1A Receptor Function Research Articles

Selective heterologous regulation of 5-HT 1A receptor-stimulated [ 35S]GTPγS binding in the anterior cingulate cortex as a result of 5-HT 2 receptor activation

Previous studies have shown that administration of the 5-HT 2 receptor agonist DOI to rats results in the heterologous desensitization of 5-HT 1A receptor-mediated behavioral and neuroendocrine responses [Neuropsychopharmacology 19 (1998) 354; J. Neurosci. 21 (2001) 7919]. We hypothesized that the basis for these changes in 5-HT 1A receptor function may involve changes in the capacity of the 5-HT 1A receptor to activate G proteins. We examined the effect of chronic administration of DOI on the regulation of 5-HT 1A receptor function at the level of receptor–G protein interaction using quantitative autoradiography of [ 35S]GTPγS binding stimulated by the 5-HT 1A receptor agonist (±)8-OH-DPAT (1 μM). Repeated administration of DOI (1 mg/kg, s.c. once daily for 8 days) resulted in a marked down-regulation in 5-HT 2A binding sites, as labeled by the antagonist radioligand [ 3H]ketanserin, throughout the cerebral cortex. Chronic DOI treatment also resulted in a significant and selective attenuation of 5-HT 1A receptor-stimulated [ 35S]GTPγS binding in the anterior cingulate cortex (vehicle-treated: 74±7.7% above basal; DOI-treated: 43±4.6% above basal). Interestingly, 5-HT 1A receptor-stimulated [ 35S]GTPγS binding was not altered in the dorsal or median raphe, or in the limbic structures and other cortical regions examined. The decrease in 5-HT 1A receptor-stimulated [ 35S]GTPγS binding in anterior cingulate cortex was not due to a decrease in 5-HT 1A receptor number, indicating that the capacity of the 5-HT 1A receptor to activate G proteins is attenuated in this cortical area following repeated DOI treatment. The heterologous regulation of 5-HT 1A receptor function by chronic 5-HT 2 receptor activation in the anterior cingulate cortex raises interesting questions as to how the regulatory interaction between these serotonin receptor subtypes influences cognition, memory and emotion.

Functional effects of chronic electroconvulsive shock on serotonergic 5-HT 1A and 5-HT 1B receptor activity in rat hippocampus and hypothalamus

The aims of this work were to determine the influence of chronic electroconvulsive shock (ECS) on presynaptic 5-HT 1A receptor function, postsynaptic 5-HT 1A receptor function in hippocampus and hypothalamus, and presynaptic 5-HT 1B receptor function in hippocampus and hypothalamus. This represents part of an on-going study of the effects of ECS on serotonergic receptor activity in selected brain areas which may be relevant to the effects of electroconvulsive therapy (ECT) in humans. Chronic ECS reduced the ability of the 5-HT 1A receptor agonist 8-hydroxy-2(di- n-propylamino)tetraline (8-OH-DPAT) (0.2 mg/kg s.c.) to decrease 5-HT levels in hypothalamus as shown by in vivo microdialysis, indicative of a reduction in sensitivity of presynaptic 5-HT 1A autoreceptors. The ability of the 5-HT 1B receptor antagonist GR 127935 (5 mg/kg s.c.) to increase 5-HT levels in both hippocampus and hypothalamus was unaffected by chronic ECS. 8-OH-DPAT (0.2 mg/kg s.c.) increased cyclic AMP levels in hippocampus measured by in vivo microdialysis approximately 2-fold. The degree of stimulation of cyclic AMP formation was not altered by chronic ECS. However the cyclic AMP response to forskolin (50 μM) administered via the microdialysis probe, which was approximately 4-fold of basal in sham-treated rats, was almost completely abolished in ECS-treated rats. Since this indicates that either adenylate cyclase catalytic unit activity or Gs protein activity is reduced in the hippocampus after chronic ECS, the lack of change in 8-OH-DPAT-induced cyclic AMP formation may be taken as possible evidence of an increase in sensitivity of postsynaptic 5-HT 1A receptors in the hippocampus by chronic ECS. Chronic ECS increased basal plasma levels of corticosterone, ACTH and oxytocin. The ACTH response to s.c. injections of 0.2 mg/kg or 0.5 mg/kg 8-OH-DPAT was reduced by chronic ECS. Postsynaptic 5-HT 1A receptor activity in the hypothalamus, in contrast to the hippocampus, thus appears to be desensitized after chronic ECS. We conclude that chronic ECS has regionally specific effects on both pre- and post-synaptic 5-HT 1A receptors, but, in contrast to some antidepressant drugs, does not affect presynaptic 5-HT 1B receptor activity.

Antidepressant-like activity of VN2222, a serotonin reuptake inhibitor with high affinity at 5-HT 1A receptors

It has been suggested that drugs combining serotonin (5-hydroxytryptamine, 5-HT) transporter blockade and 5-HT 1A autoreceptor antagonism could be a novel strategy for a shorter onset of action and higher therapeutic efficacy of antidepressants. The present study was aimed at characterizing the pharmacology of 1-(3-benzo[b]tiophenyl)-3-[4-(2-methoxyphenyl)-1-piperazinyl]-1-propanol (VN2222) a new synthetic compound with high affinity at both the 5-HT transporter and 5-HT 1A receptors and devoid of high affinity at other receptors studied, with the only exception of α 1-adrenoceptors. In keeping with the binding affinity at the 5-HT transporter, VN2222 inhibited 5-HT uptake in vitro both in rat cortical synaptosomes and in mesencephalic cultures and also in vivo when administered locally into the rat ventral hippocampus. After systemic administration, VN2222 exhibited an inverted U-shape effect so the inhibition of [ 3H]5-HT uptake ex vivo and the increase in 5-HT extracellular levels in microdialysis experiments was observed at low doses of 0.01–0.1 mg/kg whereas higher doses were ineffective. In studies related to 5-HT 1A receptor function, 0.01–0.1 μM VN2222 produced a partial inhibition of forskolin-stimulated cAMP formation behaving as a weak agonist of 5-HT 1A receptors. In body temperature studies, 5 mg/kg VN2222 produced a mild hypothermic effect in mice, suggesting a weak agonist activity at presynaptic 5-HT 1A receptors; much lower doses (0.01–0.5 mg/kg) partially antagonized the hypothermia induced by 8-hydroxy-2-(di- n-propylamino)tetralin (8-OH-DPAT) possibly through 5-HT transporter blockade. In the learned helplessness test in rats, an animal model for antidepressants, 1–5 mg/kg VN2222 reduced significantly the number of escape failures. Consequently, VN2222 is a new compound with a dual effect on the serotonergic system, as 5-HT uptake blocker and 5-HT 1A receptor partial agonist, and with a remarkable activity in an animal model of depression with high predictive validity.

5-HT 1B receptor knockout mice show no adaptive changes in 5-HT 1A receptor function as measured telemetrically on body temperature and heart rate responses

Two presynaptic receptors play an important role in the regulation of serotonergic neurotransmission, i.e., the 5-HT 1A and 5-HT 1B receptor. The present study focuses on putative adaptive changes in the 5-HT 1A receptor system in mice that lack 5-HT 1B receptors (5-HT 1B KO). 5-HT 1A receptor sensitivity was assessed in vivo in two models of presynaptic 5-HT 1A receptor activity: agonist-induced hypothermia and prevention of stress-induced hyperthermia. The effects of 5-HT 1A receptor activation by flesinoxan (0.1–3.0 mg/kg s.c.) were determined telemetrically on body temperature and heart rate in 5-HT 1B KO and wild-type (WT) mice. Flesinoxan induced hypothermia dose-dependently without affecting heart rate and prevented stress-induced hyperthermia and tachycardia equipotently in both genotypes. Specificity of these responses was confirmed by blockade with the selective 5-HT 1A receptor antagonist WAY100635 (1.0 mg/kg s.c.). The importance of continuous sampling in freely moving subjects to improve appropriate characterization of mutants is discussed. 5-HT 1B KO mice showed no shift in 5-HT 1A receptor sensitivity compared to WT mice. This study found no indications for adaptive changes in presynaptic 5-HT 1A receptor function in 5-HT 1B KO mice as measured telemetrically on body temperature and heart rate responses.

Blunted brain metabolic response to ketamine in mice lacking D 1A dopamine receptors

The interaction of glutamatergic and dopamine neurotransmission is thought to have relevance to both the pathophysiology and pharmacotherapy of schizophrenia. For example, subanesthetic doses of the N-methyl- d-aspartate receptor (NMDA-R) antagonist ketamine induce schizophrenia-like behavioral effects in humans and both behavioral and brain metabolic activation in rodents. Blockade of NMDA-R results in dopamine release, and antipsychotic drugs that block dopamine neurotransmission decrease NMDA-R antagonist-induced behavioral activation. The involvement of dopamine receptors in brain metabolic activation induced by ketamine is, however, unknown. The present study used D 1A knockout mice to determine the role of dopamine D 1A receptors in the effects of subanesthetic doses of ketamine on both behavioral responses and on alterations in regional [ 14C]2-deoxyglucose (2-DG) uptake. There was less ketamine-induced behavioral activation in D 1A knockout mice than in wild-type mice. In wild-type mice, ketamine (30 mg/kg) induced dramatic increases in 2-DG uptake in limbic cortical regions, hippocampal formation, nucleus accumbens, basolateral amygdala, and caudal parts of the substantia nigra pars reticulata. D 1A knockout mice exhibited blunted metabolic activation in response to ketamine in a neuroanatomically specific manner. The selective D 1 antagonist, SCH23390 (0.3 mg/kg), inhibited both ketamine-induced brain metabolic activation and behavioral responses in the wild-type mice, with a similar neuroanatomical specificity observed in the D 1A knockout mice. Thus, the neuroanatomically selective role that D 1A receptors play in ketamine-induced behavior and regional brain metabolic activation in mice provides a useful model for further studies of how the D 1A receptor function may be altered in schizophrenia.

The effects of compounds varying in selectivity as 5-HT 1A receptor antagonists in three rat models of anxiety

Compounds varying in selectivity as 5-HT 1A receptor antagonists have recently been reported to produce benzodiazepine-like antianxiety effects in mice. To assess the cross-species generality of these findings, the present experiments compared the effects of diazepam (0.625–5 mg/kg) with those of several non-selective (MM-77, 0.03–1 mg/kg and pindobind-5-HT 1A, 0.1–5 mg/kg) and selective (WAY100635, 0.01–10 mg/kg, p-MPPI, 0.01–3 mg/kg and SL88.0338, 0.3–10 mg/kg) 5-HT 1A receptor antagonists in three well-validated anxiolytic screening tests in rats: punished lever-pressing, punished drinking, and the elevated plus-maze. In the punished lever-pressing conflict test, none of the 5-HT 1A receptor antagonists modified rates of punished responding, whereas in the punished drinking test, WAY100635 (0.3–1 mg/kg), SL88.0338 (3–10 mg/kg), p-MPPI (1 mg/kg), MM-77 (0.03–0.3 mg/kg), but not pindobind-5-HT 1A, produced clear anticonflict activity. However, the increase in punished responding with the 5-HT 1A compounds was smaller than that produced by diazepam, indicating weaker anxiolytic-like activity. In the elevated plus-maze test, WAY100635 (0.1–0.3 mg/kg), SL88.0338 (0.3–10 mg/kg), MM-77 (0.01–3 mg/kg), pindobind-5-HT 1A (0.1–3 mg/kg), but not p-MPPI, showed anxiolytic-like activity on traditional behavioral indices, increasing the percentage of time spent in open arms and the percentage of open arm entries. As was the case in the punished drinking test, the magnitude of the positive effects of the 5-HT 1A compounds was generally smaller than that of diazepam. Of the ethological measures recorded in the plus-maze, all compounds markedly decreased risk assessment (i.e. attempts) over the entire dose-range, but only diazepam clearly increased directed exploration (i.e. head-dipping). Although the present results demonstrate that 5-HT 1A receptor antagonists elicit anxiolytic-like effects in rats, this action appears to be test-specific and, unlike previous findings in mice, smaller than that observed with benzodiazepines. The data are discussed in relation to the possible relevance of species differences in 5-HT 1A receptor function and the nature of the anxiety response studied.

Inactivation of 5-HT 1A receptors in hippocampal and cortical homogenates

5-HT 1A receptor function can be assessed in rat hippocampal and cortical membrane preparations as agonist-stimulated [ 35S]GTPγS binding. Membranes were preincubated in vitro with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). R(+)-8-hydroxy-2-(di- n-propylamino)tetralin [ R(+)-8-OH-DPAT]-stimulated [ 35S]GTPγS binding and [ 3H]8-OH-DPAT binding assays were used to assess 5-HT 1A receptor function and density, respectively. EEDQ decreased both R(+)-8-OH-DPAT-stimulated [ 35S]GTPγS and [ 3H]8-OH-DPAT binding in hippocampal and cortical membranes. The E max but not the EC 50 of R(+)-8-OH-DPAT to stimulate [ 35S]GTPγS binding was decreased by EEDQ in both preparations. Additionally, the IC 50 for EEDQ to reduce R(+)-8-OH-DPAT-stimulated [ 35S]GTPγS and [ 3H]8-OH-DPAT binding was the same for both brain regions in both assays. In contrast to EEDQ alone, agonist-stimulated [ 35S]GTPγS binding was not reduced in hippocampal membranes preincubated with EEDQ and the 5-HT 1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]- N-2-pyridinyl-cyclohexanecarboxamide maleate (WAY 100,635), suggesting that EEDQ acts directly on the receptor. Due to parallel reductions in receptor density and maximal functional response, it is concluded that there is little or no reserve for 5-HT 1A receptor coupling to G α in these preparations. In addition, the sensitivity of hippocampal and cortical 5-HT 1A receptors to inactivation by EEDQ in vitro is the same.

Region-specific changes in 5-HT 1A receptor-activated G-proteins in rat brain following chronic buspirone

5-Hydroxytryptamine 1A (5-HT 1A) receptors, which activate inhibitory G-proteins, are implicated in psychiatric disorders including anxiety and depression. Studies suggest that chronic 5-HT 1A receptor agonist administration alters 5-HT 1A receptor function, but the effect of chronic treatment on 5-HT 1A receptor-activated G-proteins is unclear. In this study, agonist-stimulated [ 35S]guanylyl-5′- O-(γ-thio)-triphosphate (GTPγS) binding was examined following chronic administration of buspirone. Brains were processed for [ 35S]GTPγS autoradiography using R(+)-8-hydroxy-2-(di- n-propylamino)tetralin (8-OH-DPAT) for 5-HT 1A receptors or baclofen for GABA B receptors. Net 8-OH-DPAT-stimulated [ 35S]GTPγS binding was decreased by 25–30% in the septum and dorsal raphe nucleus of buspirone-treated animals. No significant changes in 8-OH-DPAT-stimulated [ 35S]GTPγS binding were found in the prefrontal, entorhinal or cingulate cortices or hippocampus in buspirone-treated rats. GABA B receptor-stimulated [ 35S]GTPγS binding was increased by 25% in the hippocampus, with no significant changes in any other region examined. These results demonstrate region-specific alterations in 5-HT 1A and GABA B receptor-activated G-proteins following chronic buspirone treatment, which may contribute to the clinical effects of this drug.

Identification of a Ca 2+/calmodulin-binding domain within the carboxyl-terminus of the angiotensin II (AT 1A) receptor

To identify regulators of the type 1A angiotensin II receptor (AT 1A), we investigated the interaction of cellular proteins with a fusion protein containing the rat AT 1A receptor carboxyl-terminus. An ∼20 kDa cytoplasmic protein interacted with the fusion protein in a Ca 2+-dependent manner and was identified as calmodulin. A control peptide with high affinity for Ca 2+/calmodulin and a peptide corresponding to a membrane proximal portion of the AT 1A receptor carboxyl-terminus with analogy to known calmodulin-binding sequences were synthesised and tested for calmodulin-binding. Using in vitro binding assays combined with gel shift analysis, we demonstrated the formation of complexes between calmodulin and both peptides, which were Ca 2+-dependent and of 1:1 stoichiometry. Affinity gels produced from these peptides also purified calmodulin from cell extracts. These results suggest a novel feedback regulation of the AT 1A receptor by Ca 2+/calmodulin and identify the membrane proximal region of the carboxyl-terminus as a focal point for interactions important for AT 1A receptor function.

Hypothermic, ACTH, and cortisol responses to ipsapirone in patients with mania and healthy controls

Background: The selective 5-HT 1A receptor agonist ipsapirone causes dose-dependent decrease in body temperature and increase in adrenocorticotropic hormone (ACTH) and cortisol release in humans. These responses are attenuated by 5-HT 1A receptor antagonists, suggesting that hypothermia, ACTH and cortisol release induced by ipsapirone are indeed mediated by 5-HT 1A receptors and that these responses provide a valid index of 5-HT 1A receptor function in humans. Methods: To examine the 5-HT 1A receptor sensitivity in patients with mania, we studied six manic patients and six age and sex matched healthy controls. After obtaining a blood sample for baseline hormone levels and measuring body temperature, a single dose of 0.3 mg/kg of ipsapirone was given orally to all the subjects and further bloods and temperature reading were obtained every 30 minutes for 3 hours. Results: We found that ACTH and cortisol responses to ipsapirone were significantly increased in mania when compared to healthy controls, but there was no significant difference in hypothermic response to ipsapirone between the two groups. Limitations: A lack of placebo control, heterogeneity of patients, and a small sample size are the limitations. Conclusions: Our findings suggest that manic patients may have enhanced postsynaptic 5-HT 1A receptor sensitivity, but presynaptic 5-HT 1A receptors are unaltered in this condition. Further placebo-controlled studies with a larger number of manic patients are needed to verify this.

Onset of the Effects of the 5-HT 1A Antagonist, WAY-100635, Alone, and in Combination With Paroxetine, on Olfactory Bulbectomy and 8-OH-DPAT–Induced Changes in the Rat

5-HT 1A receptor antagonists have recently been shown to accelerate the efects of some antidepressant drugs in clinical trials. In this study we investigate the effects of combining a full antagonist at the 5-HT 1A receptor, WAY 100635 (0.2 mg/kg, SC) with the selective serotonin reuptake inhibitor (SSRI) paroxetine (5 mg/kg, IP) in the olfactory bulbectomized (OB) rat, an animal model of chronic (but not acute) antidepressant activity. Ambulation scores were measured in the open-field apparatus, following 3, 7, and 14 days of treatment. Further to the OB study, we simultaneously studied adaptive changes in 5-HT 1A receptor function, utilizing alterations in the hypothermic response to the 5-HT 1A receptor agonist 8-OH-DPAT. Paroxetine, in combination with WAY 100635, attenuated the hypothermic effects of 8-OH-DPAT as early as 3 days, with a full reversal evident following 7 days, whereas paroxetine, although attenuating the hypothermic effects in OB group by day 7, only reversed it fully after 14 days. Paroxetine alone and in combination with the antagonist reversed the olfactory bulbectomy-induced hyperactivity in the open field following 14 days of treatment only, this being the normal time of an “antidepressant” response in this model. However, there was no significant attenuation at any of the earlier time points. This further demonstrates that the reversal of this aspect of the olfactory bulbectomy-induced behavioral syndrome is insensitive to the potential faster onset of antidepressant action induced by 5-HT 1A receptor antagonists. Nonetheless, WAY 100635, unlike previous studies with pindolol, did not interfere with the effects of the antidepressant in the model. The ability of the combination group to attenuate the hypothermic effects of 8-OH-DPAT faster than paroxetine alone, further emphasizes the role of the 5-HT 1A receptor in the mechanism of action of antidepressants, and as a target for the development of faster acting antidepressants.

Chronic Fluoxetine in Tests of Anxiety in Rat Lines Selectively Bred for Differential 5-HT 1A Receptor Function

Selective breeding for high and low sensitivity to the hypothermic response induced by the 5-HT 1A receptor agonist 8- OH-DPAT has established two lines of rat (HDS and LDS, respectively) whose behavior differs in a model of depression and in the social interaction test of anxiety. The HDS line has a higher level of anxiety and, furthermore, does not display the usual anxiogenic response to intrahippocampal administration of 8-OH-DPAT. It was therefore hypothesized that this line of rat might be a useful model of high trait anxiety with a susceptibility to depression. We thus investigated whether chronic treatment with fluoxetine would result in an anxiolytic effect in the social interaction test in the LDS and HDS lines of rat. In both lines, acute fluoxetine (10 mg/kg) produced an anxiogenic effect in the social interaction test; when rats were tested 24 h after 14 days of fluoxetine treatment there were no anxiolytic effects in either line. In the social interaction test, chronic fluoxetine treatment did not change either the anxiogenic effect of 8-OH-DPAT (100 ng) injected bilaterally into the dorsal hippocampus in the LDS line or the lack of response in the HDS line. In the elevated plus-maze, chronic fluoxetine treatment resulted in a significant anxiogenic effect in the HDS line, but was without effect in the LDS line. Intrahippocampal 8-OH-DPAT was without effect in the plus-maze in either line. These results suggest that chronic treatment with fluoxetine did not modify the hippocampal 5-HT 1A receptor in either line. The anxiogenic effects observed in the plus-maze in the HDS line after chronic fluoxetine might relate to line differences in 5-HT 1A receptors in other brain regions.

Combining pindolol and paroxetine in an animal model of chronic antidepressant action—can early onset of action be detected?

The realisation that pindolol may accelerate the effects of some antidepressant drugs in clinical trials has added extra impetus to the search for faster acting antidepressants. Currently, no animal model of depression can identify potential faster acting antidepressant drugs or drug combinations. In this study, we investigate the effects of combining pindolol (2 mg/kg, s.c., bid) with the antidepressant paroxetine (2.5 mg/kg, i.p., bid) in the olfactory bulbectomised rat, an animal model of chronic (but not acute) antidepressant activity. Ambulation scores were measured in separate groups of rats, following 3, 7 and 14 days of treatment. Further, we simultaneously study adaptive changes in 5-HT 1A receptor function, utilising alterations in the hypothermic response to the 5-HT 1A receptor agonist 8-hydroxy-2-(di- n-propylamino)tetralin (8-OH-DPAT). Pindolol in combination with paroxetine attenuated the hypothermic effects of 8-OH-DPAT as early as 3 days with a full reversal evident following 7 days, whereas paroxetine alone did so after 14 days only. Likewise, paroxetine alone reversed the olfactory bulbectomy-induced hyperactivity in the open field following 14 days of treatment only, this being the normal time of an `antidepressant' response in this model. However, the group treated with both paroxetine and pindolol failed to reverse the hyperactive response. This suggests that a factor intrinsic to pindolol antagonises the behavioural effects of paroxetine in the olfactory bulbectomised rat. It also demonstrates that the reversal of this aspect of the olfactory bulbectomy-induced behavioural syndrome is insensitive to the potential faster onset of antidepressant action induced by pindolol. The ability of the combination group to attenuate the hypothermic effects of 8-OH-DPAT much faster further emphasises the role of the 5-HT 1A receptor in the mechanism of action of antidepressants and as a target for the development of faster acting antidepressants. However, an animal model sensitive to the effects of any such compound and the actions of pindolol remains elusive.

Characterization of 5-HT 1A receptor functional coupling in cells expressing the human 5-HT 1A receptor as assessed with the cytosensor microphysiometer

The functional activity of a series of 5-HT 1A receptor ligands has been evaluated in a cell line expressing the human 5-HT 1A receptor (h5-HT 1A · CHO) using the agonist-stimulated increase in extracellular acidification rate, measured with the microphysiometer, as a functional assay. Both 5-CT and 8-OH-DPAT were potent agonists in stimulating an increase in extracellular acidification rate in h5-HT 1A · CHO cells with estimated EC 50 values of 1.2 and 7.8 nM, respectively. Additionally, these two 5-HT 1A receptor agonists elicited a similar maximum response. Concentration-dependent agonist activity was also observed in the presence of buspirone, ipsapirone, BMY7378, NAN-190 and WAY100135, and each of these compounds behaved as partial 5-HT 1A receptor agonists. The selective 5-HT 1A receptor antagonist WAY100635 produced a potent (IC 50, 2.3 nM) and complete block of the 8-OH-DPAT-stimulated response. An evaluation of the inhibitory activity of a series of 5-HT 1A receptor antagonists produced the following rank order of potency; WAY100635 > LY206130 (IC 50, 7.1 nM) > WAY100135 (30.8 nM) > pindolol (76.2 nM) > (−)UH-301 (92.8 nM). Parallel studies on the inhibition of forskolin-stimulated adenylyl cyclase activity in h5-HT 1A · CHO cells revealed that agonist potencies were generally similar between the two functional assays and were in good agreement with the estimated 5-HT 1A receptor binding affinities. However, the relative efficacies determined for the partial agonists in the cAMP assay were substantially greater than those observed with the microphysiometer. Finally, antagonists were considerably weaker in the cAMP assay compared with the microphysiometer. The evaluation of 5-HT 1A ligands using the microphysiometer, which represents a very distinct indice of 5-HT 1A receptor function compared with the cAMP assay, results in a different profile of functional activity.

Dose-dependent effects of estradiol benzoate on 5-HT 1A receptor agonist action

The lordosis-inhibiting effects of the 5-HT 1A receptor agonist, (±)8-hydroxy-2-(di- n-propylamino) tetralin (8-OH-DPAT), were examined in ovariectomized rats, hormone primed with 2.5, 7.5, or 25 μg estradiol benzoate plus 500 μg progesterone. 8-OH-DPAT (50, 100 or 200 ng per bilateral site) infused into the ventromedial nucleus of the hypothalamus (VMN), inhibited lordosis behavior in all hormone-treated conditions. However, animals primed with 2.5 μg estradiol benzoate were significantly more affected by the infusion than rats primed with 7.5 or 25 μg of the hormone. These findings strengthen prior speculations that 5-HT 1A receptor function is modulated by estrogen.

Effects of lamotrigine on the 5-HT 1A receptor function in healthy human males

Background: Lamotrigine, a new anticonvulsant, has recently been reported to be effective in treating patients with bipolar mania, depression, and schizoaffective disorder, suggesting that it is perhaps a mood stabilizer with antimanic and antidepressant properties. However, the mechanism of action underlying its efficacy in mood disorders is still unknown. Methods: To explore the role of 5-HT 1A receptors in the mechanism of action of lamotrigine, we measured the body temperature and plasma cortisol responses to a challenge with a selective 5-HT 1A receptor agonist ipsapirone in ten healthy human males. Each subject received 0.3 mg/kg of ipsapirone hydrochloride tablets at time “0”. Body temperature readings and blood samples for cortisol levels were obtained at 0, 30, 60, 90, 120, 150, 180 min. The ipsapirone challenge tests were repeated after 1 week treatment with lamotrigine (100 mg/day). Results: Treatment with lamotrigine for 1 week did not significantly alter the hypothermic or cortisol responses to ipsapirone. Limitations: The limitations of this study included small sample size, low treatment dose, short treatment interval, and lack of placebo control. Conclusions: Our findings might suggest that 5-HT 1A receptor function is not involved in the mechanism of action of lamotrigine in humans. Further placebo-controlled studies with a higher lamotrigine dose and a longer treatment interval in a larger number of subjects are needed to verify this.

Selectively Bred Lines of Rats Differ in Social Interaction and Hippocampal 5-HT 1A Receptor Function: A Link Between Anxiety and Depression?

Selective breeding for high and low sensitivity to the hypothermic response of the 5-HT 1A receptor agonist 8-OH-DPAT has established two lines (HDS and LDS, respectively) whose behavior differs in a model of depression, but not in the elevated plus-maze test of anxiety. The lines also differed in postsynaptic, but not presynaptic, 5-HT 1A receptors. Based on previous evidence that postsynaptic 5-HT 1A receptors mediate anxiogenic effects in the social interaction test of anxiety, but not the elevated plus-maze, we investigated possible differences between the lines in these two tests. The HDS line had a consistently lower level of social interaction compared with the LDS line, but no differences were found on any of the measures of the anxiety on trials 1 or 2 in the elevated plus-maze. To determine whether the line differences in anxiety were mediated by different hippocampal 5-HT 1A receptor function, 8-OH-DPAT (50 and 100 ng) was applied bilaterally to the dorsal hippocampus. This elicited anxiogenic effects in the LDS line, as has been previously reported in other rat strains, but there was no response in the HDS line, thus demonstrating an abnormal 5-HT 1A receptor function in the hippocampus. The 5-HT 1A receptor antagonist WAY100635 (200 ng) was administered to the dorsal hippocampus to test for possible differences between the lines in 5-HT tone. There were no significant changes in social interaction in either the HDS or LDS rats, indicating that the different level of anxiety between lines is not due to differences in hippocampal 5-HT tone. It is proposed that the HDS line may prove a useful model of a type of high trait anxiety linked to a susceptibility to depression.

Elevated basal trough levels of corticosterone suppress hippocampal 5-hydroxytryptamine 1A receptor expression in adrenally intact rats: implication for the pathogenesis of depression

Several studies with adrenalectomized rats have shown that the suppressive effects of exogenous corticosteroids on 5-hydroxytryptamine 1A receptor function are mediated by the high-affinity mineralocorticoid receptor, rather than the lower affinity glucocorticoid receptor. In the present study, adrenally intact rats were subcutaneously implanted for six days with pellets containing a small amount of corticosterone, which leads to a flattening of the circadian rhythm in the level of circulating hormone. The peak in daily corticosterone is suppressed, the basal trough is increased, and the hormone levels remain at a constant value equivalent to the daily average of about 5 μg/dl, which is usually observed in rats. Accordingly, this regime of corticosterone treatment did not enhance exclusively glucocorticoid receptor-controlled parameters, such as the weight of the thymus. Effects involving mineralocorticoid receptor activation were enhanced, since reductions were observed in stress-induced plasma corticosterone levels and adrenal weight. 5-Hydroxytryptamine 1A receptor messenger RNA levels were found to be suppressed by approximately 25% in the dentate gyrus of the hippocampus of these corticosterone pellet-implanted rats. This suppression was reflected in significantly reduced [ 3H]8-hydroxy-2-(di- n-propylamino)tetralin binding in the hippocampal region. We propose therefore that this suppressive effect on 5-hydroxytryptamine 1A receptor expression involves enhanced occupation of mineralocorticoid receptors, under a condition of elevated basal trough corticosteroid levels as is commonly observed in human depression.

Comparison of relative potencies of i.v. and i.c.v. administered 8-OH-DPAT gives evidence of different sites of action for hypothermia, lower lip retraction and tail flicks

8-Hydroxy-2-(di- n-propylamino)tetralin (8-OH-DPAT)-induced temperature reduction, lower lip retraction and tail flick responses are widely used models of 5-HT 1A receptor function. To obtain information about the sites of receptors mediating these effects we measured these responses, parallel over wide dose ranges after intracerebroventricular (i.c.v., 0.6–67 μg/kg) and intravenous (i.v., 3–500 μg/kg) administration. Analysis of the dose–response curves provided evidence for a 9.8-fold ratio of the potency of 8-OH-DPAT following i.c.v. compared to i.v. administration on body temperature reduction (ED 50 values are 5.1 and 50 μg/kg, after i.c.v. and i.v. administration, respectively) and a 2.9-fold ratio in potency for lower lip retraction (ED 50 values are 29 and 86 μg/kg, after i.c.v. and i.v. administration, respectively). 8-OH-DPAT was less potent in the induction of tail flicks than of the other responses and had a lower potency after i.c.v. than after i.v. administration (ED 50 values, the first one extrapolated, are 526 and 246 μg/kg, after i.c.v. and i.v. administration, respectively). In addition, the i.c.v. ED 50 for temperature reduction was significantly lower than those for lower lip retraction or tail flick responses. The relative potency, that is, the ratio of i.v. and i.c.v. ED 50, was significantly higher for temperature reduction than for lower lip retraction or tail flick responses (ED 50 i.v./ED 50 i.c.v. values are 9.8, 2.9, and 0.47, respectively). These data provide evidence that distinct sites of action are involved in these models. Temperature reduction is mediated mainly by postsynaptic receptors in the close vicinity of the lateral ventricle. Receptors that mediate lower lip retraction are located more distantly in the brain, supporting previous evidence that they are somatodendritic autoreceptors, and receptors in the spinal cord are probably responsible for tail flick responses. © 1997 Elsevier Science B.V. All rights reserved.

Functional domains of the C-terminus of the rat angiotensin AT 1A receptor

Previous work has shown that truncating the carboxyl terminus (C-terminus) of the rat angiotensin AT 1A receptor to 309 amino acids abolishes G-protein coupling and receptor internalization. This suggests that domains responsible for these functions lie beyond amino acid 309 of the C-terminus. The objective of this study was to determine the effect on angiotensin AT 1A receptor function and regulation of deleting 41 amino acids from the C-terminus, which include the putative protein kinase C phosphorylation sites. Using site directed mutagenesis, the codon for Tyr 319 was converted to a stop codon and the resulting truncated receptor permanently expressed in cultured human kidney cells. The properties of the truncated receptor were compared to those of the full length receptor. Expression of the truncated receptor was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of photolabelled membrane preparations. Angiotensin II activation of both full length and truncated receptors resulted in mobilization of inositol phosphates. However, whereas this was associated with rapid internalization of the full length receptor, the truncated receptor failed to internalize. Furthermore, pretreatment of cells with phorbol 12-myristate 13-acetate, a direct activator of protein kinase C, markedly attenuated the full length, but not the truncated receptor's ability to mobilise inositol phosphates. Thus, we conclude that the domain between amino acids 309 & 318 is important for G-protein coupling; that amino acids beyond 318 regulate internalization and one or more of the putative protein kinase C phosphorylation sites, present in the C-terminus of the angiotensin AT 1A receptor, actively regulate the receptor.