Effects of acute and chronic apomorphine on sex behavior and copulation-induced neural activation in the male rat.

Apomorphine is a non-selective dopaminergic receptor agonist. Because of its pro-erectile effects, apomorphine is clinically used for treatment of erectile dysfunction. We investigated the effects of subcutaneous apomorphine administration (0.4 mg/kg rat) on sexual behavior and mating-induced Fos-expression following acute (day 1) or chronic apomorphine treatment (days 8 and 15) in sexually experienced male rats. Consistent facilitatory effects of apomorphine were observed in the reduced numbers of mounts and intromissions over time and an increased ejaculation frequency on day 1. The first post-ejaculatory interval, however, was lengthened, while other behavioral parameters were unaffected. Fos-immunoreactivity induced by acute apomorphine administration (barrel cortex, paraventricular hypothalamic nucleus, central amygdala and locus coeruleus) was strongly reduced after chronic administration. After mating, induction of Fos-immunoreactivity was observed in well-known areas like medial preoptic nucleus and the posterodorsal medial amygdaloid area. Apomorphine, however, reduced mating-induced Fos-immunoreactivity in the nucleus accumbens shell and prevented its occurrence in its core area. This remarkable apomorphine effect was not observed in any other brain area. We conclude that the behavioral (pro-erectile) effects of apomorphine are consistent over time, and that the diminished accumbens-Fos-immunoreactivity and the elongated post-ejaculatory interval may reflect a decreased response to remote cues from the estrus female.

Effects of genetic background and null mutation of 5-HT1A receptors on basal and stress-induced body temperature: modulation by serotonergic and GABAA-ergic drugs.

The stress-induced hyperthermia procedure, in which effects of drugs on basal (T(1)) and stress-induced body temperature (T(2)) are measured, predicts anxiolytic drug effect. Serotonergic drugs alter these responses and here, we studied the role of 5-HT(1A) receptors in stress-induced hyperthermia by using 5-HT(1A) receptor knockout mice. Three strains (129/Sv, Swiss Webster and C57Bl6) were used because genetic background can significantly modulate the null phenotype. We found that GABA-ergic drugs with an anxiolytic profile and stimulate alpha(2) subunit containing GABA(A) receptors, including diazepam and L838,417, result in reduced DeltaT (DeltaT=T(2)-T(1)). The alpha(1) subunit containing GABA(A) receptor was found to be primarily involved in regulation of basal body temperature T(1) and its stimulation can induce hypothermia. In addition, stimulation of 5-HT(1A) receptors by buspirone results in a reduced DeltaT, while stimulation of 5-HT(7) receptors primarily results in hypothermia. The null mutation of 5-HT(1A) receptors resulted in differences in drug-sensitivity that was further modulated by the genetic background. In particular, the null mutation on the SW and C57Bl6 backgrounds resulted in differential diazepam/L838,417 and 5-CT responses respectively. This indicates an interaction between the 5-HT(1A) receptor and genetic background and demonstrates the importance of selecting the background strain in a receptor knockout model.

5-HT1B receptors and aggression: a review.

The serotonergic (5-HT) system in the brain is involved in the modulation of offensive aggressive behavior. The dogma that activity of the 5-HT system is inversely related to aggression is obsolete now. Research on the status of the 5-HT system before, during and after the execution of aggression is ongoing but has not yet led to a clear picture about the actual functional role of the 5-HT system, the more because state versus trait aggression seems to play a pivotal role in the outcome. Pharmacological challenges pinpoint 5-HT(1A) and 5-HT(1B) receptors as key players in the modulation of offensive aggression. This review emphasizes in particular the role of postsynaptic 5-HT(1B) (hetero) receptors as a premier site to modulate offensive aggression. Modulation of the firing and 5-HT release of the serotonergic neuron, via presynaptic 5-HT(1A) (auto) receptors, presynaptic 5-HT(1B) (auto) receptors and serotonergic transporters, may also have striking influences on aggression under certain conditions. Therefore, it is hypothesized that postsynaptic 5-HT(1B) (hetero) receptors directly influence the executive, consummatory phases of agonistic behavior, whereas presynaptic serotonergic feedback systems are particularly useful in the introductory (appetitive) phases of the agonistic behavioral complex.

The selective serotonin re-uptake inhibitors fluvoxamine and paroxetine differ in sexual inhibitory effects after chronic treatment.

RATIONALE: The selective serotonin re-uptake inhibitors (SSRIs) delay orgasm and ejaculation in men. In men with rapid ejaculation it was shown that, of the SSRIs, paroxetine exerted the strongest delay in ejaculation and fluvoxamine the weakest. OBJECTIVES: In the present study, we compared the acute and chronic effects of fluvoxamine and paroxetine on sexual behavior in the male rat in order to compare their differential inhibitory effects on sexual behavior. METHODS: During a 4-week period, 48 male Wistar rats, selected on the basis of their sexual performance, were repeatedly tested for sexual behavior. All male rats received vehicle (saline, n=12), fluvoxamine (30 mg/kg, n=12), or paroxetine (10 mg/kg, n=12) daily for 2 weeks. Sexual behavioral tests were performed on days 1 (acute), 7, and 14. RESULTS: After acute oral administration, fluvoxamine and paroxetine did not inhibit sexual behavior. After 7 days and 14 days treatment, fluvoxamine mildly inhibited certain parameters of sexual behavior but ejaculation was never delayed. In contrast, paroxetine, after 7 days and particularly after 14 days treatment, strongly inhibited sexual behavior, including ejaculation. CONCLUSIONS: These results strongly concur with clinical data, suggesting that paroxetine, but not fluvoxamine, delays ejaculation. Because fluvoxamine does not delay ejaculation it may serve as an optimal treatment for depressive illness when sexual side effects, such as a delayed ejaculation, are undesired. The mechanisms whereby paroxetine and fluvoxamine, both being selective serotonin uptake inhibitors, differentially inhibit sexual behavior are unclear.

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.

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.

Lifelong CRF overproduction is associated with altered gene expression and sensitivity of discrete GABA(A) and mGlu receptor subtypes.

RATIONALE: Repeated activation of corticotropin-releasing factor (CRF) receptors is associated with increased anxiety and enhanced stress responsivity, which may be mediated via limbic GABAergic and glutamatergic transmission. OBJECTIVE: The present study investigated molecular and functional alterations in GABA(A) receptor (GABA(A)R) and metabotropic glutamate receptor (mGluR) responsivity in transgenic mice that chronically overexpress CRF. METHODS: CRF(1) receptor, GABA(A)R, and mGluR sensitivity were determined in CRF-overexpressing mice using the stress-induced hyperthermia (SIH) test. In addition, we measured mRNA expression levels of GABA(A)R α subunits and mGluRs in the amygdala and hypothalamus. RESULTS: CRF-overexpressing mice were less sensitive to the anxiolytic effects of the CRF(1) receptor antagonists CP154,526 and DMP695, the GABA(A)R α(3)-selective agonist TP003 (0-3 mg/kg) and the mGluR(2/3) agonist LY379268 (0-10 mg/kg) in the SIH test. The hypothermic effect of the non-selective GABA(A)R agonist diazepam (0-4 mg/kg) and the α(1)-subunit-selective GABA(A)R agonist zolpidem (0-10 mg/kg) was reduced in CRF-overexpressing mice. No genotype differences were found using the GABA(A)R α(5)-subunit preferential compound SH-053-2'F-R-CH(3) and mGluR(5) antagonists MPEP and MTEP. CRF-overexpressing mice showed decreased expression levels of GABA(A)R α(2) subunit and mGluR(3) mRNA levels in the amygdala, whereas these expression levels were increased in the hypothalamus. CRF-overexpressing mice also showed increased hypothalamic mRNA levels of α(1) and α(5) GABA(A)R subunits. CONCLUSIONS: We found that lifelong CRF overproduction is associated with altered gene expression and reduced functional sensitivity of discrete GABA(A) and mGluR receptor subtypes. These findings suggest that sustained over-activation of cerebral CRF receptors may contribute to the development of altered stress-related behavior via modulation of GABAergic and glutamatergic transmission.

GABA(A) receptor α subunits differentially contribute to diazepam tolerance after chronic treatment.

BACKGROUND: Within the GABA(A)-receptor field, two important questions are what molecular mechanisms underlie benzodiazepine tolerance, and whether tolerance can be ascribed to certain GABA(A)-receptor subtypes. METHODS: We investigated tolerance to acute anxiolytic, hypothermic and sedative effects of diazepam in mice exposed for 28-days to non-selective/selective GABA(A)-receptor positive allosteric modulators: diazepam (non-selective), bretazenil (partial non-selective), zolpidem (α(1) selective) and TPA023 (α(2/3) selective). In-vivo binding studies with [(3)H]flumazenil confirmed compounds occupied CNS GABA(A) receptors. RESULTS: Chronic diazepam treatment resulted in tolerance to diazepam's acute anxiolytic, hypothermic and sedative effects. In mice treated chronically with bretazenil, tolerance to diazepam's anxiolytic and hypothermic, but not sedative, effects was seen. Chronic zolpidem treatment resulted in tolerance to diazepam's hypothermic effect, but partial anxiolytic tolerance and no sedative tolerance. Chronic TPA023 treatment did not result in tolerance to diazepam's hypothermic, anxiolytic or sedative effects. CONCLUSIONS: OUR DATA INDICATE THAT: (i) GABA(A)-α(2)/α(3) subtype selective drugs might not induce tolerance; (ii) in rodents quantitative and temporal variations in tolerance development occur dependent on the endpoint assessed, consistent with clinical experience with benzodiazepines (e.g., differential tolerance to antiepileptic and anxiolytic actions); (iii) tolerance to diazepam's sedative actions needs concomitant activation of GABA(A)-α(1)/GABA(A)-α(5) receptors. Regarding mechanism, in-situ hybridization studies indicated no gross changes in expression levels of GABA(A) α(1), α(2) or α(5) subunit mRNA in hippocampus or cortex. Since selective chronic activation of either GABA(A) α(2), or α(3) receptors does not engender tolerance development, subtype-selective GABA(A) drugs might constitute a promising class of novel drugs.

5-HT1A receptor blockade reverses GABA(A) receptor alpha3 subunit-mediated anxiolytic effects on stress-induced hyperthermia.

RATIONALE: Stress-related disorders are associated with dysfunction of both serotonergic and GABAergic pathways, and clinically effective anxiolytics act via both neurotransmitter systems. As there is evidence that the GABA(A) and the serotonin receptor system interact, a serotonergic component in the anxiolytic actions of benzodiazepines could be present. OBJECTIVES: The main aim of the present study was to investigate whether the anxiolytic effects of (non-)selective alpha subunit GABA(A) receptor agonists could be reversed with 5-HT(1A) receptor blockade using the stress-induced hyperthermia (SIH) paradigm. RESULTS: The 5-HT(1A) receptor antagonist WAY-100635 (0.1-1 mg/kg) reversed the SIH-reducing effects of the non-alpha-subunit selective GABA(A) receptor agonist diazepam (1-4 mg/kg) and the GABA(A) receptor alpha(3)-subunit selective agonist TP003 (1 mg/kg), whereas WAY-100635 alone was without effect on the SIH response or basal body temperature. At the same time, co-administration of WAY-100635 with diazepam or TP003 reduced basal body temperature. WAY-100635 did not affect the SIH response when combined with the preferential alpha(1)-subunit GABA(A) receptor agonist zolpidem (10 mg/kg), although zolpidem markedly reduced basal body temperature. CONCLUSIONS: The present study suggests an interaction between GABA(A) receptor alpha-subunits and 5-HT(1A) receptor activation in the SIH response. Specifically, our data indicate that benzodiazepines affect serotonergic signaling via GABA(A) receptor alpha(3)-subunits. Further understanding of the interactions between the GABA(A) and serotonin system in reaction to stress may be valuable in the search for novel anxiolytic drugs.

Models of anxiety: stress-induced hyperthermia (SIH) in singly housed mice.

Described in this unit is the stress-induced hyperthermia (SIH) test in mice in a single-housed format. This protocol has proven reliable in detecting the anxiolytic properties of test compounds. In this test, SIH is quantified in singly housed mice using a rectal temperature measurement as the stressor. Rectal temperature is measured twice at a 10-min interval. Due to the stress experienced during the first temperature measurement, the temperature of the second measurement (T(2)) is ∼0.8° to 1.5°C higher than that of the first (T(1)). This difference in temperature (ΔT = T(2) - T(1)) is defined as the SIH response. The SIH response is reduced by different classes of anxiolytics. The SIH test is simple and robust, it does not require training of animals, and test compound effects on motor behavior, feeding, and nociception do not affect test outcome. Furthermore, it is one of few anxiety tests that focuses on the physiological component of anxiety.

Stress-induced hyperthermia is reduced by rapid-acting anxiolytic drugs independent of injection stress in rats.

BACKGROUND: Stress-induced hyperthermia (SIH) is the transient rise in body temperature after encountering a stressor. The SIH response can be blocked by administration of various anxiolytic drugs prior to inducing stress. However, a drug injection involves handling and injection stress and therefore induces a SIH response itself. In the standard SIH test, drugs are therefore injected 60 min before stress induction to allow injection-induced hyperthermia to decline. This makes it difficult to study putative anxiolytic compounds with a short half-life. The present study therefore aimed to compare the effects of standard (stressful) and stress-free anxiolytic drug administration on the subsequent SIH response with a 10-minute injection-stressor interval. METHODS: Anxiolytic drugs with short half-lives (midazolam, 8-OH-DPAT, nicotine) were injected subcutaneously in rats using either a stressful (manual injection) or stress-free injection (subcutaneous cannula) method 10 min before novel cage stress. Body temperature and locomotor activity were measured using telemetric transmitters. RESULTS: Stressful and stress-free drug administration resulted in comparable drug effects on the stress-induced hyperthermia and locomotor responses in rats. CONCLUSION: The present study shows that both stressful and stress-free drug injection shortly before a stressor results in reproducible attenuation of the SIH response in rats. In rats, a short injection-stressor interval can therefore be applied using the SIH model, enabling the study of putative anxiolytic drugs with short half-lives.

Stress-induced hyperthermia and infection-induced fever: two of a kind?

Stress exposure activates the autonomic nervous system and leads to a body temperature increase (stress-induced hyperthermia, SIH). On the other hand, an activation of the immune system in response to an infection leads to fever. Both processes increase body temperature, and the relation between SIH and infection-induced fever has been subject to debate. It is not clear whether SIH is a form of fever, or whether both processes are more or less distinct. We therefore examined the relation between SIH and infection-induced fever by looking at the effects of a GABA(A) receptor agonist (diazepam) and a prostaglandin-synthesis blocking drug (acetylsalicylic acid, aspirin) on both the SIH response and fever in rats and mice. The present study shows that the benzodiazepine diazepam but not the prostaglandin-synthesis blocking drug aspirin dose-dependently attenuated the SIH response in both rats and mice. In contrast, aspirin reduced both LPS- and IL-1beta induced fever, whereas diazepam had little effect on these fever states. Altogether, our findings support the hypothesis that stress-induced hyperthermia and infection-induced fever are two distinct processes mediated largely by different neurobiological mechanisms.

Models of anxiety: ultrasonic vocalizations of isolated rat pups.

Described in this unit is the ultrasonic distress vocalization test in rat pups. This test is a reliable method for detecting anxiolytic properties of test compounds. In this test, ultrasonic vocalizations (30 to 50 kHz) are elicited by separating rat pups of 9 to 11 days of age from their mother and littermates for a brief period of time. The test can be performed under two different stress conditions. Pups are placed in isolation in either a warm (37°C) or cold (18°C) environment for 5 min. The total number and duration of ultrasonic calls emitted by the pups during this period is used as an index of anxiety. Established anxiolytic compounds, including benzodiazepines, serotonin(1A) receptor agonists, and selective serotonin reuptake inhibitors (SSRIs), consistently reduce the number and cumulative duration of these ultrasonic distress vocalizations.