Metal Ion and Guanine Nucleotide Modulations of Agonist Interaction in G-Protein-Coupled Serotonin1A Receptors from Bovine Hippocampus

1. The serotonin type 1A (5-HT_1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to GTP-binding regulatory proteins (G-proteins). We have studied the modulation of agonist binding to 5-HT_1A receptors from bovine hippocampus by metal ions and guanine nucleotide.2. Bovine hippocampal membranes containing the 5-HT_1A receptor were isolated. These membranes exhibited high-affinity binding sites for the specific agonist [³H]OH-DPAT.3. The agonist binding is inhibited by monovalent cations Na⁺, K⁺, and Li⁺ in a concentration-dependent manner. Divalent cations such as Ca²⁺, Mg²⁺, and Mn²⁺, on the other hand, show more complex behavior and induce enhancement of agonist binding up to a certain concentration. The effect of the metal ions on agonist binding is strongly modulated in the presence of GTP--S, a nonhydrolyzable analogue of GTP, indicating that these receptors are coupled to G-proteins.4. To gain further insight into the mechanisms of agonist binding to bovine hippocampal 5-HT_1A receptors under these conditions, the binding affinities and binding sites have been analyzed by Scatchard analysis of saturation binding data. Our results are relevant to ongoing analyses of the overall regulation of receptor activity for G-protein-coupled seven transmembrane domain receptors.     

Role of Disulfides and Sulfhydryl Groups in Agonist and Antagonist Binding in Serotonin1A Receptors from Bovine Hippocampus

SUMMARY 1. The serotonin_1A (5-HT_1A) receptors are members of a superfamily of seven-transmembrane-domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions. Mutagenesis and modeling studies point out that the ligand-binding sites in serotonin receptors are located in the transmembrane domain. However, these binding sites are not very well characterized. Since disulfide bonds and sulfhydryl groups have been shown to play vital roles in the assembly, organization, and function of various G-protein-coupled receptors, we report here the effect of disulfide and sulfhydryl group modifications on the agonist and antagonist binding activity of 5-HT_1A receptors from bovine hippocampus.2. DTT or NEM treatment caused a concentration-dependent reduction in specific binding of the agonist and antagonist in 5-HT_1A receptors from bovine hippocampal native and solubilized membranes. This is supported by a concomitant reduction in binding affinity.3. Pretreatment of the receptor with unlabeled ligands prior to chemical modifications indicate that the majority of disulfides or sulfhydryl groups that undergo modification giving rise to inhibition in binding activity could be at the vicinity of the ligand-binding sites.4. In addition, ligand-binding studies in presence of GTP--S, a nonhydrolyzable analogue of GTP, indicate that sulfhydryl groups (and disulfide bonds to a lesser extent) are vital for efficient coupling between the 5-HT_1A receptor and the G-protein.5. Our results point out that disulfide bonds and sulfhydryl groups could play an important role in ligand binding in 5-HT_1A receptors.     

Interaction of Serotonin1A Receptors from Bovine Hippocampus with Tertiary Amine Local Anesthetics

1. We have examined the interaction of tertiary amine local anesthetics with the bovine hippocampal serotonin1A (5-HT1A) receptor, an important member of the G-protein-coupled receptor superfamily. 2. The local anesthetics inhibit specific agonist and antagonist binding to the 5-HT1A receptor at a clinically relevant concentration range of the anesthetics. This is accompanied by a concomitant reduction in the binding affinity of the 5-HT1A receptor to the agonist. Interestingly, the extent of G-protein coupling of the receptor is reduced in the presence of the local anesthetics. 3. Fluorescence polarization measurements using depth-dependent fluorescent probes show that procaine and lidocaine do not show any significant change in membrane fluidity. On the other hand, tetracaine and dibucaine were found to alter fluidity of the membrane as indicated by a fluorescent probe which monitors the headgroup region of the membrane. 4. The local anesthetics showed inhibition of agonist binding to the 5-HT1A receptor in membranes depleted of cholesterol more or less to the same extent as that of control membranes in all cases. This suggests that the inhibition in ligand binding to the 5-HT1A receptor brought about by local anesthetics is independent of the membrane cholesterol content. 5. Our results on the effects of the local anesthetics on the ligand binding and G-protein coupling of the 5-HT1A receptor support the possibility that G-protein-coupled receptors could be involved in the action of local anesthetics.     

The Serotonin 1A A Receptor: A Representative Member of the Serotonin Receptor Family

1. Serotonin is an intrinsically fluorescent biogenic amine that acts as a neurotransmitter and is found in a wide variety of sites in the central and peripheral nervous system. Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive and behavioral functions.2. Serotonin exerts its diverse actions by binding to distinct cell surface receptors which have been classified into many groups. The serotonin_1A (5-HT_1A) receptor is the most extensively studied of the serotonin receptors and belongs to the large family of seven transmembrane domain G-protein coupled receptors.3. The tissue and sub-cellular distribution, structural characteristics, signaling of the serotonin_1A receptor and its interaction with G-proteins are discussed.4. The pharmacology of serotonin_1A receptors is reviewed in terms of binding of agonists and antagonists and sensitivity of their binding to guanine nucleotides.5. Membrane biology of 5-HT_1A receptors is presented using the bovine hippocampal serotonin_1A receptor as a model system. The ligand binding activity and G-protein coupling of the receptor is modulated by membrane cholesterol thereby indicating the requirement of cholesterol in maintaining the receptor organization and function. This, along with the reported detergent resistance characteristics of the receptor, raises important questions on the role of membrane lipids and domains in the function of this receptor.     

Solubilization of Serotonin1A Receptors Heterologously Expressed in Chinese Hamster Ovary Cells

1. The serotonin1A (5-HT1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions. 2. We report here, for the first time, the solubilization of 5-HT1A receptors stably expressed in Chinese Hamster Ovary (CHO) cells using the zwitterionic detergent CHAPS in presence of NaCl followed by polyethylene glycol (PEG) precipitation. We show by ligand-binding assay that the 5-HT1A receptor solubilized this way is functionally active. We have optimized the efficiency of solubilization with respect to total protein and NaCl concentration. 3. Our results show that careful control of salt and protein concentration is crucial in optimal solubilization of membrane receptors heterologously expressed in cells in culture. The effective solubilization of important neurotransmitter receptors such as 5-HT1A receptors which are present in very low amounts in the native tissue may represent an important step in characterizing membrane receptors expressed in mammalian cells in culture.     

Ligand Binding and G-protein Coupling of the Serotonin1A Receptor in Cholesterol-enriched Hippocampal Membranes

The serotonin_1A receptor is the most extensively studied member of the family of seven transmembrane domain G-protein coupled serotonin receptors. Since a large portion of such transmembrane receptors remains in contact with the membrane lipid environment, lipid–protein interactions assume importance in the structure-function analysis of such receptors. We have earlier reported the requirement of cholesterol for serotonin_1A receptor function in native hippocampal membranes by specific depletion of cholesterol using methyl- β-cyclodextrin. In this paper, we monitored the serotonin_1A receptor function in membranes that are enriched in cholesterol using a complex prepared from cholesterol and methyl-β-cyclodextrin. Our results indicate that ligand binding and receptor/G-protein interaction of the serotonin_1A receptor do not exhibit significant difference in native and cholesterol-enriched hippocampal membranes indicating that further enrichment of cholesterol has little functional consequence on the serotonin_1A receptor function. These results therefore provide new information on the effect of cholesterol enrichment on the hippocampal serotonin_1A receptor function.     

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.     

The Human Serotonin 1A Receptor Expressed in Neuronal Cells: Toward a Native Environment for Neuronal Receptors

1. The serotonin_1A (5-HT_1A) receptor is an important representative of G-protein coupled family of receptors. It is the most extensively studied among the serotonin receptors, and appears to be involved in various behavioral and cognitive functions.2. We report here the pharmacological and functional characterization of the human serotonin_1A receptor stably expressed in HN2 cell line, which is a hybrid cell line between hippocampal cells and mouse neuroblastoma.3. Our results show that serotonin_1A receptors in HN2-5-HT_1AR cells display ligand-binding properties that closely mimic binding properties observed with native receptors. We further demonstrate that the differential discrimination of G-protein coupling by the specific agonist and antagonist, a hallmark of the native receptor, is maintained for the receptor in HN2-5-HT_1AR cells. Importantly, the serotonin_1A receptor in HN2-5-HT_1AR cells shows efficient downstream signalling by reducing cellular cyclic AMP levels.4. We conclude that serotonin_1A receptors expressed in HN2-5-HT_1AR cells represent a useful model system to study serotonin_1A receptor biology, and is a potential system for solubilization and purification of the receptor in native-like membrane environment.     

The sterol-binding antibiotic nystatin differentially modulates ligand binding of the bovine hippocampal serotonin1A receptor

We have monitored the ligand binding of the bovine hippocampal 5-HT1A receptor following treatment with the sterol-binding antifungal antibiotic nystatin. Nystatin considerably inhibits the specific binding of the antagonist to 5-HT1A receptors in a concentration-dependent manner. However, the specific agonist binding does not show significant changes. Fluorescence polarization measurements of membrane probes incorporated at different locations in the membrane revealed a substantial decrease in the membrane order in the interior of the bilayer. Experiments with cholesterol-depleted membranes indicate that the action of nystatin is mediated through membrane cholesterol. These results represent the first report on the effect of a cholesterol-perturbing agent on the ligand-binding activity of this important neurotransmitter receptor.     

Effect of Mg2+ on guanine nucleotide sensitivity of ligand binding to serotonin1A receptors from bovine hippocampus

The serotonin1A (5-HT1A) receptor is an important member of the superfamily of seven transmembrane domain G-protein coupled receptors (GPCRs). We report here that guanine nucleotide sensitivity of agonist binding to hippocampal 5-HT1A receptors is dependent on the concentration of Mg2+. Our results show that agonist binding to 5-HT1A receptors is relatively insensitive to guanine nucleotides in the absence of Mg2+. In contrast to this, the specific antagonist binding is insensitive to guanine nucleotides, even in the presence of Mg2+. These results point out the requirement of an optimal concentration of Mg2+ which could be used in assays toward determining guanine nucleotide sensitivity of ligand binding to GPCRs such as the 5-HT1A receptor. Our results provide novel insight into the requirement and concentration dependence of Mg2+ in relation to guanine nucleotide sensitivity for the 5-HT1A receptor in particular, and GPCRs in general.     

Seizure Activity Produces Differential Changes in Adenosine A1 Receptors within Rat Hippocampus

Specific ligand binding to rat hippocampal adenosine A₁ receptor after administration of the convulsant drug 3-mercaptopropionic acid (MP) was studied by means of a quantitative autoradiographic method. 2-Chloro-N6-[cyclopentyl-2,3,4,5-³H adenosine] ([³H]CCPA), a potent and selective A₁ receptor ligand, was selected for binding studies. MP administration (150 mg/kg, i.p.), at seizure, caused significant increases in the following CA1 layers: pyramidal (45%), radiatum (18%) and lacunosum molecular (35%); in CA2 area, a significant decrease in stratum oriens (36%) and an increase in stratum radiatum (14%) and lacunosum molecular (33%) layers was observed. In CA3 area a rise in pyramidal (40%) and radiatum layers (26%), as well as in hillus (97%) was found. At postseizure, changes were restricted to CA1, CA2 and CA3 pyramidal layers and to CA1 lacunosum molecular layer, with increases ranging from 22 to 50%. These results show that [³H]CCPA binding is modified diversely in intrahippocampal layers and areas, thus indicating their dissimilar role in seizure activity.     

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.     

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.     

Further Characterization of 5-HT1A Receptors in the Goldfish Retina: Role of Cyclic AMP in the Regulation of the In Vitro Outgrowth of Retinal Explants

The presence of serotonin 5-HT_1A receptors and their physiological role were further characterized in the goldfish retina. The effects of the 5-HT_6/7 receptor antagonists pimozide, fluphenazine and amoxapine, the 5-HT_1A receptor antagonist WAY-100,135, and the alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, on the 5-HT_1A receptor agonist [³H]8-hydroxy-2-(di-n-propylamino)tetralin binding to retinal membranes, were evaluated. In addition, the effects of serotonin, 8-hydroxy-2-(di-n-propylamino)tetralin, WAY-100,135, the adenylate cyclase inhibitors SQ22536 and MDL12330A, and the cyclic AMP analog 8-bromoadenosine-3:5 cyclic monophosphate were also studied on neuritic outgrowth from retinal explants. WAY-100,135 but not 5-HT_6/7receptor antagonists inhibited [³H]8-hydroxy-2-(di-n-propylamino)tetralin binding to retinal membranes N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline decreased [³H]8-hydroxy-2-(di-n-propylamino)tetralin binding sites up to 70%, while receptor turnover was similar to that reported in other tissues. Serotonin and 8-hydroxy-2-(di-n-propylamino)tetralin stimulated cyclic AMP production, both ex vivo and in vitro, and these increases were related to inhibition of neuritic outgrowth. The inhibitory effect was reduced by SQ22536 and by WAY-100,135, and was mimicked by 8-bromoadenosine-3:5cyclic monophosphate. This study supports previous findings about the role of serotonin as a regulator of axonal outgrowth during in vitro regeneration of the goldfish retina and demonstrates that this effect is mediated, at least in part, by 5-HT_1A receptors through a mechanism which involves an increase of cyclic AMP levels.     

Ethanol Inhibits the Binding of Substance P to Rat Brain Cortex NK1 Receptors

The binding of ¹²⁵I-labeled substance P (SP) to rat brain cortex membranes has been studied Under control conditions and in the presence of ethanol. The binding of SP at low concentrations (20–1000 pM) gave two components, one with a K _D value of 80 pM and another one with a K _D of 500 pM. The higher-affinity component is due to NK₁ receptors, as confirmed by the inhibition Of the SP binding by the rodent NK1 specific agonist [Sar₉ Met(O₂)₁₁]SP. Ethanol (1.7 mM) added to the binding assays inhibited by more than 50% the specific binding at a very low SP concentration (20 pM); however, it had no effect at SP concentrations ranging from 50 to 120 pM. This suggests a decrease by ethanol of the affinity of SP to the NK₁ receptors involved in this binding component. The ethanol effect disappeared at [EtOH] 0.17 mM.     

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.     

Decreased Hepatic 5-HT<Subscript>1A</Subscript> Receptors During Liver Regeneration and Neoplasia in Rats

In the present study we investigated the role of 5-hydroxytryptamine (5-HT) and 5-HT_1A receptor during liver regeneration after partial hepatectomy (PH) and N-nitrosodiethylamine (NDEA) induced hepatocellular carcinoma in male Wistar rats. 5-HT content was significantly increased during liver regeneration after PH and NDEA induced hepatocellular carcinoma. Scatchard analysis using 8-OH-DPAT, a 5-HT_1A specific agonist showed a decreased receptor during liver regeneration after PH and NDEA induced hepatocellular carcinoma. 5-HT when added alone to primary hepatocyte culture did not increase DNA synthesis but was able to increase the EGF mediated DNA synthesis and inhibit the TGFβ1 mediated DNA synthesis suppression in vitro. This confirmed the co-mitogenic activity of 5-HT. 8-OH-DPAT at a concentration of 10⁻⁴ M inhibited the basal and EGF-mediated DNA synthesis in primary hepatocyte cultures. It also suppressed the TGFβ1-mediated DNA synthesis suppression. This clearly showed that activated 5-HT_1A receptor inhibited hepatocyte DNA synthesis. Our results suggest that decreased hepatic 5-HT_1A receptor function during hepatocyte regeneration and neoplasia has clinical significance in the control of cell proliferation.     

Alterations in 5-HT(1A) receptors and adenylyl cyclase response by trazodone in regions of rat brain

The in vivo effect of trazodone on the density of [(3)H]5-HT binding sites and 5-HT(1A) receptors and adenylyl cyclase (AC) response was studied in regions of rat brain. The chronic administration of trazodone (10 mg/Kg body wt, 40 days) resulted in a significant downregulation of [(3)H]5-HT binding sites and 5-HT(1A) receptors in cortex and hippocampus. Trazodone significantly (p < 0.0001) decreased the density of [(3)H]5-HT binding sites in cortex (42.6 +/- 3.6 fmol/mg protein, 65%) and hippocampus (12.6 +/- 1.6 fmol/mg protein, 87%) when compared to control values of 121.9 +/- 5.4 and 99.3 +/- 7.5 fmol/mg protein in these regions, respectively. Similarly there was a significant (p < 0.0001) decrease in the density of 5-HT(1A) receptors in both cortex (7.2 +/- 0.5 fmol/mg protein, 70%) and hippocampus (6.3 +/- 1.2 fmol/mg protein, 79%) when compared to control values of 24.2 +/- 2.1 and 30.6 +/- 3.7 fmol/mg protein, in these regions respectively. However, the affinity of [(3)H]5-HT to 5-HT binding sites (1.83 +/- 0.26 nM, p < 0.0001) and [(3)H]8-OH-DPAT to 5-HT(1A) receptors (0.60 +/- 0.06 nM, p < 0.05) was significantly decreased only in cortex when compared to the control K(d) values of 0.88 +/- 0.04 nM and 0.47 +/- 0.02 nM in these regions, respectively.The basal AC activity did not alter in treated rats, where as, the inhibition of forskolin-stimulated AC activity by 5-HT (10 microM) was significantly (p < 0.0001) decreased both in cortex (43%) and hippocampus (40%) when compared to control levels. In conclusion, chronic treatment with trazodone results in downregulation of 5-HT(1A) receptors in cortex and hippocampus along with concomitant increased AC response, suggesting the involvement of 5-HT(1A) receptor-mediated AC response in the mechanism of action of trazodone.     

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.     

Reduced Efficacy of 8-OH-DPAT's Inhibition of Lordosis Behavior by Prior Estrogen Treatment

The effects of bilateral VMN infusion with the 5-HT1A receptor agonist, (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 200, 1000, or 2000 ng), on lordosis behavior were examined in hormonally primed ovariectomized rats. When rats were given a single injection with 25 microg estradiol benzoate followed 48 h later with 500 microg progesterone, inhibition of lordosis behavior was evident at all doses of 8-OH-DPAT. However, when rats were treated with 25 microg estradiol benzoate followed 7 days later with a second injection of 25 microg estradiol benzoate and then progesterone, none of the doses of 8-OH-DPAT effectively inhibited lordosis behavior. In some rats, cannulae were located near the most rostral portion of the VMN. In these rats, there was no effect of the second estrogen treatment on the response to 8-OH-DPAT. Therefore, a second experiment was performed to specifically evaluate the effects of two estradiol benzoate treatments on the response to bilateral 8-OH-DPAT infusion in the rostral VMN. In contrast to the reduced effectiveness of the 8-OH-DPAT infusion in the mid to caudal VMN in rats given two injections with estradiol benzoate, 2000 ng 8-OH-DPAT continued to effectively inhibit lordosis behavior following the 5-HT1A receptor agonist's infusion into the more rostral areas. These findings are discussed in relation to earlier studies in which the potency, but not the efficacy, of 8-OH-DPAT was reduced following systemic treatment with the 5-HT1A receptor agonist.