Acetaminophen-induced antinociception via central 5-HT(2A) receptors.

Acetaminophen is one of the most widely used analgesic drugs. Although the mechanism of analgesic action of acetaminophen is still not known, the involvement of the central serotonin (5-hydroxytryptamine: 5-HT) system is one possibility. In the present study, we examined the antinociceptive effect of acute and chronic intraperitoneally (i.p.) administered acetaminophen by tail flick latency measurements in the rat. A significantly increased tail flick latency was observed in acute and 15-day acetaminophen-treated rats, but not in 30-day acetaminophen-treated rats, at a dose of 400 mg/kg/day. To investigate the plasticity of receptors at postsynaptic membrane, we conducted a series of experiments by radioligand binding method on frontal cortex and brainstem membrane. The technique involved radioligand binding with [phenyl-4-³H]spiperone and ketanserin for studying 5-HT_2A receptor characteristics. A significant decrease in the maximum number of 5-HT_2A binding sites (B_max) was demonstrated in all treatment groups with acetaminophen 300 and 400 mg/kg on frontal cortex membrane, whereas the value of the dissociation equilibrium constant (K_d) remained unchanged. The down-regulation of 5-HT_2A binding sites in frontal cortex was of a lesser magnitude after 30 days of treatment and the tail flick latency was as in the control animals. These results suggest that down-regulation of 5-HT_2A receptor in response to 5-HT release is a major step in the mechanism underlying analgesia produced by this agent. On the contrary, chronic use of acetaminophen may result in 5-HT depletion, which in turn produces re-adaptation of postsynaptic 5-HT_2A receptors. These data provide further evidence for a central 5-HT-dependent antinociceptive effect of acetaminophen.     

The Serotonin 5-HT2C Receptor Is a Prominent Serotonin Receptor in Basal Ganglia: Evidence from Functional Studies on Serotonin-Mediated Phosphoinositide Hydrolysis

Abstract: Serotonin (5-hydroxytryptamine; 5-HT) 5-HT_2A and 5-HT_2C receptors belong to the class of phosphoinositide-specific phospholipase C (PLC)-linked receptors. Conditions were established for measuring 5-HT_2A-linked and 5-HT_2C-linked PLC activity in membranes prepared from previously frozen rat frontal cortex and caudate. In the presence of Ca²⁺ (300 nM) and GTPγS (1 µM), 5-HT increased PLC activity in caudate membranes. Pharmacological analysis using the selective 5-HT_2A antagonist, spiperone, and the nonselective 5-HT_2A/2C antagonist, mianserin, demonstrated that over half of the 5-HT-stimulated PLC activity was due to stimulation of 5-HT_2C receptors as opposed to 5-HT_2A receptors. Radioligand binding assays with [³H]RP 62203 and [³H]-mesulergine were used to quantify 5-HT_2A and 5-HT_2C sites, respectively, in caudate. From these data, the B_max for caudate 5-HT_2A sites and 5-HT_2C sites was 165.4 ± 9.7 fmol/mg of protein and 49.7 ± 3.3 fmol/mg of protein, respectively. In contrast to that in caudate, PLC activity in frontal cortex was stimulated by 5-HT in a manner that was inhibited by the 5-HT_2A-selective antagonists, spiperone and ketanserin. Taken together, the results indicate that 5-HT_2A- and 5-HT_2C-linked PLC activity can be discerned in brain regions possessing both receptor subtypes using membranes prepared from previously frozen tissue. More importantly, significant 5-HT_2C-mediated phosphoinositide hydrolysis was observed in caudate, despite the relatively low density of 5-HT_2C sites. The significance of these observations with respect to the physiological function of 5-HT_2C receptors is discussed.     

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.     

Characterization of Multiple [3H]5–Hydroxytryptamine Binding Sites in Rat Spinal Cord Tissue

Abstract: High-affinity [³H]5-hydroxytryptamine ([³H]5-HT) binding in the rat spinal cord is similar to that demonstrated in the frontal cortex. [³H]5-HT binds with nearly the same affinity to sites in both tissues. Furthermore, similar patterns of displacement of [³H]5–HT were seen in both tissues, with either spiperone or LSD as the unlabeled ligand. This high-affinity binding appears to be to multiple sites, since displacement studies using 2 nM [³H]5–HT result in Hill coefficients less than unity for spiperone, LSD, and quipazine [Hill coefficients (n_H): 0.44, 0.39, 0.40, respectively]. These sites apparently have an equal affinity for [³H]5-HT, since unlabeled 5-HT did not discriminate between them. Thus, the high-affinity [³H]5-HT binding in the spinal cord may be analogous to that observed in the frontal cortex, where two populations of sites have previously been described (5-HT_IA, 5-HT_IB). In addition to the multiple high-affinity spinal cord binding sites, a low-affinity [³H]5-HT binding component was also identified. A curvilinear Scatchard plot results from saturation studies using [³H]5-HT (0.5–100 nM) in the spinal cord. The plot can be resolved into sites having apparent dissociation constants of 1.4 nM and 57.8 nM for the high-and low-affinity components, respectively. Additional support for a change in affinity characteristics at higher radioligand concentrations comes from the displacement of 30 nM [³H]5-HT by the unlabeled ligand. A nonparallel shift in the dissociation curve was seen, resulting in a Hill coefficient less than unity (0.32). None of the specifically bound [³H]5-HT in the spinal cord is associated with the 5-HT uptake carrier, since fluoxetine, an inhibitor of 5-HT uptake, does not alter binding characteristics. In addition, a 5-HT binding site analogous to the site designated 5-HT, was not apparent in the spinal cord. Ketanse-rin and cyproheptadine, drugs that are highly selective for 5-HT, sites, did not displace [³H]5-HT from spinal tissue, and [³H]spiperone, a radioligand that binds with high affinity to 5-HT₂ sites, did not exhibit saturable binding in the tissue. Thus, the 5-HT₂ binding site reported in other regions of the central nervous system, and the serotonin uptake carrier do not appear to contribute to the multiple binding sites demonstrated in the spinal cord.     

[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).     

Pharmacological characterization of the 5-hydroxytryptamine receptor coupled to adenylyl cyclase stimulation in human brain

Recently, a 5-hydroxytryptamine (5-HT) receptor has been described, whose pharmacology was distinct from that of the already known serotonergic receptors, so that it has been called 5-HT₄. Because the lack of a high affinity radioligand, the identification of this receptor depends entirely on functional pharmacological analysis. Its stimulation leads to an increase in cyclic AMP accumulation in mouse embryo colliculi neurons, in guinea pig hippocampus and in human heart. We studied the effect of two indoleamines, 5-HT and 5-methoxytryptamine (5-MeO-T), and a benzimidazolone derivative, BIMU 8, in stimulating basal adenylyl cyclase activity in human frontal cortex, and characterized the receptor subtype involved. In membranes prepared from this tissue, 5-HT, 5-MeO-T and BIMU 8 dose-dependently stimulated (13–25 %) the basal enzyme activity (220 pmoles cyclic AMP/min/mg protein). 5-MeO-T behaved as a full agonist, BIMU 8 elicited about 60 % of the maximal 5-HT effect. The selective 5-HT_1A agonist 8-OH-DPAT, was devoid of any stimulating activity. ICS 205–930, a low affinity 5-HT₄ receptor antagonist, completely reversed the effect of all three agonists at high concentrations. Therefore, the present data are consistent with the 5-HT-mediated stimulation of adenylyl cyclase in human frontal cortex resulting by the activation of a 5-HT₄ receptor subtype.     

Decreased 5-HT<Subscript>1A</Subscript> Receptor Gene Expression and 5-HT<Subscript>1A</Subscript> Receptor Protein in the Cerebral Cortex and Brain Stem during Pancreatic Regeneration in Rats

The present study was to investigate the role of central 5-HT and 5-HT_1A receptor binding and gene expression in a rat model of pancreatic regeneration using 60% pancreatectomy. The pancreatic regeneration was evaluated by 5-HT content and 5-HT_1A receptor gene expression in the cerebral cortex (CC) and brain stem (BS) of sham operated, 72 h and 7 days pancreatectomised rats. 5-HT content significantly increased in the CC (P < 0.01) and BS (P < 0.05) of 72 h pancreatectomised rats. Sympathetic activity was decreased as indicated by the significantly decreased norepinephrine (NE) and epinephrine (EPI) level (P < 0.001 and P < 0.05) in the plasma of 72 h pancreatectomised rats. 5-HT_1A receptor density and affinity was decreased in the CC (P < 0.01) and BS (P < 0.01). These changes correlated with a diminished 5-HT_1Areceptor mRNA expression in the brain regions studied. Our results suggest that the brain 5-HT through 5-HT_1A receptor has a functional role in the pancreatic regeneration through the sympathetic regulation.     

The influence of isoflurane on the synaptic activity of 5-hydroxytryptamine

The effects of isoflurane on uptake of 5-hydroxytryptamine(serotonin; 5-HT) by rat brain synaptosomes and binding of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and ketanserin to 5-HT_1A and 5-HT₂ receptors were examined. Isoflurane caused a concentration-dependent decrease in synaptosomal 5-HT uptake that was kinetically defined as non-competitive; exposure to isoflurane decreased V_max but had no effect on the apparent K_m. Removal of the drug from the reaction mixture resulted in the return of 5-HT accumulation rates to control levels. Isoflurane inhibited 8-OH-DPAT binding to hippocampal membranes by up to 27±6% at 4.5 mM, but did not significantly affect ketanserin binding to 5-HT₂ receptors. These findings suggest that presynaptic actions are more important than postsynaptic actions in the modulation of serotonergic neutrotransmission by isoflurane.     

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.     

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.     

Molecular basis for cis-urocanic acid as a 5-HT2A receptor agonist

The underlying mechanisms of urocanic acid (UA) to induce immune suppression remain elusive until the recent finding that cis-UA acts via the serotonin, 5-hydroxytryptamine (5-HT) receptor subtype 5-HT_2A. In the present study, the interactions of cis-UA to 5-HT_2A receptor were explored and compared with those of 5-HT to the same receptor using computational docking. Similar binding modes were observed for cis-UA and 5-HT with 5-HT_2A receptor and the former possessed relatively higher binding affinity, which may account for cis-UA being a serotonin receptor agonist. Moreover, the molecular basis for the distinct binding affinities between the trans- and cis-UA with 5-HT_2A receptor was also provided.     

Characterisation of Human 5-Hydroxytryptamine2A and 5-Hydroxytryptamine2C Receptors Expressed in the Human Neuroblastoma Cell Line SH-SY5Y: Comparative Stimulation by Hallucinogenic Drugs

Abstract: Stable transfection of the human neuroblastoma cell line SH-SY5Y with the human 5-hydroxytryptamine_2A (5-HT_2A) or 5-HT_2C receptor cDNA produced cell lines demonstrating ligand affinities that correlated closely with those for the corresponding endogenous receptors in human frontal cortex and choroid plexus, respectively. Stimulation of the recombinant receptors by 5-HT induced phosphoinositide hydrolysis with higher potency but lower efficacy at the 5-HT_2C receptor (pEC₅₀ = 7.80 ± 0.06) compared with the 5-HT_2A receptor (pEC₅₀ = 7.30 ± 0.08). Activation of the 5-HT_2A receptor caused a transient fourfold increase in intracellular Ca²⁺ concentration. Whole-cell recordings of cells clamped at ?50 mV demonstrated a small inward current (2 pA) in response to 10 µM 5-HT for both receptors. There were no differences in potency or efficacy of phosphoinositide hydrolysis among four hallucinogenic [d-lysergic acid diethylamide (LSD), 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI), 5-methoxy-N,N-dimethyltryptamine, and mescaline] and three nonhallucinogenic drugs (m-chlorophenylpiperazine, quipazine, and ergotamine). Comparison of equipotent doses producing 20% of the maximal response induced by 5-HT revealed selective activation of the 5-HT_2A receptor by LSD and to a lesser degree by DOI, mescaline, and ergotamine. Quipazine and 5-methoxy-N,N-dimethyltryptamine were relatively nonselective, whereas m-chlorophenylpiperazine selectively activated the 5-HT_2C receptor. It is unlikely therefore that hallucinosis is mediated primarily by activity at the 5-HT_2C receptor, whereas activity at the 5-HT_2A receptor may represent an important but not unique mechanism associated with hallucinogenic drug action.     

Involvement of 5-HT<Subscript>2A</Subscript> receptors in genetic mechanisms of autoregulation of brain 5-HT system

The brain serotonin (5-HT) system has been implicated in the pathophysiology of anxiety, depression, drug addiction, and schizophrenia. 5-HT_2A receptors are involved in the mechanisms of stressinduced psychopathology and impulsive behavior. In this work, we investigated the role of 5-HT_2A receptors in the autoregulation of the brain 5-HT system. Chronic treatment with DOI, a 5-HT_2A receptor agonist (1.0 mg/kg, i.p./14 days), produced a considerable decrease in the number of 5-HT_2A receptor-mediated head twitches in AKR/J mice, indicating the desensitization of 5-HT_2A receptors. Chronic DOI treatment did not affect the expression of the 5-HT_2A receptor gene in the midbrain, hippocampus and frontal cortex. At the same time, an increase in the expression of the gene encoding a key enzyme of 5-HT synthesis, tryptophan hydroxylase-2 (TPH-2), accompanied with an increase in TPH-2 activity and 5-HT levels, and decreased expression of the serotonin transporter (5-HTT) gene were observed in the midbrain of DOI-treated mice. These results provide new evidence of receptor-gene cross-talk in the brain 5-HT system and implication 5-HT_2A receptors in the autoregulation of the brain 5-HT system.     

Modulation of Antagonist Binding to Serotonin1A Receptors from Bovine Hippocampus by Metal Ions

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. Although specific 5-HT_1Aagonists have been discovered more than a decade back, the development of selective 5-HT_1Aantagonists has been achieved only recently.2. We have examined the modulation of the specific antagonist [³H]p-MPPF binding to 5-HT_1Areceptors from bovine hippocampal membranes by monovalent and divalent metal ions. Our results show that the antagonist binding to 5-HT_1Areceptors is inhibited by both monovalent and divalent cations in a concentration-dependent manner. This is accompanied by a concomitant reduction in binding affinity.3. Our results also show that the specific antagonist p-MPPF binds to all available receptors in the bovine hippocampal membrane irrespective of their state of G-protein coupling and other serotonergic ligands such as 5-HT and OH-DPAT effectively compete with the specific antagonist [³H]p-MPPF.4. These results are relevant to ongoing analyses of the overall modulation of ligand binding in G-protein-coupled seven transmembrane domain receptors.     

Regulation of GABAergic inhibition by serotonin signaling in prefrontal cortex: molecular mechanisms and functional implications

Serotonergic neurotransmission in prefrontal cortex (PFC) plays a key role in regulating emotion and cognition under normal and pathological conditios. Increasing evidence suggests that serotonin receptors are involved in the complex regulation of GABAergic inhibitory transmission in PFC. Activation of postsynaptic 5-HT₂ receptors in PFC pyramidal neurons inhibits GABA_A-receptor currents via phosphorylation of GABA_A receptor γ2 subunits by RACK1-anchored PKC. In contrast, activation of postsynaptic 5-HT₄ receptors produces an activity-dependent bi-directional regulation of GABA-evoked currents in PFC pyramidal neurons, which is mediated through phosphorylation of GABA_A-receptor β subunits by anchored PKA. On the presynaptic side, GABAergic inhibition is regulated by 5-HT through the activation of 5-HT₂, 5-HT₁, and 5-HT₃ receptors on GABAergic intereneurons. These data provide a molecular and cellular mechanism for serotonin to dynamically regulate synaptic transmission and neuronal excitability in the PFC network, which may underlie the actions of many antidepressant and antipsychotic drugs.     

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     

Interaction of the D1 Receptor Antagonist Sch 23390 with the Central 5-HT System: Radioligang Binding Studies,Measurements of Biochemical Parameters and Effects on L-5-HTP Syndrome

Abstract

The interaction of SCH 23390 with dopamine (DA) and serotonin (5-HT) systems has been examined in vivo and in vitro. Like selective 5-HT₂ blockers, SCH 23390 inhibited in vivo [³H]spiperone binding in the rat frontal cortex (ID₅₀: 1.5 mg/kg) without interacting at D₂ sites. SCH 23390 was equipotent to cinanserin and methysergide. In vitro, SCH 23390 inhibited [³H]ketanserin binding to 5-HT₂ sites (IC₅₀ = 30 nM). Biochemical parameters linked to DA and 5-HT were not changed excepted in striatum where SCH 23390 increased HVA and DOPAC. In the L-5-HTP syndrome model, SCH 23390 clearly showed antagonism of 5-HT₂ receptors. SCH 23390 had weak affinity for 5-HT_1B (IC₅₀ = 0.5 μM), 5-HT_1A (IC₅₀ = 2.6 μM) and α;₁-adenergic receptors (IC₅₀ = 4.4 μM).     

Alterations in hippocampal serotonergic and INSR function in streptozotocin induced diabetic rats exposed to stress: neuroprotective role of pyridoxine and <Emphasis Type="Italic">Aegle marmelose</Emphasis>

Diabetes and stress stimulate hippocampal 5-HT synthesis, metabolism and release. The present study was carried out to find the effects of insulin, Aegle marmelose alone and in combination with pyridoxine on the hippocampal 5-HT, 5-HT_2A receptor subtype, gene expression studies on 5-HT_2A, 5-HTT, INSR, immunohistochemical studies and elevated plus maze in streptozotocin induced diabetic rats. 5-HT content showed a significant decrease (p < 0.001) and a significant increase (p < 0.001) in 5-HIAA in hippocampus of diabetic rats compared to control. 5-HT receptor binding parameters B_max and K_d showed a significant decrease (p < 0.001) whereas 5-HT_2A receptor binding parameters B_max showed a significant decrease (p < 0.001) with a significant increase (p < 0.05) in K_d in hippocampus of diabetic rats compared to control. Gene expression studies of 5-HT_2A, 5-HTT and INSR in hippocampus showed a significant down regulation (p < 0.001) in diabetic rats compared to control. Pyridoxine treated in combination with insulin and A. marmelose to diabetic rats reversed the 5-HT content, B_max , K_d of 5-HT, 5-HT_2A and gene expression of 5-HT_2A, 5-HTT and INSR in hippocampus to near control. The gene expression of 5-HT_2A and 5-HTT were confirmed by immunohistochemical studies. Behavioural studies using elevated plus maze showed that serotonin through its transporter significantly increased (p < 0.001) anxiety-related traits in diabetic rats which were corrected by combination therapy. Our results suggest that pyridoxine treated in combination with insulin and A. marmelose has a role in the regulation of insulin synthesis and release, normalising diabetic related stress and anxiety through hippocampal serotonergic function. This has clinical significance in the management of diabetes.