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Psychopharmacology
 
Psychopharmacology (Berl). 2010 August; 211(2): 123–130.
Published online 2010 June 10. doi:  10.1007/s00213-010-1895-7
PMCID: PMC2892061

5-HT1A receptor blockade reverses GABAA receptor α3 subunit-mediated anxiolytic effects on stress-induced hyperthermia

Abstract

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 GABAA 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 α subunit GABAA receptor agonists could be reversed with 5-HT1A receptor blockade using the stress-induced hyperthermia (SIH) paradigm.

Results

The 5-HT1A receptor antagonist WAY-100635 (0.1–1 mg/kg) reversed the SIH-reducing effects of the non-α-subunit selective GABAA receptor agonist diazepam (1–4 mg/kg) and the GABAA receptor α3-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 α1-subunit GABAA receptor agonist zolpidem (10 mg/kg), although zolpidem markedly reduced basal body temperature.

Conclusions

The present study suggests an interaction between GABAA receptor α-subunits and 5-HT1A receptor activation in the SIH response. Specifically, our data indicate that benzodiazepines affect serotonergic signaling via GABAA receptor α3-subunits. Further understanding of the interactions between the GABAA and serotonin system in reaction to stress may be valuable in the search for novel anxiolytic drugs.

Keywords: Interaction, Zolpidem, TP003, Benzodiazepine, Serotonin antagonist, Serotonergic, WAY-100635, WAY100,635, alpha3, alpha 3, Stress, Anxiety

Introduction

Stress-related disorders are associated with dysfunction of both serotonergic and GABAergic pathways (Akimova et al. 2009; Kalueff and Nutt 2007; Nemeroff 2003). The clinical anxiolytic effects of selective serotonin reuptake inhibitors, 5-HT1A receptor agonists and GABAA receptor agonists, indicate that both the GABAAergic as well as the serotonergic system may be involved in the pathological basis underlying anxiety disorders (Nutt 2005; Zohar and Westenberg 2000). There is evidence that the GABA and the serotonergic system interact (Fernandez-Guasti and Lopez-Rubalcava 1998; Gao et al. 1993; Lista et al. 1989), although the evidence whether it plays a role in the stress response is inconsistent (Shephard et al. 1982; Thiebot 1986). Specifically, a serotonergic component in the anxiolytic actions of benzodiazepines has been suggested (Harandi et al. 1987; Stein et al. 1977; Thiebot et al. 1984). Hence, studying the interactions of the GABAA and serotonin system in stress and anxiety could be valuable in the search for novel anxiolytic drugs.

Here, we investigate whether the anxiolytic effects of GABAA receptor agonists are dependent on 5-HT1A receptor activation using the stress-induced hyperthermia (SIH) paradigm. The SIH response is the transient rise in body temperature in response to acute stress that is mediated by the autonomic nervous system (Bouwknecht et al. 2007; Vinkers et al. 2008). Both classical benzodiazepines and 5-HT1A receptor agonists consistently reduce the SIH response (as well as basal body temperature at higher doses), whereas dopaminergic and noradrenergic systems are generally ineffective (Olivier et al. 2003). Classical (non-subunit selective) benzodiazepines bind to GABAA receptor α1-, α2-, α3-, or α5-subunits, and the various benzodiazepine effects are thought to be mediated through different GABAA receptor subtypes (Rudolph and Mohler 2006). Interactions with the serotonergic system may thus depend on the GABAA receptor composition. In the present study, we investigated whether the silent 5-HT1A receptor antagonist WAY-100635 (WAY) could alter the SIH-reducing and hypothermic effects of the non-subunit selective GABAA receptor agonist diazepam, the selective GABAA receptor α3-subunit agonist TP003 (Dias et al. 2005), and the preferential GABAA receptor α1-subunit agonist zolpidem.

Materials and methods

Animals

Eighty-four male NMRI mice (Charles River, The Netherlands) were housed in Macrolon type 3 cages enriched with bedding and nesting material under a 12-h light/12-h dark cycle (lights on from 0600 to 1800 hours) at controlled temperature (22 ± 2°C) and relative humidity (40–60%) with free access to standard food pellets and tap water. Experiments were carried out with approval of the ethical committee on animal experiments of the Faculties of Sciences, Utrecht University, The Netherlands, and in accordance with the Declaration of Helsinki.

The stress-induced hyperthermia (SIH) procedure

The SIH tests were carried out one time per week according to standard procedures (Groenink et al. 2009). A between-subject design was used, and animals were randomly allocated to an experimental group. Cages were randomly and evenly allocated over daytimes (morning to afternoon). The temperature of mice was measured by rectally inserting a thermistor probe by a length of 2 cm. Digital temperature recordings were obtained with an accuracy of 0.1°C using a Keithley 871A digital thermometer (NiCr–NiAl thermocouple). The probe, dipped into silicon oil before inserting, was held in the rectum until a stable rectal temperature had been obtained for 20 s. Animals were injected intraperitoneally with vehicle or WAY-100635 on the left flank and with vehicle, diazepam, zolpidem, or TP003 on the right flank. All drugs were injected 60 min before the first temperature measurement (T1). This first temperature measurement, representing the basal body temperature, functioned as an adequate stressor as well. The temperature was again measured 10 min later (T2), representing the stress-induced body temperature. The SIH response was calculated by subtracting T1 from T2.

Drugs

Diazepam (base), zolpidem (tartaric acid), and WAY-100635 (maleate) (N-{2-[4-(2-methoxyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl) cyclohexanecarboxamide tri-chloride) were obtained from Sigma Aldrich. TP003 was synthesized according to published methods (Dias et al. 2005; Humphries et al. 2006). An injection volume of 10 ml/kg was used for intraperitoneal injections of all drugs. WAY-100635 was dissolved in saline. Diazepam, zolpidem, and TP003 were suspended in gelatin–mannitol 0.5%/5%. Fresh solutions and suspensions were prepared each testing day. Doses of TP003 and zolpidem were based on previous SIH studies (Dias et al. 2005; Olivier et al. 2002, 2003).

Data analysis

All experiments were carried out using a between-subject design. For each individual mouse, a basal temperature (T1), an end temperature (T2), and the difference (SIH response = T2  T1) were determined. Treatment effects were evaluated using a two-way analysis of variance with explanatory factors drug1 (WAY-100635 or vehicle) and drug2 (diazepam/zolpidem/TP003 or vehicle). In addition, a post-hoc analysis was carried out comparing diazepam/zolpidem/TP003 with vehicle under both WAY-100635 and vehicle conditions using a Tukey's Honestly Significant Difference (HSD) test. A probability level of p < 0.05 was set as statistically significant.

Results

Effects on the stress-induced hyperthermia response

Diazepam 1 mg/kg and WAY-100635 (n=8–9)

WAY-100635 (WAY) significantly reversed the diazepam effect on the SIH response at all three WAY doses tested (WAY 0.1 mg/kg, WAY × diazepam interaction, F1,31 = 5.02, p < 0.05; WAY 0.3 mg/kg, WAY × diazepam interaction, F1,31 = 4.71, p < 0.05; WAY 1.0 mg/kg, WAY × diazepam interaction, F1,32 = 5.76, p < 0.05; Fig. 1a). Post-hoc analysis showed that diazepam reduced the SIH response when it was co-administered with vehicle in one out of three experiments (veh-veh vs. diazepam-veh: WAY 0.1 mg/kg, p < 0.05; WAY 0.3 mg/kg, p = 0.53, NS; WAY 1.0 mg/kg, p = 0.17, NS), but that WAY altered the diazepam-induced reduction of the SIH response (diazepam-veh vs. diazepam-WAY: WAY 0.1 mg/kg, p < 0.01; WAY 0.3 mg/kg, p < 0.05; WAY 1.0 mg/kg, p < 0.01). In contrast, diazepam had no effects when it was combined with WAY (veh-WAY vs. diazepam-WAY: WAY 0.1 mg/kg, p = 0.77, NS; WAY 0.3 mg/kg, p = 0.10, NS; WAY 1.0 mg/kg, p = 0.61, NS).

Fig. 1
Effects of (a, b) diazepam (1 and 4 mg/kg, IP, n = 8–10), (c) TP003 (1 mg/kg, IP, n = 7–10), and (d) zolpidem (10 mg/kg, IP, n = 8–10) administration in combination ...

Diazepam 4 mg/kg and WAY-100635 (n=8–10)

WAY significantly reversed the diazepam effects on the SIH response at higher doses (WAY 0.3 mg/kg, WAY × diazepam interaction, F1,32 = 3.88, p = 0.05; WAY 1.0 mg/kg, WAY × diazepam interaction, F1,36 = 4.74, p < 0.05; Fig. 1b). In contrast, WAY did not alter the diazepam effects at the lowest dose (WAY 0.1 mg/kg, WAY × diazepam interaction, F1,35 = 1.20, p = 0.28, NS). Post-hoc analysis showed that diazepam reduced the SIH response when co-administered with vehicle (veh-veh vs. diazepam-veh: WAY 0.1 mg/kg, p < 0.01; WAY 0.3 mg/kg, p < 0.05; WAY 1.0 mg/kg, p < 0.01), and that WAY altered the diazepam-induced reduction of the SIH response at the highest dose (diazepam-veh vs. diazepam-WAY: WAY 0.1 mg/kg, p = 0.31, NS; WAY 0.3 mg/kg, p = 0.28, NS; WAY 1.0 mg/kg, p < 0.05). Diazepam did not reduce the SIH response combined with WAY (veh-WAY vs. diazepam-WAY: WAY 0.1 mg/kg, p = 0.06, NS; WAY 0.3 mg/kg, p = 0.98, NS; WAY 1.0 mg/kg, p = 0.74, NS).

TP003 (1 mg/kg) and WAY-100635 (n=7–10)

WAY influenced the TP003 effect on the SIH response at the lower WAY doses (WAY 0.1 mg/kg, WAY × TP003 interaction, F1,30 = 4.56, p < 0.05; WAY 0.3 mg/kg, WAY × TP003 interaction, F1,30 = 4.87, p < 0.05; WAY 1.0 mg/kg, WAY × TP003 interaction, F1,31 = 3.38, p = 0.06, NS; Fig. 1c). Post-hoc analysis showed that TP003 reduced the SIH response when co-administered with vehicle in one out of three experiments (veh-veh vs. TP003-veh: WAY 0.1 mg/kg, p = 0.11, NS; WAY 0.3 mg/kg, p < 0.05; WAY 1.0 mg/kg, p = 0.27, NS). However, WAY altered the TP003 effects on the SIH response (TP003-veh vs. TP003-WAY: WAY 0.1 mg/kg, p < 0.01; WAY 0.3 mg/kg, p < 0.05; WAY 1.0 mg/kg, p < 0.05). In contrast, TP003 did not alter the SIH response combined with WAY (veh-WAY vs. TP003-WAY: WAY 0.1 mg/kg, p = 0.52, NS; WAY 0.3 mg/kg, p = 0.77, NS; WAY 1.0 mg/kg, p = 0.47, NS).

Zolpidem 10 mg/kg and WAY-100635 (n=8–10)

Zolpidem did not affect the SIH response in all three experiments (WAY 0.1 mg/kg, zolpidem effect, F1,32 = 0.77, p = 0.39, NS; WAY 0.3 mg/kg, zolpidem effect, F1,31 = 0.30, p = 0.87, NS; WAY 1 mg/kg, zolpidem effect, F1,32 = 0.72, p = 0.40, NS; Fig. 1d). WAY did not change the zolpidem effects at any dose (WAY 0.1 mg/kg, WAY × zolpidem interaction F1,32 = 0.001, p = 0.94, NS; WAY 0.3 mg/kg, WAY × zolpidem interaction, F1,31 = 1.63, p = 0.21, NS; WAY 1 mg/kg, WAY × zolpidem interaction, F1,32 = 0.29, p = 0.59, NS). Post-hoc analysis indicated that zolpidem did not reduce the SIH response, regardless whether it was co-administered with vehicle (veh-veh vs. zolpidem-veh: WAY 0.1 mg/kg, p = 0.95, NS; WAY 0.3 mg/kg, p = 0.99, NS; WAY 1.0 mg/kg, p = 0.97, NS) or with WAY (veh-WAY vs. zolpidem-WAY: WAY 0.1 mg/kg, p = 0.90, NS; WAY 0.3 mg/kg, p = 0.76, NS; WAY 1.0 mg/kg, p = 0.72, NS).

Effects on basal body temperature

Diazepam 1 mg/kg and WAY-100635 (n=8–9)

At higher doses, WAY appeared to enhance the temperature-reducing effects of diazepam (WAY 0.1 mg/kg, WAY × diazepam interaction, F1,31 = 0.23, p = 0.63, NS; WAY 0.3 mg/kg, WAY × diazepam interaction, F1,31 = 4.40, p < 0.05; WAY 1.0 mg/kg, WAY × diazepam interaction, F1,32 = 3.43, p = 0.07; Fig. 2a). Post-hoc analysis confirmed that diazepam reduced basal body temperature combined with WAY at higher doses (veh-WAY vs. diazepam-WAY: WAY 0.1 mg/kg, p = 0.31, NS; WAY 0.3 mg/kg, p < 0.01; WAY 1.0 mg/kg, p < 0.01) and that WAY enhanced the diazepam effects on basal body temperature at higher doses (diazepam-veh vs. diazepam-WAY: WAY 0.1 mg/kg, p = 0.80, NS; WAY 0.3 mg/kg, p < 0.01; WAY 1.0 mg/kg, p < 0.05). In contrast, diazepam did not reduce basal body temperature when co-administered with vehicle (veh-veh vs. diazepam-veh: WAY 0.1 mg/kg, p = 0.73, NS; WAY 0.3 mg/kg, p = 0.96, NS; WAY 1.0 mg/kg, p = 0.12, NS).

Fig. 2
Effects of (a, b) diazepam (1 and 4 mg/kg, IP, n = 8–10), (c) TP003 (1 mg/kg, IP, n = 7–10), and (d) zolpidem (10 mg/kg, IP, n = 8–10) administration in combination ...

Diazepam 4 mg/kg and WAY-100635 (n=8–10)

WAY enhanced the effect of diazepam on basal body temperature at higher doses (WAY 0.1 mg/kg, WAY × diazepam interaction, F1,35 = 0.91, p = 0.35, NS; WAY 0.3 mg/kg, WAY × diazepam interaction, F1,32 = 4.86, p < 0.05; WAY 1 mg/kg, WAY × diazepam interaction, F1,36 = 18.47, p < 0.001; Fig. 2b). Post-hoc analysis showed that diazepam reduced basal body temperature when it was combined with WAY (veh-WAY vs. diazepam-WAY: WAY 0.1 mg/kg, p < 0.001; WAY 0.3 mg/kg, p < 0.001; WAY 1.0 mg/kg, p < 0.001), and that WAY enhanced the diazepam effects on basal body temperature at the highest WAY dose (diazepam-veh vs. diazepam-WAY: WAY 0.1 mg/kg, p = 0.21, NS; WAY 0.3 mg/kg, p = 0.20, NS; WAY 1.0 mg/kg, p < 0.001). In contrast, diazepam did not reduce basal body temperature when it was co-administered with vehicle in two out of three experiments (veh-veh vs. diazepam-veh: WAY 0.1 mg/kg, p < 0.01; WAY 0.3 mg/kg, p = 0.37, NS; WAY 1.0 mg/kg, p = 0.65, NS).

TP003 (1 mg/kg) and WAY-100635 (n=7–10)

WAY altered the effects of TP003 on basal body temperature at all WAY doses tested (WAY 0.1 mg/kg, WAY × TP003 interaction, F1,30 = 4.54, p < 0.05; WAY 0.3 mg/kg, WAY × TP003 interaction, F1,30 = 9.01, p < 0.01; WAY 1 mg/kg, WAY × TP003 interaction, F1,31 = 4.28, p < 0.05; Fig. 2c). Post-hoc analysis showed that TP003 reduced basal body temperature when it was combined with WAY in two out of three experiments (veh-WAY vs. TP003-WAY: WAY 0.1 mg/kg, p = 0.13, NS; WAY 0.3 mg/kg, p < 0.05; WAY 1.0 mg/kg, p < 0.05) and that the TP003-WAY combination reduced basal body temperature compared to the TP003-vehicle combination (TP003-veh vs. TP003-WAY: WAY 0.1 mg/kg, p < 0.01; WAY 0.3 mg/kg, p = 0.06, NS; WAY 1.0 mg/kg, p < 0.05). In contrast, TP003 did not reduce basal body temperature when co-administered with vehicle (veh-veh vs. TP003-veh: WAY 0.1 mg/kg, p = 0.87, NS; WAY 0.3 mg/kg, p = 0.47, NS; WAY 1.0 mg/kg, p = 0.91, NS).

Zolpidem 10 mg/kg and WAY-100635 (n=8–10)

In all experiments, zolpidem reduced basal body temperature (WAY 0.1 mg/kg, zolpidem effect, F1,32 = 25.07, p < 0.001; WAY 0.3 mg/kg, zolpidem effect, F1,31 = 136.20, p < 0.001; WAY 1 mg/kg, zolpidem effect, F1,32 = 41.39, p < 0.001; Fig. 2d). WAY did not alter the zolpidem effects on body temperature (WAY 0.1 mg/kg, WAY × zolpidem interaction, F1,32 = 0.01, p = 0.98, NS; WAY 0.3 mg/kg, WAY × zolpidem interaction, F1,31 = 0.38, p = 0.54, NS; WAY 1 mg/kg, WAY × zolpidem interaction, F1,32 = 2.28, p = 0.14, NS). Post-hoc analysis indicated that zolpidem reduced basal body temperature, regardless whether it was co-administered with vehicle (WAY 0.1 mg/kg, p < 0.01; WAY 0.3 mg/kg, p < 0.01; WAY 1.0 mg/kg, p < 0.05) or with WAY (WAY 0.1 mg/kg, p < 0.01; WAY 0.3 mg/kg, p < 0.001; WAY 1.0 mg/kg, p < 0.01). Furthermore, WAY did not alter the zolpidem effect on basal body temperature (zolpidem-veh vs. zolpidem-WAY: WAY 0.1 mg/kg, p = 0.56, NS; WAY 0.3 mg/kg, p = 0.43, NS; WAY 1.0 mg/kg, p = 0.07, NS).

Discussion

The present study investigated putative GABA-serotonin interactions using the SIH paradigm. Our main finding is that the non-selective GABAA receptor agonist diazepam and the α3-subunit selective GABAA receptor agonist TP003 no longer reduced the SIH response and augmented hypothermia in the presence of the 5-HT1A receptor antagonist WAY-100635, suggesting an interaction between the activation of the GABAA receptor α3-subunits and 5-HT1A receptors. In contrast, WAY-100635 did not have any effect when it was combined with the preferential α1-subunit GABAA receptor agonist zolpidem. As WAY-100635 has no affinity for GABAA receptors (Fletcher et al. 1996), our data suggest that in the SIH paradigm, anxiolytic effects of GABAA receptor agonists may be mediated via the serotonin system. Thus, benzodiazepines may affect serotonergic signaling via α3-subunits on a distinct group of serotonergic neurons. In support, the vast majority of serotonergic neurons express GABAA receptor α3-subunit immunoreactivity but not GABAA receptor α1-subunit staining (Gao et al. 1993). This is remarkable as the α1-subunit is highly prevalent in the central nervous system.

The effects of the GABAergic drugs diazepam, TP003, and zolpidem on the SIH response and body temperature are generally in line with earlier SIH studies (Olivier et al. 2002; Vinkers et al. 2008, 2009). Diazepam effects on basal body temperature slightly varied over the experiments, which may be attributed to fluctuations in body temperature under vehicle conditions due to physiological variance, differences in environmental temperature, or the time of testing. Classical non-selective benzodiazepines enhance the inhibitory actions of GABA by binding to an allosteric site on GABAA receptors that contain α1-, α2-, α3-, or α5-subunits in combination with a β and a γ2 subunit (Rudolph and Mohler 2006). Zolpidem is approximately five- to tenfold more selective for α1-subunit-containing GABAA receptors than α23-subunit-containing receptors (Petroski et al. 2006), whereas TP003 is α3-subunit selective with low modulation via α1-, α2-, and α5-containing subtypes (Dias et al. 2005). Recently, genetic and pharmacological evidence has indicated that α-subunits may differentially contribute to the various classical benzodiazepine-induced effects such as anxiolysis, dependence, anticonvulsant activity, sedation, and amnesia (Crestani et al. 2001; Rudolph et al. 1999). Here, we confirm and extend our earlier findings suggesting a role for the GABAA receptor α1 subunit in hypothermia and a role for the GABAA receptor α2/3 subunit in reduction of the SIH response (Vinkers et al. 2009).

In the present study, WAY-100635 did not affect the SIH response in any of the experiments when it was administered alone which is in line with earlier studies (Olivier et al. 2003, 2008). The 5-HT1A receptor antagonist WAY-100635 is generally assumed to act as silent antagonist but has also been reported to exert anxiolytic or even anxiogenic effects depending on the experimental design (Cao and Rodgers 1997; Fletcher et al. 1996; Forster et al. 1995; Griebel et al. 2000; Groenink et al. 1996; Joordens et al. 1998; Stanhope and Dourish 1996). WAY-100635 has also been shown to reverse the SIH-reducing effects of 5-HT1A receptor agonists such as buspirone and flesinoxan, confirming that WAY-100635 targets 5-HT1A receptors (Iijima et al. 2007; Olivier et al. 1998). Interestingly, WAY-100635 has also been able to reverse the SIH reduction caused by the mGluR2/3 receptor antagonist MGS0039, suggesting that the 5-HT1A receptors may also be involved in the effects of glutamate receptor antagonists (Iijima et al. 2007).

WAY-100635 could reverse the α3-induced effects in the SIH paradigm by blocking presynaptic 5-HT1A receptors that disinhibit serotonin release and turnover at synaptic levels (Wesolowska et al. 2003), which may then activate postsynaptic 5-HT receptors. Electrophysiological studies show that WAY-100635 increases serotonergic neuronal activity probably by blocking 5-HT1A autoreceptors (Corradetti et al. 1996; Fornal et al. 1996; Mundey et al. 1996). In support, serotonergic raphe nuclei receive a prominent GABAergic input via distant sources as well as interneurons (Bagdy et al. 2000; Gervasoni et al. 2000; Harandi et al. 1987; Varga et al. 2001). However, this can only provide a partial explanation as WAY-100635 also augmented the benzodiazepine-induced hypothermia, putatively via an activation of α1-subunits (Vinkers et al. 2009). Also, raphe lesions did not attenuate the anticonflict activity of peripherally administered benzodiazepines, suggesting that difficulties may exist in generalizing findings from one paradigm to another (Green and Hodges 1986). Furthermore, it is striking that WAY-100635 reverses the SIH response while it augments the hypothermia. It may be hypothesized that GABAA–serotonin interactions relevant for thermoregulation exist in the preoptic area or dorsomedial hypothalamus (Dimicco and Zaretsky 2007) or, alternatively, that dorsal and median raphe projections differentially affect basal and stress-induced body temperature levels. In support, 5-HT was found to presynaptically inhibit GABA release in the thermoregulatory hypothalamic medial preoptic area, which could be blocked by a 5-HT1A receptor antagonist (Lee et al. 2008). We also cannot exclude the possibility that downstream activation of hypothalamic 5-HT1A receptors, subsequent to the direct activation of GABAA receptors, is needed to maintain the core body temperature. Thus, detailed hypotheses on the potential interactive sites of GABAA receptors and 5-HT1A receptors and their functional relevance must await further experimental analysis. Some studies have found decreased serotonin activity and turnover after benzodiazepine administration (Chase et al. 1970; Pratt et al. 1979; Stein et al. 1977; Trulson et al. 1982; Wright et al. 1992), although others have not found such effects (Shephard et al. 1982; Thiebot 1986; Thiebot et al. 1984). The present study used the SIH paradigm as an anxiolytic assay as it can repeatedly be used over the weeks without any habituation. However, the use of a single paradigm prevents a direct generalization of our results to other anxiolytic tests.

In conclusion, the present study shows that 5-HT1A receptor blockade reversed the anxiolytic effects of the non-selective GABAA receptor agonist diazepam and the α3-selective GABAA receptor agonist TP003, whereas it enhanced benzodiazepine-induced hypothermia. In contrast, these effects were not present in combination with the preferential GABAA receptor α1-subunit agonist zolpidem. Together, these data suggest that the GABAA receptor α3-subunit functionally interacts with 5-HT1A receptors of the serotonin system to exert its anxiolytic effects in the SIH paradigm.

Acknowledgments

Conflicts of interest The authors declare no financial disclosures or conflicts of interest.

Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

References

  • Akimova E, Lanzenberger R, Kasper S. The serotonin-1A receptor in anxiety disorders. Biol Psychiatry. 2009;66(7):627–635. doi: 10.1016/j.biopsych.2009.03.012. [PubMed] [Cross Ref]
  • Bagdy E, Kiraly I, Harsing LG., Jr Reciprocal innervation between serotonergic and GABAergic neurons in raphe nuclei of the rat. Neurochem Res. 2000;25:1465–1473. doi: 10.1023/A:1007672008297. [PubMed] [Cross Ref]
  • Bouwknecht JA, Olivier B, Paylor RE. The stress-induced hyperthermia paradigm as a physiological animal model for anxiety: a review of pharmacological and genetic studies in the mouse. Neurosci Biobehav Rev. 2007;31:41–59. doi: 10.1016/j.neubiorev.2006.02.002. [PubMed] [Cross Ref]
  • Cao BJ, Rodgers RJ. Influence of 5-HT1A receptor antagonism on plus-maze behaviour in mice. II. WAY 100635, SDZ 216-525 and NAN-190. Pharmacol Biochem Behav. 1997;58:593–603. doi: 10.1016/S0091-3057(97)00279-7. [PubMed] [Cross Ref]
  • Chase TN, Katz RI, Kopin IJ. Effect of diazepam on fate of intracisternally injected serotonin-C14. Neuropharmacology. 1970;9:103–108. doi: 10.1016/0028-3908(70)90053-5. [PubMed] [Cross Ref]
  • Corradetti R, Poul E, Laaris N, Hamon M, Lanfumey L. Electrophysiological effects of N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexane carboxamide (WAY 100635) on dorsal raphe serotonergic neurons and CA1 hippocampal pyramidal cells in vitro. J Pharmacol Exp Ther. 1996;278:679–688. [PubMed]
  • Crestani F, Low K, Keist R, Mandelli M, Mohler H, Rudolph U. Molecular targets for the myorelaxant action of diazepam. Mol Pharmacol. 2001;59:442–445. [PubMed]
  • Dias R, Sheppard WF, Fradley RL, Garrett EM, Stanley JL, Tye SJ, Goodacre S, Lincoln RJ, Cook SM, Conley R, Hallett D, Humphries AC, Thompson SA, Wafford KA, Street LJ, Castro JL, Whiting PJ, Rosahl TW, Atack JR, McKernan RM, Dawson GR, Reynolds DS. Evidence for a significant role of alpha 3-containing GABAA receptors in mediating the anxiolytic effects of benzodiazepines. J Neurosci. 2005;25:10682–10688. doi: 10.1523/JNEUROSCI.1166-05.2005. [PubMed] [Cross Ref]
  • Dimicco JA, Zaretsky DV. The dorsomedial hypothalamus: a new player in thermoregulation. Am J Physiol Regul Integr Comp Physiol. 2007;292:R47–R63. [PubMed]
  • Fernandez-Guasti A, Lopez-Rubalcava C. Modification of the anxiolytic action of 5-HT1A compounds by GABA-benzodiazepine agents in rats. Pharmacol Biochem Behav. 1998;60:27–32. doi: 10.1016/S0091-3057(97)00482-6. [PubMed] [Cross Ref]
  • Fletcher A, Forster EA, Bill DJ, Brown G, Cliffe IA, Hartley JE, Jones DE, McLenachan A, Stanhope KJ, Critchley DJ, Childs KJ, Middlefell VC, Lanfumey L, Corradetti R, Laporte AM, Gozlan H, Hamon M, Dourish CT. Electrophysiological, biochemical, neurohormonal and behavioural studies with WAY-100635, a potent, selective and silent 5-HT1A receptor antagonist. Behav Brain Res. 1996;73:337–353. doi: 10.1016/0166-4328(96)00118-0. [PubMed] [Cross Ref]
  • Fornal CA, Metzler CW, Gallegos RA, Veasey SC, McCreary AC, Jacobs BL. WAY-100635, a potent and selective 5-hydroxytryptamine1A antagonist, increases serotonergic neuronal activity in behaving cats: comparison with (S)-WAY-100135. J Pharmacol Exp Ther. 1996;278:752–762. [PubMed]
  • Forster EA, Cliffe IA, Bill DJ, Dover GM, Jones D, Reilly Y, Fletcher A. A pharmacological profile of the selective silent 5-HT1A receptor antagonist, WAY-100635. Eur J Pharmacol. 1995;281:81–88. doi: 10.1016/0014-2999(95)00234-C. [PubMed] [Cross Ref]
  • Gao B, Fritschy JM, Benke D, Mohler H. Neuron-specific expression of GABAA-receptor subtypes: differential association of the alpha 1- and alpha 3-subunits with serotonergic and GABAergic neurons. Neuroscience. 1993;54:881–892. doi: 10.1016/0306-4522(93)90582-Z. [PubMed] [Cross Ref]
  • Gervasoni D, Peyron C, Rampon C, Barbagli B, Chouvet G, Urbain N, Fort P, Luppi PH. Role and origin of the GABAergic innervation of dorsal raphe serotonergic neurons. J Neurosci. 2000;20:4217–4225. [PubMed]
  • Green S, Hodges H. Differential effects of dorsal raphe lesions and intraraphe GABA and benzodiazepines on conflict behavior in rats. Behav Neural Biol. 1986;46:13–29. doi: 10.1016/S0163-1047(86)90861-7. [PubMed] [Cross Ref]
  • Griebel G, Rodgers RJ, Perrault G, Sanger DJ. The effects of compounds varying in selectivity as 5-HT(1A) receptor antagonists in three rat models of anxiety. Neuropharmacology. 2000;39:1848–1857. doi: 10.1016/S0028-3908(00)00074-5. [PubMed] [Cross Ref]
  • Groenink L, Mos J, Gugten J, Olivier B. The 5-HT1A receptor is not involved in emotional stress-induced rises in stress hormones. Pharmacol Biochem Behav. 1996;55:303–308. doi: 10.1016/S0091-3057(96)00088-3. [PubMed] [Cross Ref]
  • Groenink L, Vinkers CH, Oorschot R, Olivier B. Models of anxiety: stress-induced hyperthermia (SIH) in singly housed mice. Curr Protoc Pharmacol. 2009;S45:5.16.1–5.16.12.
  • Harandi M, Aguera M, Gamrani H, Didier M, Maitre M, Calas A, Belin MF. Gamma-aminobutyric acid and 5-hydroxytryptamine interrelationship in the rat nucleus raphe dorsalis: combination of radioautographic and immunocytochemical techniques at light and electron microscopy levels. Neuroscience. 1987;21:237–251. doi: 10.1016/0306-4522(87)90336-8. [PubMed] [Cross Ref]
  • Humphries AC, Gancia E, Gilligan MT, Goodacre S, Hallett D, Merchant KJ, Thomas SR. 8-Fluoroimidazo[1, 2-a]pyridine: synthesis, physicochemical properties and evaluation as a bioisosteric replacement for imidazo[1, 2-a]pyrimidine in an allosteric modulator ligand of the GABA A receptor. Bioorg Med Chem Lett. 2006;16:1518–1522. doi: 10.1016/j.bmcl.2005.12.037. [PubMed] [Cross Ref]
  • Iijima M, Shimazaki T, Ito A, Chaki S. Effects of metabotropic glutamate 2/3 receptor antagonists in the stress-induced hyperthermia test in singly housed mice. Psychopharmacology (Berl) 2007;190:233–239. doi: 10.1007/s00213-006-0618-6. [PubMed] [Cross Ref]
  • Joordens RJ, Hijzen TH, Olivier B. The effects of 5-HT1A receptor agonists, 5-HT1A receptor antagonists and their interaction on the fear-potentiated startle response. Psychopharmacology (Berl) 1998;139:383–390. doi: 10.1007/s002130050729. [PubMed] [Cross Ref]
  • Kalueff AV, Nutt DJ. Role of GABA in anxiety and depression. Depress Anxiety. 2007;24:495–517. doi: 10.1002/da.20262. [PubMed] [Cross Ref]
  • Lee JJ, Hahm ET, Lee CH, Cho YW. Serotonergic modulation of GABAergic and glutamatergic synaptic transmission in mechanically isolated rat medial preoptic area neurons. Neuropsychopharmacology. 2008;33:340–352. doi: 10.1038/sj.npp.1301396. [PubMed] [Cross Ref]
  • Lista A, Blier P, Montigny C. In vivo presynaptic modulation of serotonergic neurotransmission in the rat hippocampus by diazepam. Eur J Pharmacol. 1989;171:229–231. doi: 10.1016/0014-2999(89)90111-8. [PubMed] [Cross Ref]
  • Mundey MK, Fletcher A, Marsden CA. Effects of 8-OHDPAT and 5-HT1A antagonists WAY100135 and WAY100635, on guinea-pig behaviour and dorsal raphe 5-HT neurone firing. Br J Pharmacol. 1996;117:750–756. [PMC free article] [PubMed]
  • Nemeroff CB. The role of GABA in the pathophysiology and treatment of anxiety disorders. Psychopharmacol Bull. 2003;37:133–146. [PubMed]
  • Nutt DJ. Overview of diagnosis and drug treatments of anxiety disorders. CNS Spectr. 2005;10:49–56. [PubMed]
  • Olivier B, Zethof TJ, Ronken E, Heyden JA. Anxiolytic effects of flesinoxan in the stress-induced hyperthermia paradigm in singly-housed mice are 5-HT1A receptor mediated. Eur J Pharmacol. 1998;342:177–182. doi: 10.1016/S0014-2999(97)01482-9. [PubMed] [Cross Ref]
  • Olivier B, Bouwknecht JA, Pattij T, Leahy C, Oorschot R, Zethof TJ. GABAA-benzodiazepine receptor complex ligands and stress-induced hyperthermia in singly housed mice. Pharmacol Biochem Behav. 2002;72:179–188. doi: 10.1016/S0091-3057(01)00759-6. [PubMed] [Cross Ref]
  • Olivier B, Zethof T, Pattij T, Boogaert M, Oorschot R, Leahy C, Oosting R, Bouwknecht A, Veening J, Gugten J, Groenink L. Stress-induced hyperthermia and anxiety: pharmacological validation. Eur J Pharmacol. 2003;463:117–132. doi: 10.1016/S0014-2999(03)01326-8. [PubMed] [Cross Ref]
  • Olivier JD, Cools AR, Olivier B, Homberg JR, Cuppen E, Ellenbroek BA. Stress-induced hyperthermia and basal body temperature are mediated by different 5-HT(1A) receptor populations: a study in SERT knockout rats. Eur J Pharmacol. 2008;590:190–197. doi: 10.1016/j.ejphar.2008.06.008. [PubMed] [Cross Ref]
  • Petroski RE, Pomeroy JE, Das R, Bowman H, Yang W, Chen AP, Foster AC. Indiplon is a high-affinity positive allosteric modulator with selectivity for alpha1 subunit-containing GABAA receptors. J Pharmacol Exp Ther. 2006;317:369–377. doi: 10.1124/jpet.105.096701. [PubMed] [Cross Ref]
  • Pratt J, Jenner P, Reynolds EH, Marsden CD. Clonazepam induces decreased serotoninergic activity in the mouse brain. Neuropharmacology. 1979;18:791–799. doi: 10.1016/0028-3908(79)90024-8. [PubMed] [Cross Ref]
  • Rudolph U, Mohler H. GABA-based therapeutic approaches: GABAA receptor subtype functions. Curr Opin Pharmacol. 2006;6:18–23. doi: 10.1016/j.coph.2005.10.003. [PubMed] [Cross Ref]
  • Rudolph U, Crestani F, Benke D, Brunig I, Benson JA, Fritschy JM, Martin JR, Bluethmann H, Mohler H. Benzodiazepine actions mediated by specific gamma-aminobutyric acid(A) receptor subtypes. Nature. 1999;401:796–800. doi: 10.1038/44579. [PubMed] [Cross Ref]
  • Shephard RA, Buxton DA, Broadhurst PL. Drug interactions do not support reduction in serotonin turnover as the mechanism of action of benzodiazepines. Neuropharmacology. 1982;21:1027–1032. doi: 10.1016/0028-3908(82)90117-4. [PubMed] [Cross Ref]
  • Stanhope KJ, Dourish CT. Effects of 5-HT1A receptor agonists, partial agonists and a silent antagonist on the performance of the conditioned emotional response test in the rat. Psychopharmacology (Berl) 1996;128:293–303. doi: 10.1007/s002130050137. [PubMed] [Cross Ref]
  • Stein L, Belluzzi JD, Wise CD. Benzodiazepines: behavioral and neurochemical mechanisms. Am J Psychiatry. 1977;134:665–669. [PubMed]
  • Thiebot MH. Are serotonergic neurons involved in the control of anxiety and in the anxiolytic activity of benzodiazepines? Pharmacol Biochem Behav. 1986;24:1471–1477. doi: 10.1016/0091-3057(86)90214-5. [PubMed] [Cross Ref]
  • Thiebot MH, Soubrie P, Hamon M, Simon P. Evidence against the involvement of serotonergic neurons in the anti-punishment activity of diazepam in the rat. Psychopharmacology (Berl) 1984;82:355–359. doi: 10.1007/BF00427685. [PubMed] [Cross Ref]
  • Trulson ME, Preussler DW, Howell GA, Frederickson CJ. Raphe unit activity in freely moving cats: effects of benzodiazepines. Neuropharmacology. 1982;21:1045–1050. doi: 10.1016/0028-3908(82)90120-4. [PubMed] [Cross Ref]
  • Varga V, Szekely AD, Csillag A, Sharp T, Hajos M. Evidence for a role of GABA interneurones in the cortical modulation of midbrain 5-hydroxytryptamine neurones. Neuroscience. 2001;106:783–792. doi: 10.1016/S0306-4522(01)00294-9. [PubMed] [Cross Ref]
  • Vinkers CH, Bogaert MJ, Klanker M, Korte SM, Oosting R, Hanania T, Hopkins SC, Olivier B, Groenink L. Translational aspects of pharmacological research into anxiety disorders: the stress-induced hyperthermia (SIH) paradigm. Eur J Pharmacol. 2008;585:407–425. doi: 10.1016/j.ejphar.2008.02.097. [PubMed] [Cross Ref]
  • Vinkers CH, Klanker M, Groenink L, Korte SM, Cook JM, Linn ML, Hopkins SC, Olivier B. Dissociating anxiolytic and sedative effects of GABAAergic drugs using temperature and locomotor responses to acute stress. Psychopharmacology (Berl) 2009;204:299–311. doi: 10.1007/s00213-009-1460-4. [PMC free article] [PubMed] [Cross Ref]
  • Wesolowska A, Paluchowska M, Chojnacka-Wojcik E. Involvement of presynaptic 5-HT(1A) and benzodiazepine receptors in the anticonflict activity of 5-HT(1A) receptor antagonists. Eur J Pharmacol. 2003;471:27–34. doi: 10.1016/S0014-2999(03)01814-4. [PubMed] [Cross Ref]
  • Wright IK, Upton N, Marsden CA. Effect of established and putative anxiolytics on extracellular 5-HT and 5-HIAA in the ventral hippocampus of rats during behaviour on the elevated X-maze. Psychopharmacology (Berl) 1992;109:338–346. doi: 10.1007/BF02245882. [PubMed] [Cross Ref]
  • Zohar J, Westenberg HG. Anxiety disorders: a review of tricyclic antidepressants and selective serotonin reuptake inhibitors. Acta Psychiatr Scand Suppl. 2000;403:39–49. doi: 10.1111/j.1600-0447.2000.tb10947.x. [PubMed] [Cross Ref]

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