits the structure activated by high doses of Meth, which underlies the exaggerated responses to high doses of Meth at the presence of a high dose of SB. We conclude that both excitatory and inhibitory components in temperature responses to Meth administration and stress are mediated by orexinergic pathways. Non-specificity of SB at high doses to ORX1 receptors manifests itself in additional suppression of inhibition resulting in facilitation of the responses to high-doses of Meth

Automatic analysis of treadmill running to estimate times to fatigue and exhaustion in rodents

Introduction: The determination of fatigue and exhaustion in experimental animals is complicated by the subjective nature of the measurement. Typically, it requires an observer to watch exercising animals, e.g. rats running on the treadmill, and to identify the time of the event. In this study, we hypothesized that automatic analysis of the time-averaged position of a rat on a treadmill could be an objective way for estimating times to fatigue and exhaustion. To test this hypothesis, we compared these times measured by a human observer to the results of an automated video tracking system. Methods: Rats, previously familiarized to running on the treadmill, ran at a fixed speed with zero incline, until exhaustion. The experiments were performed at either room temperature (24 °C) or in a hot environment (32 °C). Each experiment was video recorded. A trained observer estimated the times to fatigue and exhaustion. Then, video tracking software was used to determine the position of the animals on the treadmill belt. The times to fatigue and exhaustion were determined, based on the position on the treadmill using predefined criteria. Results: Manual scores and the average position on the treadmill had significant correlation. Both the observer and the automated video tracking determined that exercise in a hot environment, compared with the exercise at room temperature, results in shorter times to exhaustion and fatigue. Also, estimates of times made by the observer and the automated video tracking were not statistically different from each other. Discussion: A similarity between the estimates of times to fatigue and exhaustion made by the observer and the automated technique suggests that video tracking of rodents running on a treadmill can be used to determine both parameters in experimental studies. Video tracking technique allows for a more objective measure and would allow for an increased performance in experimentation. The Supplemental information to this manuscript contains an Excel file, which includes the code in Virtual Basic with freeware license, to process and visualize running data and automatically estimate the times to fatigue and exhaustion. Instructions for the software are also included

Treadmill running restores MDMA-mediated hyperthermia prevented by inhibition of the dorsomedial hypothalamus

The contribution of exercise to hyperthermia mediated by MDMA is not known. We recently showed that inhibiting the dorsomedial hypothalamus (DMH) attenuated spontaneous locomotion and hyperthermia and prevented deaths in rats given MDMA in a warm environment. The goal of this study was to confirm that restoring locomotion through a treadmill would reverse these effects thereby confirming that locomotion mediated by the DMH contributes to MDMA-mediated hyperthermia. Rats were randomized to receive bilateral microinjections, into the region of the DMH, of muscimol (80pmol/100nl) or artificial CSF followed by a systemic dose of either MDMA (7.5mg/kg, i.v.) or saline. Immediately after the systemic injection, rats were placed on a motorized treadmill maintained at 32°C. Rats were exercised at a fixed speed (10m/min) until their core temperature reached 41°C. Our results showed that a fixed exercise load abolished the decreases in temperature and mortality, seen previously with inhibition of the DMH in freely moving rats. Therefore, locomotion mediated by neurons in the DMH is critical to the development of hyperthermia from MDMA

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 100635. 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 1 mg/kg significantly activate these receptors

Inhibition of the dorsomedial hypothalamus, but not the medullary raphe pallidus, decreases hyperthermia and mortality from MDMA given in a warm environment.

The central mechanisms through which MDMA mediates life-threatening hyperthermia when taken in a warm environment are not well described. It is assumed that MDMA alters normal thermoregulatory circuits resulting in increased heat production through interscapular brown adipose tissue (iBAT) and decreased heat dissipation through cutaneous vasoconstriction. We studied the role of the dorsomedial hypothalamus (DMH) and medullary raphe pallidus (mRPa) in mediating iBAT, tail blood flow, and locomotor effects produced by MDMA. Rats were instrumented with guide cannulas targeting either the DMH or the mRPa-brain regions involved in regulating iBAT and cutaneous vascular beds. In all animals, core temperature and locomotion were recorded with surgically implanted telemetric transmitters; and additionally either iBAT temperature (via telemetric transmitter) or tail artery blood flow (via tail artery Doppler cuff) were also recorded. Animals were placed in an environmental chamber at 32°C and microinjected with either control or the GABA agonist muscimol (80pmol) followed by an intravenous injection of saline or MDMA (7.5 mg kg-1). To prevent undue suffering, a core temperature of 41°C was chosen as the surrogate marker of mortality. Inhibition of the DMH, but not the mRPa, prevented mortality and attenuated hyperthermia and locomotion. Inhibition of either the DMH or the mRPa did not affect iBAT temperature increases or tail blood flow decreases. While MDMA increases iBAT thermogenesis and decreases heat dissipation through cutaneous vasoconstriction, thermoregulatory brain regions known to mediate these effects are not involved. Rather, the finding that inhibiting the DMH decreases both locomotion and body temperature suggests that locomotion may be a key central contributor to MDMA-evoked hyperthermia

Amphetamine enhances endurance by increasing heat dissipation

Athletes use amphetamines to improve their performance through largely unknown mechanisms. Considering that body temperature is one of the major determinants of exhaustion during exercise, we investigated the influence of amphetamine on the thermoregulation. To explore this, we measured core body temperature and oxygen consumption of control and amphetamine‐trea ted rats running on a treadmill with an incrementally increasing load (both speed and incline). Experimental results showed that rats treated with amphetamine (2 mg/kg) were able to run significantly longer than control rats. Due to a progressively increasing workload, which was matched by oxygen consumption, the control group exhibited a steady increase in the body temperature. The administration of amphetamine slowed down the temperature rise (thus decreasing core body temperature) in the beginning of the run without affecting oxygen consumption. In contrast, a lower dose of amphetamine (1 mg/kg) had no effect on measured parameters. Using a mathematical model describing temperature dynamics in two compartments (the core and the muscles), we were able to infer what physiological parameters were affected by amphetamine. Modeling revealed that amphetamine administration increases heat dissipation in the core. Furthermore, the model predicted that the muscle temperature at the end of the run in the amphetamine‐treated group was significantly higher than in the control group. Therefore, we conclude that amphetamine may mask or delay fatigue by slowing down exercise‐induced core body temperature growth by increasing heat dissipation. However, this affects the integrity of thermoregulatory system and may result in potentially dangerous overheating of the muscles

MUC1 gene overexpressed in breast cancer: structure and transcriptional activity of the MUC1 promoter and role of estrogen receptor alpha (ERα) in regulation of the MUC1 gene expression

BACKGROUND: The MUC1 gene encodes a mucin glycoprotein(s) which is basally expressed in most epithelial cells. In breast adenocarcinoma and a variety of epithelial tumors its transcription is dramatically upregulated. Of particular relevance to breast cancer, steroid hormones also stimulate the expression of the MUC1 gene. The MUC1 gene directs expression of several protein isoforms, which participate in many crucial cell processes. Although the MUC1 gene plays a critical role in cell physiology and pathology, little is known about its promoter organization and transcriptional regulation. The goal of this study was to provide insight into the structure and transcriptional activity of the MUC1 promoter. RESULTS: Using TRANSFAC and TSSG soft-ware programs the transcription factor binding sites of the MUC1 promoter were analyzed and a map of transcription cis-elements was constructed. The effect of different MUC1 promoter regions on MUC1 gene expression was monitored. Different regions of the MUC1 promoter were analyzed for their ability to control expression of specific MUC1 isoforms. Differences in the expression of human MUC1 gene transfected into mouse cells (heterologous artificial system) compared to human cells (homologous natural system) were observed. The role of estrogen on MUC1 isoform expression in human breast cancer cells, MCF-7 and T47D, was also analyzed. It was shown for the first time that synthesis of MUC1/SEC is dependent on estrogen whereas expression of MUC1/TM did not demonstrate such dependence. Moreover, the estrogen receptor alpha, ERα, could bind in vitro estrogen responsive cis-elements, EREs, that are present in the MUC1 promoter. The potential roles of different regions of the MUC1 promoter and ER in regulation of MUC1 gene expression are discussed. CONCLUSION: Analysis of the structure and transcriptional activity of the MUC1 promoter performed in this study helps to better understand the mechanisms controlling transcription of the MUC1 gene. The role of different regions of the MUC1 promoter in expression of the MUC1 isoforms and possible function of ERα in this process has been established. The data obtained in this study may help in development of molecular modalities for controlled regulation of the MUC1 gene thus contributing to progress in breast cancer gene therapy

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

Disinhibiting neurons in the dorsomedial hypothalamus delays the onset of exertional fatigue and exhaustion in rats exercising in a warm environment

Stimulants cause hyperthermia, in part, by increasing heat generation through exercise. Stimulants also delay the onset of fatigue and exhaustion allowing animals to exercise longer. If used in a warm environment, this combination (increased exercise and decreased fatigue) can cause heat stroke. The dorsomedial hypothalamus (DMH) is involved in mediating locomotion from stimulants. Furthermore, inhibiting the DMH decreases locomotion and prevents hyperthermia in rats given stimulants in a warm environment. Whether the DMH is involved in mediating exercise-induced fatigue and exhaustion is not known. We hypothesized that disinhibiting neurons in the dorsomedial hypothalamus (DMH) would delay the onset of fatigue and exhaustion in animals exercising in a warm environment. To test this hypothesis, we used automated video tracking software to measure fatigue and exhaustion. In rats, using wearable mini-pumps, we demonstrated that disinhibiting the DMH, via bicuculline perfusion (5 µM), increased the duration of exercise in a warm environment as compared to control animals (25 ± 3 min vs 15 ± 2 min). Bicuculline-perfused animals also had higher temperatures at exhaustion (41.4 ± 0.2 °C vs 40.0 ± 0.4 °C). Disinhibiting neurons in the DMH also increased the time to fatigue. Our data show that the same region of the hypothalamus that is involved in mediating locomotion to stimulants, is also involved in controlling exhaustion and fatigue. These findings have implications for understanding the cause and treatment of stimulant-induced-hyperthermia