Towards the Integrative Theory of Alzheimer's Disease: Linking Molecular Mechanisms of Neurotoxicity, Beta-amyloid Biomarkers, and the Diagnosis.

INTRODUCTION: A major gap in amyloid-centric theories of Alzheimer's disease (AD) is that even though amyloid fibrils per se are not toxic in vitro, the diagnosis of AD clearly correlates with the density of beta-amyloid (Aß) deposits. Based on our proposed amyloid degradation toxicity hypothesis, we developed a mathematical model explaining this discrepancy. It suggests that cytotoxicity depends on the cellular uptake of soluble Aß rather than on the presence of amyloid aggregates. The dynamics of soluble beta-amyloid in the cerebrospinal fluid (CSF) and the density of Aß deposits is described using a system of differential equations. In the model, cytotoxic damage is proportional to the cellular uptake of Aß, while the probability of an AD diagnosis is defined by the Aß cytotoxicity accumulated over the duration of the disease. After uptake, Aß is concentrated intralysosomally, promoting the formation of fibrillation seeds inside cells. These seeds cannot be digested and are either accumulated intracellularly or exocytosed. Aß starts aggregating on the extracellular seeds and, therefore, decreases in concentration in the interstitial fluid. The dependence of both Aß toxicity and aggregation on the same process-cellular uptake of Aß-explains the correlation between AD diagnosis and the density of amyloid aggregates in the brain. METHODS: We tested the model using clinical data obtained from the Alzheimer's Disease Neuroimaging Initiative (ADNI), which included records of beta-amyloid concentration in the cerebrospinal fluid (CSF-Aß42) and the density of beta-amyloid deposits measured using positron emission tomography (PET). The model predicts the probability of AD diagnosis as a function of CSF-Aß42 and PET and fits the experimental data at the 95% confidence level. RESULTS: Our study shows that existing clinical data allows for the inference of kinetic parameters describing beta-amyloid turnover and disease progression. Each combination of CSF-Aß42 and PET values can be used to calculate the individual's cellular uptake rate, the effective disease duration, and the accumulated toxicity. We show that natural limitations on these parameters explain the characteristic distribution of the clinical dataset for these two biomarkers in the population. CONCLUSION: The resulting mathematical model interprets the positive correlation between the density of Aß deposits and the probability of an AD diagnosis without assuming any cytotoxicity of the aggregated beta-amyloid. To the best of our knowledge, this model is the first to mechanistically explain the negative correlation between the concentration of Aß42 in the CSF and the probability of an AD diagnosis. Finally, based on the amyloid degradation toxicity hypothesis and the insights provided by mathematical modeling, we propose new pathophysiology-relevant biomarkers to diagnose and predict AD.

Patients with Alzheimer's disease have an increased removal rate of soluble beta-amyloid-42.

Senile plaques, which are mostly composed of beta-amyloid peptide, are the main signature of Alzheimer's disease (AD). Two main forms of beta-amyloid in humans are 40 and 42-amino acid, long; the latter is considered more relevant to AD etiology. The concentration of soluble beta-amyloid-42 (Aß42) in cerebrospinal fluid (CSF-Aß42) and the density of amyloid depositions have a strong negative correlation. However, AD patients have lower CSF-Aß42 levels compared to individuals with normal cognition (NC), even after accounting for this correlation. The goal of this study was to infer deviations of Aß42 metabolism parameters that underlie this difference using data from the Alzheimer's Disease Neuroimaging Initiative cohort. Aß42 is released to the interstitial fluid (ISF) by cells and is removed by several processes. First, growth of insoluble fibrils by aggregation decreases the concentration of soluble beta-amyloid in the ISF. Second, Aß42 is physically transferred from the brain to the CSF and removed with the CSF flow. Finally, there is an intratissue removal of Aß42 ending in proteolysis, which can occur either in the ISF or inside the cells after the peptide is endocytosed. Unlike aggregation, which preserves the peptide in the brain, transfer to the CSF and intratissue proteolysis together represent amyloid removal. Using a kinetic model of Aß42 turnover, we found that compared to NC subjects, AD patients had dramatically increased rates of amyloid removal. A group with late-onset mild cognitive impairment (LMCI) also exhibited a higher rate of amyloid removal; however, this was less pronounced than in the AD group. Estimated parameters in the early-onset MCI group did not differ significantly from those in the NC group. We hypothesize that increased amyloid removal is mediated by Aß42 cellular uptake; this is because CSF flow is not increased in AD patients, while most proteases are intracellular. Aß cytotoxicity depends on both the amount of beta-amyloid internalized by cells and its intracellular conversion into toxic products. We speculate that AD and LMCI are associated with increased cellular amyloid uptake, which leads to faster disease progression. The early-onset MCI (EMCI) patients do not differ from the NC participants in terms of cellular amyloid uptake. Therefore, EMCI may be mediated by the increased production of toxic amyloid metabolites.

Intracellular ion changes induced by the exposure to beta-amyloid can be explained by the formation of channels in the lysosomal membranes.

In this manuscript, we reassess the data on beta-amyloid-induced changes of intracellular ions concentrations published previously by Abramov et al. (2003, 2004). Their observations made using high-resolution confocal microscopy with fast temporal resolution of images formed by fluorescent ion-sensitive fluorescent probes in living cells represent an unequivocal support for the amyloid channel theory. However, closer look reveals multiple facts which cannot be explained by channel formation in plasma membrane. Recently proposed amyloid degradation toxicity hypothesis provides the interpretation to these facts by considering that channels are formed in the lysosomal membranes.

Membrane channel hypothesis of lysosomal permeabilization by beta-amyloid.

Alzheimer's disease (AD) is the most common cause of dementia affecting millions of people. Neuronal death in AD is initiated by oligomeric amyloid-ß (Aß) peptides. Recently, we proposed the amyloid degradation toxicity hypothesis, which explains multiple major observations associated with AD including autophagy failure and a decreased metabolism. According to the hypothesis, the key event in the cellular toxicity of amyloid is the formation of non-selective membrane channels in lysosomal membranes by amyloid fragments that are produced by the digestion of Aß previously absorbed by endocytosis. Electrophysiological data suggest that amyloid-formed channels have different sizes, which can be explained by the fact that channel creating barrel-shaped amyloid aggregates can consist of different number of monomers. To estimate the ability of channels to leak molecules of various molecular weights, we modeled the channels as saline-filled cylinders in non-conductive membranes that pass spheres with a density of average globular proteins. As a basis, we used the conductance distribution taken from the previously published experimental dataset, in which single channels with electrical conductance of up to one nanosiemens were registered. Our calculations show that channels with such a giant conductance can allow for passing macromolecules such as large as lysosomal cathepsins implicated in the activation of apoptosis. The formation of giant channels is disproportionally promoted in an acidic environment. Also, amyloid fragments leaking from permeabilized lysosomes can reach the internal leaflet of the plasma membrane and permeabilize it. We conclude that while dissipation of the proton gradient by any (even smallest) amyloid channels readily explains lysosomal failure, the relatively rare events of lysosomal permeabilization to large macromolecules can be an additional mechanism of cellular death induced by exposure to Aß.

Mini-review: Amyloid degradation toxicity hypothesis of Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of dementia affecting millions of people. Neuronal death in AD is initiated by oligomeric amyloid-ß (Aß) peptides. The amyloid channel hypothesis readily explains the primary molecular damage but does not address major observations associated with AD such as autophagy failure and decreased metabolism. The amyloid degradation toxicity hypothesis provides the interpretation as a sequence of molecular events. Aß enters a cell by endocytosis, and the endocytic vesicle is merged with a lysosome. Lysosomal peptidases degrade the peptide. Fragments form membrane channels in lysosomal membranes that have a significant negative charge due to the presence of acidic phospholipids. Amyloid channels can transfer various ions (including protons) and even relatively large compounds, which explains lysosomal permeabilization. The neutralization of lysosomal content inactivates degradation enzymes, results in an accumulation of undigested amyloid, and stalls autophagy. Inadequate quality control of mitochondria is associated with an increased production of reactive oxygen species and decreased energy production. Also, the passage of lysosomal proteases through rare extremely large channels results in cell death. Proposed hypothesis identifies biochemical pathways involved in the initiation and progression of cellular damage induced by beta-amyloid and provides new potential pharmacological targets to treat Alzheimer's disease.

Flow cytometry method to quantify the formation of beta-amyloid membrane ion channels.

It is accepted that the cytotoxicity of beta-amyloid is mediated by its oligomers. Amyloid peptides can form ion channels in cell membranes and allow calcium and other ions to enter cells. In this project, we developed a technique to quantify the appearance of calcium in liposomes and applied this technique to study the effect of amyloid peptides on the permeability of membranes. Calcium influx was monitored in liposomes made of phosphatidylcholine (PC) or phosphatidylserine (PS) with an addition of a lipid-soluble dye DiD and containing fluorescent calcium-sensitive probe Fluo-3. The intensity of fluorescence of individual liposomes was measured using a flow cytometer. Calcium ionophore ionomycin served as a positive control. The addition of micromolar concentrations of short fragments of amyloid-beta (Aß25-35) permeabilized a significant number of PS liposomes. This effect was not observed in PC liposomes. Our data supports the hypothesis that the ion channel formation by amyloid peptide is dependent on electrostatic interactions. High concentrations of Aß25-35 (above 20 µM) increased signal intensity in a recording channel corresponding to the calcium-sensing probe. However, this phenomenon was also observed in Ca2+-free conditions and even in liposomes without Fluo-3, so we interpreted it as an artifact. Using the described technique, we were not able to detect the formation of calcium channels by several other amyloid peptides. Considering that liposomes appeared resistant to reasonable concentrations of solvents, we expect that described flowmetric technique can be used in high-throughput screening applications.

Degradation Products of Amyloid Protein: Are They The Culprits?

OBJECTIVES: Beta-amyloid (Aß) peptides are most toxic to cells in oligomeric form. It is commonly accepted that oligomers can form ion channels in cell membranes and allow calcium and other ions to enter cells. The activation of other mechanisms, such as apoptosis or lipid peroxidation, aggravates the toxicity, but it itself can result from the same initial point, that is, ion disturbance due to an increased permeability of membranes. However, experimental studies of membrane channels created by Aß are surprisingly limited. METHODS: Here, we report a novel flow cytometry technique which can be used to detect increased permeability of membranes to calcium induced by the exposure to amyloid peptides. Calcium entry into the liposome is monitored using calcium-sensitive fluorescent probe. Undamaged lipid membranes are not permeable to calcium. Liposomes that are prepared in a calcium-free medium become able to accumulate calcium in a calcium-containing medium only after the formation of channels. RESULTS: Using this technique, we demonstrated that the addition of short amyloid fragment Ab25-35, which is known for its extreme toxicity on cultured neurons, readily increased membrane permeability to calcium. However, neither similarly sized peptide Ab22-35 nor full-length peptide Aß1-42 were producing channels. The formation of channels was observed in the membranes made of phosphatidylserine, a negatively charged lipid, but not in membranes made of the neutral phosphatidylcholine. DISCUSSION: We have analyzed several issues which could be critical for understanding the pathogenesis of Alzheimer's disease, specifically 1) the need for a negatively charged membrane to produce the ion channel; 2) the potential role of the aggregated form in cellular toxicity of Aß peptides; 3) channelforming ability of multiple degradation products of amyloid; 4) non-specificity of ion channels formed by amyloid peptides. Potential targets of channel-forming oligomers appear to be intracellular and are organelles well-known for dysfunction in Alzheimer's disease (mitochondria and lysosomes). In fact, lysosomes can also be the producers of degraded amyloid. Provided speculations support the hypothesis that neuronal toxicity can be caused by the degradation products of beta-amyloid.

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.

Tissue oxidative metabolism can increase the difference between local temperature and arterial blood temperature by up to 1.3oC: Implications for brain, brown adipose tissue, and muscle physiology.

Tissue temperature increases, when oxidative metabolism is boosted. The source of nutrients and oxygen for this metabolism is the blood. The blood also cools down the tissue, and this is the only cooling mechanism, when direct dissipation of heat from the tissue to the environment is insignificant, e.g., in the brain. While this concept is relatively simple, it has not been described quantitatively. The purpose of the present work was to answer two questions: 1) to what extent can oxidative metabolism make the organ tissue warmer than the body core, and, 2) how quickly are changes in the local metabolism reflected in the temperature of the tissue? Our theoretical analysis demonstrates that, at equilibrium, given that heat exchange with the organ is provided by the blood, the temperature difference between the organ tissue and the arterial blood is proportional to the arteriovenous difference in oxygen content, does not depend on the blood flow, and cannot exceed 1.3oC. Unlike the equilibrium temperature difference, the rate of change of the local temperature, with respect to time, does depend on the blood flow. In organs with high perfusion rates, such as the brain and muscles, temperature changes occur on a time scale of a few minutes. In organs with low perfusion rates, such changes may have characteristic time constants of tens or hundreds of minutes. Our analysis explains, why arterial blood temperature is the main determinant of the temperature of tissues with limited heat exchange, such as the brain.

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 ±â€¯3 min vs 15 ±â€¯2 min). Bicuculline-perfused animals also had higher temperatures at exhaustion (41.4 ±â€¯0.2 °C vs 40.0 ±â€¯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.

Circadian variability of body temperature responses to methamphetamine.

Vital parameters of living organisms exhibit circadian rhythmicity. Although rats are nocturnal animals, most of the studies involving rats are performed during the day. The objective of this study was to examine the circadian variability of the body temperature responses to methamphetamine. Body temperature was recorded in male Sprague-Dawley rats that received intraperitoneal injections of methamphetamine (Meth, 1 or 5 mg/kg) or saline at 10 AM or at 10 PM. The baseline body temperature at night was 0.8°C higher than during the day. Both during the day and at night, 1 mg/kg of Meth induced monophasic hyperthermia. However, the maximal temperature increase at night was 50% smaller than during the daytime. Injection of 5 mg/kg of Meth during the daytime caused a delayed hyperthermic response. In contrast, the same dose at night produced responses with a tendency toward a decrease of body temperature. Using mathematical modeling, we previously showed that the complex dose dependence of the daytime temperature responses to Meth results from an interplay between inhibitory and excitatory drives. In this study, using our model, we explain the suppression of the hyperthermia in response to Meth at night. First, we found that the baseline activity of the excitatory drive is greater at night. It appears partially saturated and thus is additionally activated by Meth to a lesser extent. Therefore, the excitatory component causes less hyperthermia or becomes overpowered by the inhibitory drive in response to the higher dose. Second, at night the injection of Meth results in reduction of the equilibrium body temperature, leading to gradual cooling counteracting hyperthermia.

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.

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.

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.

Exercise activates compensatory thermoregulatory reaction in rats: a modeling study.

The importance of exercise is increasingly emphasized for maintaining health. However, exercise itself can pose threats to health such as the development of exertional heat shock in warm environments. Therefore, it is important to understand how the thermoregulation system adjusts during exercise and how alterations of this can contribute to heat stroke. To explore this we measured the core body temperature of rats (Tc) running for 15 min on a treadmill at various speeds in two ambient temperatures (Ta = 25°C and 32°C). We assimilated the experimental data into a mathematical model that describes temperature changes in two compartments of the body, representing the muscles and the core. In our model the core body generates heat to maintain normal body temperature, and dissipates it into the environment. The muscles produce additional heat during exercise. According to the estimation of model parameters, at Ta = 25°C, the heat generation in the core was progressively reduced with the increase of the treadmill speed to compensate for a progressive increase in heat production by the muscles. This compensation was ineffective at Ta = 32°C, which resulted in an increased rate of heat accumulation with increasing speed, as opposed to the Ta = 25°C case. Interestingly, placing an animal on a treadmill increased heat production in the muscles even when the treadmill speed was zero. Quantitatively, this "ready-to-run" phenomenon accounted for over half of the heat generation in the muscles observed at maximal treadmill speed. We speculate that this anticipatory response utilizes stress-related circuitry.

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.

Orexinergic neurotransmission in temperature responses to methamphetamine and stress: mathematical modeling as a data assimilation approach.

EXPERIMENTAL DATA: Orexinergic neurotransmission is involved in mediating temperature responses to methamphetamine (Meth). In experiments in rats, SB-334867 (SB), an antagonist of orexin receptors (OX1R), at a dose of 10 mg/kg decreases late temperature responses (t > 60 min) to an intermediate dose of Meth (5 mg/kg). A higher dose of SB (30 mg/kg) attenuates temperature responses to low dose (1 mg/kg) of Meth and to stress. In contrast, it significantly exaggerates early responses (t < 60 min) to intermediate and high doses (5 and 10 mg/kg) of Meth. As pretreatment with SB also inhibits temperature response to the stress of injection, traditional statistical analysis of temperature responses is difficult. MATHEMATICAL MODELING: We have developed a mathematical model that explains the complexity of temperature responses to Meth as the interplay between excitatory and inhibitory nodes. We have extended the developed model to include the stress of manipulations and the effects of SB. Stress is synergistic with Meth on the action on excitatory node. Orexin receptors mediate an activation of on both excitatory and inhibitory nodes by low doses of Meth, but not on the node activated by high doses (HD). Exaggeration of early responses to high doses of Meth involves disinhibition: low dose of SB decreases tonic inhibition of HD and lowers the activation threshold, while the higher dose suppresses the inhibitory component. Using a modeling approach to data assimilation appears efficient in separating individual components of complex response with statistical analysis unachievable by traditional data processing methods.

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.

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.