Theo J.J. Zethof

Stress-induced hyperthermia and anxiety: pharmacological validation.

When mammals, including man, are confronted with a stressful event, their core body temperature rises, stress-induced hyperthermia. In mice, the stress-induced hyperthermia procedure has been developed to measure antistress or anxiolytic-like effects of psychoactive drugs. Group-housed and singly housed versions of the stress-induced hyperthermia generate comparable results. Because the number of animals needed to perform an experiment is much lower in the singly housed versus the group-housed procedure, the former is the test of choice for pharmacological testing. A typical stress-induced hyperthermia test starts with an injection 60 min before the first rectal temperature measurement (T(1)), followed by a second temperature measurement (T(2)) 10-15 min later. The difference DeltaT (=T(2)-T(1)) is the stress-induced hyperthermia. The procedure also measures the intrinsic activity of drugs on the basal body temperature and DeltaT is relatively independent from the intrinsic temperature effects of drugs. Anxiolytic drugs (benzodiazepines, 5-HT(1A) receptor agonists, alcohol) reduce DeltaT suggestive of anxiolytic-like effects. Because the parameter measured for anxiety in the stress-induced hyperthermia procedure is not dependent on locomotor activity, like in almost all other anxiety tests, the stress-induced hyperthermia procedure is an attractive addition to tests in the anxiety field. Because the stress-induced hyperthermia is also present with a comparable pharmacological profile in females, this procedure has a wide species and gender validity. The procedure was applied in various genetically modified mice [5-HT(1A) and 5-HT(1B) receptor knockout (KO) mice and corticotropin-releasing hormone overexpressing (CRH-OE) mice] to study phenotypic influences of the various mutations on aspects of anxiety. The stress-induced hyperthermia test in singly housed male and female mice appears a useful and extremely simple test to measure effects of drugs on certain aspects of anxiety or to help to determine phenotypic differences in mutant mice.

Stress-Induced Hyperthermia in Singly Housed Mice

The stress-induced hyperthermia (SIH) paradigm in group-housed mice allows screening of putative anxiolytic drugs. The group-housed SIH was adapted to singly housed animals in order to drastically reduce the number of animals used. The effect of various stressors on rectal temperature was measured in order to find a simple and reliable test procedure. Repeated, but not single disturbance of animals resulted in a strong hyperthermia (deltaT) within 10 min. Similar hyperthermic responses were obtained after immobilization for 1 min or rectal temperature measurement itself. Neither a 120 dB acoustic stimulus, nor repeated 1 mA footshocks led to a temperature change, but 2 mA electric footshocks led to hyperthermia. The final test paradigm chosen involved repeated temperature measurement at a 10 min interval, thus providing both information on basal temperature and deltaT in each animal within a short time frame. Repeated temperature measurements at 10 min intervals revealed a maximum hyperthermia after approximately 30 min, but up to 70% of the hyperthermia is already present 10 min after the first measurement. Repeated use of animals at successive daily or weekly intervals resulted in a gradual increase of both the basal temperature and the temperature 10 min later. At short inter-test intervals (one day) deltaT also decreased, whereas weekly intervals did not affect the amplitude of deltaT. Prior injection of the animals resulted in modest hyperthermia, that returned to baseline after 60 min. The anxiolytics diazepam and 5-HT1A receptor agonist flesinoxan dose-dependently suppressed SIH. The antidepressant amitriptyline lowered temperature levels but did not affect deltaT. The SIH model in singly housed mice appears a fast and reproducible screening test for anxiolytic activity. Compared to the group-housed version, the singly-housed SIH enabled a drastic reduction in the number of animals used.

Stress-induced hyperthermia in mice: A methodological study

When the rectal temperature of group-housed mice is measured sequentially, the temperature of the last measured mouse is higher than that of the first mouse. This phenomenon is called stress-induced hyperthermia (SIH). We varied several experimental parameters to elucidate the mechanism behind this SIH. SIH was stable and found by all technicians performing the experiments. The large intertechnician difference in the mean rectal temperature could be eliminated by training in an identical fixation and handling technique. SIH was both independent of the number of handling days preceding the experiment and of the number of disturbances (0, 1, 2, or 5) implied on the mice per minute. The percentage of hyperthermic mice in 10-mice cages increased when the time interval between the individual measurements increased from 1 to 2, 5 or 10 min. In all groups the maximum increase was reached after an interval of approximately 10 min. SIH of mouse 10 returned time dependently in approximately 60 min to basal temperature. When SIH was tested on 2 or 5 successive days no tolerance developed. When animals were reused after 7 or 14 days SIH did not differ from day 1, implying that animals can be reused. When the number of mice was decreased from 10 to 5 mice per cage, the SIH of vehicle-treated mice was slightly lower in 5-mice cages compared to 10-mice cages. The blocking effects on SIH by anxiolytics was also less clear in 5-mice cages compared to 10-mice cages.(ABSTRACT TRUNCATED AT 250 WORDS)

Stress-induced hyperthermia in mice: Hormonal correlates

In the stress-induced hyperthermia (SIH) paradigm in group-housed male mice, the rectal temperature of last measured mice is approximately 1.5 degrees C higher than the first measured one when the temperature of each mouse is measured sequentially with an interval of 1 min. In the present study it is demonstrated that SIH is accompanied by increases in plasma ACTH, corticosterone, and glucose levels that return to baseline more or less parallel to the temperature. The simultaneous increases in temperature and plasma stress hormones strongly support the use of the SIH paradigm in mice as an animal model to study putative anti-stress or anxiolytic properties of drugs.

New animal models of anxiety

In an attempt to develop new animal models of anxiety with face and predictive validity for the spectrum of human anxiety disorders, two new animal paradigms have been described, stress-induced hyperthermia (SIH) in mice and ultrasonic pup vocalizations (UV) in rats. In SIH mice develop enhanced body temperature in anticipation of an aversive event. This SIH can be antagonized by benzodiazepines, alcohol and 5-HT1A receptor agonists, but not by specific 5-HT reuptake inhibitors (SSRIs) or 5-HT3 receptor antagonists. When rat pups are separated from their mother and littermates they produce ultrasonic sounds, indicative of a separation distress. Benzodiazepines, 5-HT1A receptor agonists and SSRIs decrease this calling, whereas 5-HT3 receptor antagonists have no effect. Antidepressants in general do not decrease pup calling because in contrast to the SSRIs, noradrenergic uptake blockers enhance calling. These two animal models of anxiety can be added to the range of anxiety models and will be of help in predicting new putative anxiolytic drugs.

Anxiolytic effects of flesinoxan in the stress-induced hyperthermia paradigm in singly-housed mice are 5-HT1A receptor mediated

In the stress-induced hyperthermia paradigm in singly-housed male mice, two sequential rectal temperature measurements reveal the basal temperature (T1) and, 10 min later, an enhanced body temperature (T2), due to the stress of the first rectal measurement. The difference T2 - T1 (deltaT) is the stress-induced hyperthermia and putatively reflects a stress-induced anxiogenic response. The full 5-HT1A receptor agonist flesinoxan ((+)-enantiomer), its (-)-enantiomer and the racemic mixture reduced stress-induced hyperthermia effects, indicating putative anxiolytic properties. The ratio of their potencies to reduce stress-induced hyperthermia was similar to their potency in receptor binding affinities for 5-HT1A receptors, supporting that the anti-hyperthermia effects are mediated by the 5-HT1A receptor. This was further substantiated when the 5-HT1A receptor antagonists WAY 100635 ((N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclo-hexane carboxamine trihydrochloride) and DU 125530 (2-[4-[4-(7-chloro-2,3-dihydro-1,4-benzodioxin-5-yl)-1-piperazinyl ]butyl]-1,2-benzisothiazol-3(2H)-one-1,1-dioxide, monomesylate) both were able to antagonize the anti-stress-induced hyperthermia effects of flesinoxan. The stress-induced hyperthermia paradigm in singly-housed mice represents a simple and robust paradigm to measure putative anxiolytic effects of drugs.

Neuroendocrine effects of diazepam and flesinoxan in the stress-induced hyperthermia test in mice

In the stress-induced hyperthermia (SIH) paradigm in mice, both a benzodiazepine receptor agonist, diazepam, and a 5-HT1A receptor agonist, flesinoxan, reduced the stress-induced increase in rectal temperature. The SIH procedure itself enhanced plasma ACTH and corticosterone levels but not plasma glucose levels. Diazepam (3, 6, and 12 mg/kg p.o.) did neither affect basal plasma ACTH, corticosterone, or glucose levels, nor did it suppress the stress-induced rises in these parameters. Flesinoxan (1, 3, and 10 mg/kg p.o.) enhanced plasma ACTH and corticosterone concentrations under nonstress conditions but did not affect the stress-induced increases in ACTH and corticosterone secretion. No clear effects of flesinoxan on plasma glucose levels were found. Our results indicate that in mice the anxiolytic effects of diazepam and flesinoxan in the SIH paradigm are not paralleled by a blockade of stress-induced increases in plasma ACTH, corticosterone, and glucose levels.

GABAA–benzodiazepine receptor complex ligands and stress-induced hyperthermia in singly housed mice

Stress-induced hyperthermia (SIH) in singly housed mice, in which the rectal temperature of a mouse is measured twice with a 10-min interval, enables to study the effects of a drug on the basal (T1) and on the stress-enhanced temperature (T2), 10 min later, using the rectal procedure as stressor. SIH (T2-T1) reflects a stress-induced phenomenon sensitive to stress- or anxiety-modifying effects of drugs. Several benzodiazepine agonists (diazepam, chlordiazepoxide, oxazepam and alprazolam) dose-dependently antagonized SIH either in NMRI mice from two different breeders or in BALB/c mice. No major differences in the sensitivity for any of the drugs tested were found between strains or between substrains from different breeders. The selective BZ1 receptor agonists alpidem and zolpidem only at relatively high doses antagonized SIH, whereas flumazenil, FG7142, pentylenetetrazol and phenobarbital did not affect SIH. Alcohol antagonized SIH, and the effects of diazepam could be antagonized by flumazenil. The findings that full BZ receptor agonists have anxiolytic-like effects in the singly housed SIH paradigm are comparable to those previously found in the group-housed version. The singly housed SIH is proposed as a simple and reliable screen for detecting anxiety-like properties of drugs that is valid in every mouse strain tested so far.

A New Animal Model for Anticipatory Anxiety

Mice removed last from their home cage have a higher rectal temperature (hyperthermia) compared to those removed first from the same cage. This phenomenon was interpreted as a state of anxiety: anticipatory anxiety. This increase in rectal temperature was prevented in a dose dependent way by diazepam and serotonin-1A agonists. Several non-anxiolytic compounds did not affect the hyperthermia.

Preclinical Evidence for the Role of Serotonin Receptors in Anxiety

Animal paradigms are important to detect anxiolytic properties of psychoactive compounds. Two animal anxiety paradigms are presented, ultrasonic distress vocalizations (USV) in rat pups and stress-induced hyperthermia (SIH) in mice, to describe the putative anxiolytic properties of serotonergic ligands. Both paradigms indicate that 5-HTAreceptor agonists are potent anxiolytics, whereas specific 5-HT reuptake blockers also exert some anxiolytic properties. 5-HT02antagonists may have some anxiogenic properties (in USV), whereas 5-HT, antagonists seem to be devoid of anxiolytic properties in the paradigms used.