Cortical 5-HT1A receptor downregulation by antidepressants in rat brain

Total 5-HT binding sites and 5-HT_1A receptor density was measured in brain regions of rats treated with imipramine (5 mg/kg body wt), desipramine (10 mg/kg body wt) and clomipramine (10 mg/kg body wt), for 40 days, using [³H]5-HT and [³H]8-OH-DPAT, respectively. It was observed that chronic exposure to tricyclic antidepressants (TCAs) results in significant downregulation of total [³H]5-HT binding sites in cortex (42–76%) and hippocampus (35–67%). The 5-HT_1A receptor density was, however, decreased significantly (32–60%) only in cortex with all the three drugs. Interestingly, in hippocampus imipramine treatment increased the 5-HT_1A receptor density (14%). The affinity of [³H]8-OH-DPAT was increased only with imipramine treatment both in cortex and hippocampus. The affinity of [³H]5-HT to 5-HT binding sites in cortex was increased with imipramine treatment and decreased with desipramine and clomipramine treatment. 5-HT sensitive adenylyl cyclase (AC) activity was significantly increased in cortex with imipramine (72%) and clomipramine (17%) treatment, whereas in hippocampus only imipramine treatment significantly increased AC activity (50%). In conclusion, chronic treatment with TCAs results in downregulation of cortical 5-HT_1A receptors along with concomitant increase in 5-HT stimulated AC activity suggesting the involvement of cortical 5-HT_1A receptors in the mechanism of action of TCAs.     

Inactivation of 5-HT1A and [3H]5-HT Binding Sites by N-Ethoxycarbonyl-2-Ethoxy-1, 2-Dihydroquinoline (EEDQ) in Rat Brain

Inactivation of 5-HT_1A and [³H]5-HT binding sites by N-Ethoxycarbonyl-2-ethoxy-1, 2-dihydro-quinoline (EEDQ) was studied in regions of rat brain. After exposure to EEDQ (4 mg/kg body wt.) for 7 days, it is observed that the density of 5-HT₁ receptor sites was decreased by nearly 20% in both cortex and hippocampus. The decrease, however, in 5-HT_1A sites was more significant (70%) in both the regions. The affinity of [³H]5-HT to 5-HT₁ sites was decreased significantly in both cortex and hippocampus after exposure to EEDQ, without affecting the Kd of 5-HT_1A sites. Displacement studies suggested that EEDQ has high affinity to 5-HT₁ sites with a Ki of 42.9 ± 2.4 nM. After exposure neither basal nor 5-HT stimulated adenylyl cyclase activity was changed in cortex. The results of this study suggest that EEDQ decreases the density of 5-HT₁ and 5-HT_1A receptor sites but does not cause functional downregulation of these sites in rat brain.     

Alkylation of [3H]8-OH-DPAT binding sites in rat cerebral cortex and hippocampus

The binding of tritiated 8-hydroxy-2-(di-n-propyl-amino)tetralin, or [³H]8-OH-DPAT, to membranes from rat cerebral cortex and hippocampus could be inhibited by serotonin (5-HT) and buspirone, and by the 5-HT antagonists propranolol, NAN-190, pindolol, pindobind-5-HT_1A, WAY100135, spiperone and ritanserin. All competition curves, except for ritanserin, best fitted a two-site model. In vitro treatment of the membranes withN-ethylmaleimide (NEM), to alkylate sulfhydryl groups, caused dose-dependent decreases of binding; the inhibition curves were biphasic, and the effects irreversible. Reduction of disulfide bonds withl-dithiothreitol (L-DTT) also decreased binding, but in a monophasic way; these effects were fully reversible in cortex, but only partially reversible in hippocampus. In the latter region, but not in cerebral cortex, previous occupancy by [³H]8-OH-DPAT partially protected binding from the effects of bothL-DTT and NEM, suggesting that the thiol groups in the receptor recognition site(s) of this brain region are readily accessible. The binding characteristics were examined with the aid of saturation curves, carried out with increasing concentrations, up to 140 nM, of [³H]8-OH-DPAT. The saturation data were suggestive of a two-site receptor model incorporating a high-affinity site (Kh of 0.3–0.5 nM) corresponding to the 5-HT_1A receptor, and a low-affinity site (Kl ofca 25 nM). After in vivo alkylations, carried out by treating rats withN-ethoxycarbonyl-2-ethoxy-1,2-dihydro-quinoline (EEDQ), the saturation curves from both control and EEDQ-treated rats were again best fitted to a two-site model. For EEDQ-treated animals, a drastic decrease of 5-HT_1A receptor activity was noted; this loss was greater in hippocampus than in cerebral cortex. Since the decrease in 5-HT_1A receptors was not associated with changes in low-affinity binding, the results suggest independent regulations of the two [³H]8-OH-DPAT binding proteins. Altogether, the present data further supports the notion that [³H]8-OH-DPAT, besides labelling 5-HT_1A receptors, also binds to other structures in rat cerebral cortex and hippocampus. Special issue dedicated to Dr. Kinya Kuriyama     

5-HT<Subscript>1A</Subscript> receptor expression in pyramidal neurons of cortical and limbic brain regions

We studied expression of the 5-HT_1A receptor in cortical and limbic areas of the brain of the tree shrew. In situ hybridization with a receptor-specific probe and immunocytochemistry with various antibodies was used to identify distinct neurons expressing the receptor. In vitro receptor autoradiography with ³H-8-OH-DPAT (³H-8-hydroxy-2-[di-n-propylamino]tetralin) was performed to visualize receptor-binding sites. In the prefrontal, insular, and occipital cortex, 5-HT_1A receptor mRNA was expressed in pyramidal neurons of layer 2, whereas ³H-8-OH-DPAT labeled layers 1 and 2 generating a columnar-like pattern in the prefrontal and occipital cortex. In the striate and ventral occipital cortex, receptor mRNA was present within layers 5 and 6 in pyramidal neurons and Meynert cells. Pyramid-like neurons in the claustrum and anterior olfactory nucleus also expressed the receptor. Principal neurons in hippocampal region CA1 expressed 5-HT_1A receptor mRNA, and ³H-8-OH-DPAT labeled both the stratum oriens and stratum radiatum. CA3 pyramidal neurons displayed low 5-HT_1A receptor expression, whereas granule neurons in the dentate gyrus revealed moderate expression of this receptor. In the amygdala, large pyramid-like neurons in the basal magnocellular nucleus strongly expressed the receptor. Immunocytochemistry with antibodies against parvalbumin, calbindin, and gamma aminobutyric acid (GABA) provided no evidence for 5-HT_1A receptor expression in GABAergic neurons in cortical and limbic brain areas. Our data agree with previous findings showing that the 5-HT_1A receptor mediates the modulation of glutamatergic neurons. Expression in the limbic and cortical areas suggested an involvement of 5-HT_1A receptors in emotional and cognitive processes.This work was supported by the German Science Foundation (SFB 406; C4 to G.F.).     

Effects of Chronic Administration of Interferon α A/D on Serotonergic Receptors in Rat Brain

The effects of chronic administration of interferon (IFN; recombinant human IFN -A/D) on serotonergic binding sites in rat brain were investigated. IFN was injected daily for 2 weeks at a dose of 100000 I.U./kg, (i.p.) in male Wistar rats. IFN did not alter either [³H]ketanserin binding to 5-HT_2A receptors or [³H]paroxetine binding to 5-HT transporters. Scatchard analysis of [³H]8-hydroxy-dipropylaminotetraline (8-OH-DPAT) binding to 5-HT_1A receptors demonstrated the presence of high- and low-affinity binding sites in both treatment and control groups. IFN significantly increased both Kd and Bmax measures of [³H]8-OH-DPAT binding at low-affinity binding sites, but not at the high-affinity sites. These results suggest that IFN affects the low-affinity 5-HT_1A receptors sites and may be involved in the development of IFN-induced psychiatric disturbances.     

Activation of 5-HT1A receptors inhibits carbachol-stimulated inositol 1,4,5-trisphosphate mass accumulation in the rodent hippocampus

We have previously demonstrated that 5-HT_1A receptor agonists partially prevent the stimulation by carbachol of [³H]-phosphoinositide hydrolysis in immature rat hippocampal slices. This negative modulation has been investigated further by measuring, using a radioreceptor assay, the mass accumulation of IP₃. In hippocampal slices from developing rats and in hippocampal neurons, carbachol enhanced the accumulation of IP₃ and this response was partially inhibited by 8-OH-DPAT with a potency compatible with the affinity of this agonist for 5-HT_1A receptors. The inhibition of the carbachol response by 8-OH-DPAT was non-competitive in nature and 8-OH-DPAT did not affect the inhibitory potency of pirenzepine. The inhibitory effect of 8-OH-DPAT was maintained after washing the slices preincubated with this compound but was not observed on the carbachol-stimulated PIP₂ hydrolysis in hippocampal membranes, suggesting that this compound induces long lasting changes of nuscarinic receptors and/or their effector mechanism by an indirect action.     

BMY 7378: Partial agonist at spinal cord 5-HT1A receptors

Recent data indicate that BMY 7378 demonstrates high affinity, selectivity and low intrinsic activity at hippocampal 5-HT_1A receptors, suggesting that BMY 7378 may represent the first selective 5-HT_1A functional antagonist. The present study examined the agonist and antagonist properties of BMY 7378 at spinal cord 5-HT_1A receptors. In electrophysiological studies, iontophoretic administration of either the 5-HT_1A agonist 8-OH-DPAT (43.8 ± 5.4 nA) or BMY 7378 (46.3 ± 5.2 nA) significantly inhibited the firing rate of wide-dynamic-range dorsal horn units indicating that BMY 7378 demonstrates significant intrinsic activity at spinal cord 5-HT_1A receptors. Concomitant BMY 7378 and 8-OH-DPAT administration identified no BMY 7378 ejection current (20–100 nA) which antagonized the 8-OH-DPAT induced inhibition of dorsal horn unit activity. In behavioral studies in the spinal rat, 8-OH-DPAT increased the animals' sensitivity to noxious levels of mechanical stimulation (ED₅₀ = 269 ± 24 nmol/kg) as did BMY 7378 (ED₅₀ = 295 ± 70 nmol/kg) with no statistically significant difference in the maximal response (Y_max) observed. Concomitant BMY 7378 and 8-OH-DPAT administration identified no BMY 7378 dose (10–1100 nmol/kg) which blocked the hyperalgesic effect of 8-OH-DPAT, rather, each drug produced similar effects which were additive. Further, the 5-HT_1A agonist effects of BMY 7378 were blocked by the 5-HT_1A antagonist, spiperone. Therefore, both the electrophysiologic and reflex data indicate that BMY 7378 possesses significant intrinsic activity at spinal cord 5-HT_1A receptors, and like 8-OH-DPAT is a partial agonist at these receptors. Differences in BMY 7378 intrinsic activity at spinal cord as opposed to hippocampal 5-HT_1A receptors are discussed in terms of regional differences in G-proteins coupled to 5-HT_1A receptors in these two CNS regions.     

Effects of a Chronic Lithium Treatment on Cortical Serotonin Uptake Sites and 5-HT1A Receptors

The objectives of this study were to characterize the effects of a chronic lithium (Li⁺) treatment on serotonin (5-HT) uptake sites and on 5-HT_1A receptors, and to determine the eventual reversibility of the treatment. The experiments were carried out with membranes from rat cerebral cortex using 8-hydroxy-2-(propylamino)tetralin, or [³H]8-OH-DPAT, and [³H]citalopram to label 5-HT_1A receptors and 5-HT uptake sites, respectively. Endogenous levels of 5-HT and 5-hydroxyindole-3-acetic acid (5-HIAA) were measured by high-performance liquid chromatography in the cingulate cortex. The saturation curves with [³H]8-OH-DPAT were always best fitted a two-site model. After a treatment with Li⁺ for 28 days, no alterations in the binding parameters of [³H]8-OH-DPAT to the high- and low-affinity binding sites could be documented. However, competition curves with 5-HT to inhibit [³H]8-OH-DPAT binding revealed a decreased proportion of sites with high affinity for the agonist, together with an increased density of sites with low affinity for 5-HT, suggesting an alteration in the coupling efficacy between 5-HT_1A receptors and their transduction systems. Saturation studies with [³H]citalopram showed an increase (>40%) in the density of 5-HT uptake sites after chronic Li⁺, suggesting a more efficient 5-HT uptake process for the treated animals, in accord with clinical observations. Although 5-HT contents in cingulate cortex remained unchanged after the treatment, 5-HIAA levels decreased (>30%), leading to a diminished (almost 50%) 5-HT turnover; and also reflecting a more efficient uptake in the treated rats, so that less 5-HT could be degraded by extracellular monoamine oxidase. All the effects revealed by [³H]8-OH-DPAT and [³H]citalopram were reversed following a recovery period of two days without Li⁺. Since symptoms of bipolar affective disorders may reappear if the chronic Li⁺ treatment is interrupted, the reversibility of the observed effects further supports the importance of central 5-HT synaptic transmission in the pathophysiology and treatment of human affective disorders.     

Unilateral lesion of the nigrostriatal pathway decreases the response of fast-spiking interneurons in the medial prefrontal cortex to 5-HT1A receptor agonist and expression of the receptor in parvalbumin-positive neurons in the rat

5-Hydroxytryptamine_1A (5-HT_1A) receptors are expressed in the prefrontal cortical interneurons. Among these interneurons, calcium-binding protein parvalbumin (PV)-positive fast spiking (FS) interneurons play an important role in regulatory function of the prefrontal cortex. In the present study, the response of medial prefrontal cortex (mPFC) FS interneurons to the selective 5-HT_1A receptor agonist 8-OH-DPAT and change in expression of 5-HT_1A receptor on PV-positive neurons were examined in rats with 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra pars compacta (SNc) by using extracellular recording and double-labeling immunofluorescence histochemistry. Systemic administration of 8-OH-DPAT (1-243 μg/kg, i.v.) dose-dependently inhibited the mean firing rate of the FS interneurons in sham-operated and the lesioned rats, respectively. The cumulative doses producing inhibition in the lesioned rats (243 μg/kg) was significantly higher than that of sham-operated rats (27 μg/kg). Furthermore, the local application of 8-OH-DPAT (0.01 μg) in the mPFC inhibited the FS interneurons in sham-operated rats, while having no effect on firing rate of the FS interneurons in the lesioned rats. In contrast to sham-operated rats, the lesion of the SNc in rats did not cause the change of PV-positive neurons in the prelimbic prefrontal cortex, a subregion of the mPFC, whereas the lesion of the SNc markedly reduced in percentage of PV-positive neurons expressing 5-HT_1A receptors. Our results indicate that degeneration of the nigrostriatal pathway results in the decreased response of FS interneurons in the mPFC to 5-HT_1A receptor stimulation, which attributes to down-regulation of 5-HT_1A receptor expression in these interneurons.     

The role of various calcium and potassium channels in the regulation of somatodendritic serotonin release

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [³H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT_1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium effux from raphe nuclei slices preloaded with [³H]serotonin, and this inhibition was reversed by 5-HT_1A receptor antagonist (+)WAY-100135. The 5-HT_1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [³H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubate with [³H]serotonin was completely external Ca²⁺-dependent, and omega-conotoxin GVIA and Cd²⁺, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced trititum release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT_1A and 5-HT_1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca²⁺ channels; (3) the 5-HT_1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K⁺ channels.     

Effects of novel 5-HT1A receptor antagonists on measures of postsynaptic 5-HT1A receptor activation in vivo

The effects of two putative 5-HT_1A antagonists, 4-(2′-methoxyphenyl)-1-[2′-[N-(2″-pyridinyl)-p-iodobenzamido]ethyl]piperazine (p-MPPI) and 4-(2′-methoxyphenyl)-1-[2′-[N-(2″-pyridinyl)-p-flourobenzamido]ethyl]piperazine (p-MPPF), were examined in vivo in two tests of postsynaptic 5-HT_1A receptor activation, hypothermia and reciprocal forepaw treading, in the rat. Both p-MPPI (10 mg/Kg, I.p.) and p-MPPF (10 mg/Kg, I.p.) antagonized the hypothermia induced by the 5-HT_1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (0.5 mg/Kg, S.c.). Neither p-MPPI nor p-MPPF administered alone at a dose of 10 mg/kg (i.p.) induced hypothermia. Similarly, both p-MPPI (10 mg/Kg, I.p.) and p-MPPF (2.5 mg/Kg, I.p.) completely antagonized 8-OH-DPAT (2 mg/Kg, S.c.)-induced forepaw treading in rats pretreated with reserpine (1 mg/Kg, S.c., 18–24 hours prior to the experiment). p-MPPI and p-MPPF, at doses of 10 mg/kg (i.p.) did not induce forepaw treading in reserpine pretreated animals. The results of the present study demonstrate that p-MPPI and p-MPPF act as 5-HT_1A receptor antagonists in these measures of postsynaptic 5-HT_1A a receptor activation.     

Subchronic Treatment with Methamphetamine and Phencyclidine Differentially Alters the Adenosine A1 and A2A Receptors in the Prefrontal Cortex,Hippocampus, and Striatum of the Rat

Subchronic treatment with MAP (4.6 mg/kg, i.p., once daily for 11 days) significantly decreased the K_d, but not B_max, values of [³H]1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) binding to adenosine A₁ receptors in the prefrontal cortex and hippocampus, but not striatum, of rat brain. However, subchronic treatment with PCP (10 mg/kg, i.p., once daily for 11 days) did not alter the K_d and B_max values of [³H]DPCPX binding to adenosine A₁ receptors in these three regions. Subchronic treatment with MAP or PCP did not alter the B_max and K_d values of [³H]2-p-(2-carboxyehyl)phenethylamino-5-N-ethylcarboxyamidoadenosine ([³H]CGS21680) binding to adenosine A_2A receptors in the striatum. Furthermore, subchronic treatment with MAP or PCP significantly decreased the specific binding of [³H]CGS21680 to adenosine A_2A receptors in the hippocampus, but not in the prefrontal cortex. Thus, these results suggest that MAP and PCP may produce differential effects on the adenosine A_2A receptors, but not adenosine A₁ receptors in rat brain.     

Dissociation of the plasticity of 5-HT1A sites and 5-HT transporter sites

To study the early effects of neonatal 5,7-dihydroxytryptamine lesions on 5-hydroxytryptamine_1A (5-HT_1A) receptors, we measured regional [³H]8-OH-DPAT-labeled 5-HT_1A sites in binding assays and compared them to our previous studies of [³H]paroxetine-labeled 5-HT transporter sites during the first month in the same rats. While there were significant time- and dose-dependent effects of 5,7-DHT on 5-HT transporter sites, there were no significant changes in 5-HT_1A sites in cortex, hippocampus, diencephalon, brainstem, cerebellum, or spinal cord. 5,7-DHT lesions also did not alter the K_i of Gpp(NH)p at brainstem 5-HT_1A sites or the K_i of 5-HT in cortex or brainstem in the presence or absence of GTPS or Gpp(NH)p. There were significant regional differences between the density of 5-HT_1A sites and 5-HT transporter sites. The ontogeny of brainstem 5-HT_1A sites was a pattern of increases until three weeks postnatal, and 5,7-DHT lesions did not alter the ontogeny of 5-HT_1A sites. These data suggest differential plasticity of 5-HT_1A and 5-HT transporter binding sites during the first month after neonatal 5,7-DHT lesions.     

Serotonin 5-HT1A Receptor Activation Increases Cyclic AMP Formation in the Rat Hippocampus In Vivo

Abstract: In vivo microdialysis was used to examine the efflux of cyclic AMP (cAMP) into the extracellular fluid of the ventral hippocampus in the freely moving rat. The changes in extracellular cAMP concentration were monitored in response to forskolin and the serotonin 5-HT_1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The basal level of hippocampal extracellular cAMP was 2.3 ± 0.2 pmol/ml (n = 6), after a 3-h postsur- gery stabilisation period. Perfusion of forskolin (100 μM) through the probe for 30 min significantly increased the efflux of cAMP, which returned to baseline levels within 90 min. 8-OH-DPAT (0.3 mg/kg s.c.) also significantly increased cAMP efflux, whereas a similar volume of saline had no effect. Desensitisation of the 8-OH-DPAT-induced increase in cAMP efflux was observed following a second administration of 8-OH-DPAT after a 4-h interval. Administration of 8-OH-DPAT did not alter the efflux of cAMP when forskolin was perfused through the probe. Pretreatment with WAY 100135 [N-tert-butyl 3–4-(2-methoxyphenyl)piperazine-1 -yl-2-phenylpropanamide dihydrochloride] (5 mg/kg s.c.), a specific 5-HT_1A receptor antagonist, prevented the 8-OH-DPAT-induced increase in cAMP efflux. The data indicate that the 8-OH-DPAT-induced increase in cAMP efflux in vivo is mediated by a 5-HT_1A receptor.     

[11C]WAY 100635: A radioligand for imaging 5-HT1A receptors with positron emission tomography

The potent and selective 5-HT_1A antagonist WAY 100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide) was radiolabeled with ¹¹C in high specific activity, and the in vivo properties of this radioligand were assessed in the brains of rats and monkeys. Following i.v. tail vein injection in rats, [¹¹C]WAY 100635 rapidly penetrated into brain tissue and was retained over a 30–90 min time period in a manner consistent with the known distribution of 5-HT_1A receptors. Pretreatment of rats with the selective 5-HT_1A agonist (±)-8-OH-DPAT effectively blocked the retention of radioactivity in brain regions known to contain high densities of 5-HT_1A receptors. The hippocampus-to-cerebellum radioactivity concentration ratio reached a maximum of 16:1 at 60 min post injection. Following i.v. injection of [¹¹C]WAY 100635 in rhesus monkeys, the concentrations of radioactivity in brain regions were consistent with the reported distribution of 5-HT_1A receptors in primates, and the frontal cortex-to-cerebellum ratio reached 5.5:1 at 80 min post injection. Pretreatment of the monkeys with (±)-8-OH-DPAT reduced this ratio to 1.4:1, and injection of (±)-8-OH-DPAT 20 min after the injection of [¹¹C]WAY 100635 significantly displaced frontal cortex binding. The in vivo properties of [¹¹C]WAY 100635 in rats and monkeys strongly support the future utility of this radioligand for imaging 5-HT_1A receptors using positron emission tomography (PET).     

Brainstem 5-Hydroxytrytamine1A Binding Sites are not Down-Regulated by Agonists which Induce Tolerance in the Rat: Myoclonus and other Serotonergic Behaviors

Abstract

To study the regulation of 5-HT_1A receptors in the brainstem, the region most relevant to the serotonin syndrome and to serotonin-responsive human myoclonic disorders, we chronically treated rats with various 5-HT_1A agonists and labeled 5-HT_1A sites with [³H]8-OH-DPAT. Daily injection for 30 consecutive days of 10 mg/kg ip 8-OH-DPAT (pre- and post-synaptic 5-HT_1A agonist) significantly decreased 8-OH-DPAT-evoked flat body posture, forelimb myoclonus, and hypothermia compared to chronic vehicle injection. There was no cross tolerance to 8-OH-DPAT in rats chronically injected with ipsapirone or buspirone (presynaptic 5-HT_1A agonists). However, none of the 5HT_1A agonists significantly altered B_max of brainstem 5-HT_1A binding sites. Chronic injection with other drugs such as 1-propranolol, (±) pindolol and spiperone (5-HT_1A and 5-HT₂ antagonists), methysergide (5-HT₁ and 5-HT₂ antagonist), and agonists and antagonists at various other 5-HT receptors also had no effect on binding parameters. These data demonstrate lack of cross-tolerance between pre- and post-synaptically acting 5-HT_1A agonists and absence of down-regulation of presynaptic 5-HT_1A sites at doses which induced tolerance of 5-HT_1A-mediated behaviors of the serotonin syndrome. They suggest changes in the post-synaptic cell rather than the receptor recognition site as the mechanism of tolerance.     

Heterogeneity of Cortical and Hippocampal 5-HT1A Receptors: A Reappraisal of Homogenate Binding with 8-[3H]Hydroxydipropylaminotetralin

Abstract: The selective serotonin (5-HT) agonist 8-hydroxydipropylaminotetralin (8-OH-DPAT) has been extensively used to characterize the physiological, biochemical, and behavioral features of the 5-HT_1A receptor. A further characterization of this receptor subtype was conducted with membrane preparations from rat cerebral cortex and hippocampus. The saturation binding isotherms of [³H]8- OH-DPAT (free ligand from 200 pM to 160 nM) revealed high-affinity 5-HT_1A receptors (K_H= 0.7–0.8 nM) and lowaffinity (K_L= 22–36 nM) binding sites. The kinetics of [³H]8-OH-DPAT binding were examined at two ligand concentrations, i.e., 1 and 10 nM, and in each case revealed two dissociation rate constants supporting the existence of high- and low-affinity binding sites. When the high-affinity sites were labeled with a 1 nM concentration of [³H]8- OH-DPAT, the competition curves of agonist and antagonist drugs were best fit to a two-site model, indicating the presence of two different 5-HT_1A binding sites or, alternatively, two affinity states, tentatively designated as 5-HT_1AHIGH and 5-HT_1A^LOW. However, the low correlation between the affinities of various drugs for these sites indicates the existence of different and independent binding sites. To determine whether 5-HT_1A sites are modulated by 5′-guanylylimidodiphosphate, inhibition experiments with 5-HT were performed in the presence or in the absence of 100 μM 5′-guanylylimidodiphosphate. The binding of 1 nM [³H]8-OH-DPAT to the 5-HT_1A^HIGH site was dramatically (80%) reduced by 5′-guanylylimidodiphosphate; in contrast, the low-affinity site, or 5-HT_1A^LOW, was seemingly insensitive to the guanine nucleotide. The findings suggest that the high-affinity 5-HT_1A^HIGH site corresponds to the classic 5-HT_1A receptor, whereas the novel 5-HT_1A^LOW binding site, labeled by 1 nM [³H]8-OH-DPAT and having a micromolar affinity for 5-HT, may not belong to the G protein family of receptors. To further investigate the relationship of 5-HT_1A sites and the 5-HT innervation, rats were treated with p-chlorophenylalanine or with the neurotoxin p-chloroamphetamine. The inhibition of 5-HT synthesis by p-chlorophenylalanine did not alter either of the two 5-HT_1A sites, but deafferentation by p-chloroamphetamine caused a loss of the low-affinity [³H]8-OH- DPAT binding sites, indicating-that these novel binding sites may be located presynaptically on 5-HT fibers and/or nerve terminals.     

Regulation of Glial Na+/K+-ATPase by Serotonin: Identification of Participating Receptors

The purpose of the present study was the characterization of the receptors participating in the regulatory mechanism of glial Na⁺/K⁺-ATPase by serotonin (5-HT) in rat brain. The activity of the Na⁺ pump was measured in four brain regions after incubation with various concentrations of serotoninergic agonists or antagonists. A concentration-dependent increase in enzyme activity was observed with the 5-HT_1A agonist R (+)-2-dipropylamino-8-hydroxy-1,2,3, 4-tetrahydronaphthalene hydrobromide (8-OH-DPAT) in homogenates or in glial membrane enriched fractions from cerebral cortex and in hippocampus. Spiperone, a 5-HT_1A antagonist, completely inhibited the response to 8-OH-DPAT but had no effect on Na⁺/K⁺-ATPase activity in cerebellum where LSD, a 5-HT₆ agonist, elicited a dose-dependent response similar to that of 5-HT. In brainstem, a lack of reponse to 5-HT and other agonists was confirmed. Altogether, these results show that serotonin modulates glial Na⁺/K⁺-ATPase activity in the brain, apparently not through only one type of 5-HT receptor. It seems that the receptor system involved is different according to the brain region. In cerebral cortex, the response seems to be mediated by 5-HT_1A as well as in hippocampus but not in cerebellum where 5-HT₆ appears as the receptor system involved.     

Photoaffinity Labelling and Solubilization of the Central 5-HT1A Receptor Binding Site

Abstract

Two complementary approaches, covalent labelling and solubilization, have been used to study the biochemical properties of the central 5-HT_1A receptor binding site. We have first designed a photoaffinity ligand containing the structure of 8-OH-DPAT, a potent and specific agonist of 5-HT_1A sites. Thus, 8-methoxy-2[N-n-propyl,N-3-(2-nitro-4-azido-phenyl)- aminopropyl]aminotetralin or 8-methoxy-3'-NAP-amino-PAT, was found to displace, in the dark, [³H]8-OH-DPAT from 5-HT_1A sites in rat hippocampal membranes with an IC₅₀ of 6.6 nM. Under two cumulative UV irradiations (366 nm, for 20 min at 4°C), 8-methoxy-3-'-NAP-amino-PAT (30 nM) blocked irreversibly 55-60% of 5-HT_1A binding sites. This blockade was specific of 5-HT_1A sites since the other serotoninergic sites, 5-HT_1B, 5-HT₂ and also the presynaptic 5-HT₃ sites were not affected by the treatment. In addition, the binding of [³H]Spiperone and [³H]7-OH-DPAT to striatal dopamine sites remained unchanged under similar photolysis conditions. The tritiated derivative of the photoaffinity ligand (92 Ci/mmol) was then synthesized for the identification of the covalently bound protein(s). SDS-PAGE of solubilized membranes irradiated in the presence of 20 nM ³H-8-methoxy-3'-NAP-amino-PAT allowed the detection of a 63 kD protein whose labelling appeared specific. Thus, ³H-incorporation into the 63 kD band could be prevented by uM concentrations of 5-HT, 8-OH-DPAT and other selective 5-HT_1A ligands such as isapirone. In contrast, the 5-HT₂ antagonist ketanserin, norepinephrine and dopamine-related ligands (including 7-OH-DPAT) were ineffective. Direct solubilization of 5-HT_1A receptor binding sites was also attempted from rat hippocampal membranes. The best results were obtained using CHAPS (10 mM) plus NaCl (0.2 M), which led to 50 % recovery of 5-HT_1A sites in the 100,000 g supernatant. The pharmacological properties and sensitivity to N-ethyl-maleimide and GppNHp of soluble sites appeared near identical to those of membrane-bound 5-HT_1A sites.     

Functional Responses to the Chronic Activation of 5-HT<Subscript>1A</Subscript> Receptors in Mice with Genetic Predisposition to Catalepsy

The effects of chronic 5-HT_1A receptor activation on the behavior, functional activity of 5-HT_1A receptors, and expression of key genes of the brain 5-HT system were studied in mice of the catalepsy-prone CBA strain and the catalepsy-resistant C57BL/6 strain. Chronic treatment with 8-Hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) (1.0 mg/kg i.p., 14 days) led to a significant decrease in the hypothermic response to acute administration of 8-OH-DPAT in CBA and C57BL/6 mice, which indicates the desensitization of 5-HT_1A receptors in both strains. Pretreatment with the 5-HT⁷ receptor agonist SB 269970 did not affect the hypothermic response to the acute administration of 8-OH-DPAT, which suggests an independent functional response of 5-HT_1A receptors. The treatment did not induce any changes in the behavior in the open field paradigm in CBA mice, but significantly increased the total path, the time spent in the center, and the number of rearings in C57BL/6 mice, which indicates the enhancement of locomotor and exploratory activity in C57BL/6 mice. The chronic activation of 5-HT_1A receptor downregulated 5-HT_1A gene expression, as well as the expression of the gene that encodes tryptophan hydroxylase 2, a key enzyme of 5-HT biosynthesis, in the midbrain and the expression of the gene that encodes the 5-HT_2A receptor in the frontal cortex of CBA, but not C57BL/6 mice. The obtained data provide a new evidence on the receptor–gene cross talk in the brain 5-HT system that may underlie the loss of pharmacological efficacy of 5-HT_1A receptor agonists. In turn, the loss of the behavioral response and compensatory alterations in key genes of the brain 5- HT system in CBA mice suggests that catalepsy-prone and -resistant genotypes demonstrate different sensibility to the effects of drugs.