Platelet 5-HT uptake sites in depression: three concurrent measures using [3H] imipramine and [3H] paroxetine

Platelet 5-HT uptake sites were measured in 40 depressed patients and 40 controls using [³H] imipramine binding, defined with desmethylimipramine (DMI) and Na⁺ dependence, and [³H] paroxetine binding. In control subjects the B_max of DMI defined [³H] imipramine binding was significantly higher than both Na⁺ dependent [³H] imipramine (by 30%) and [³H] paroxetine binding (by 22%). The B_max of Na⁺ dependent [³H] imipramine and [³H] paroxetine binding did not differ significantly. The K_d of Na⁺ dependent [³H] imipramine binding was significantly lower than the K_d of DMI defined [³H] imipramine binding. The binding of DMI defined and Na⁺ dependent [³H] imipramine and [³H] paroxetine did not differ significantly between depressed patients and controls in the total group, in those depressed patients who had never taken antidepressants or in those depressed patients who had been recently with-drawn from antidepressants. This study provides no support for the view that the number of platelet 5-HT uptake sites are reduced in depression.     

Unaltered 5-HT- and desipramine-sensitive [3H]imipramine binding and [3H]5-HT uptake in rat brain after chronic imipramine and norzimeldine treatment

Several reports have shown heterogeneity of [³H]imipramine binding to brain membranes. Recently, a high affinity and 5-HT sensitive [³H]imipramine binding site of protein nature, that was suggested to be identical to the substrate recognition site for 5-HT uptake, was demonstrated. Since most studies on the regulation of the [³H]imipramine binding sites by antidepressants have used desipramine displaceable binding, which is heterogenous in nature and contains binding not related to 5-HT uptake sites, the present report studies the possible effects of chronic (3 weeks) administration of imipramine or norzimeldine (10 mg/kg intraperitoneally twice daily) on 5-HT sensitive [³H]imipramine binding sites. For comparison, desipramine sensitive binding was also studied, as well as the physiological correlate 5-HT uptake. There were no changes in either [³H]imipramine binding or 5-HT uptake after the antidepressant treatment.Supported by the Swedish Medical Research Council Offprint requests to: J. Marcusson at Dept. of Geriatric Medicine     

Effect of acute and prolonged tianeptine administration on the 5-HT transporter: electrophysiological,biochemical and radioligand binding studies in the rat brain

In the present study, in vivo extracellular unitary recordings, in vitro [³H]5-HT uptake and [³H]cyanoimipramine binding assays were used to assess the effect of acute and prolonged administration of the putative antidepressant tianeptine, on the 5-hydroxytryptamine (5-HT) transporter. Microiontophoretic application of tianeptine onto dorsal hippocampus CA₃ pyramidal neurons, as well as its intravenous administration (2 mg/kg), increased their firing frequency. Following intracerebroventricular administration of 5,7-dihydroxytryptamine, the activation induced by the microiontophoretic application of tianeptine remained unchanged, thus suggesting that the 5-HT carrier is not involved in this effect. Furthermore, in spite of its activating effect on CA₃ pyramidal neuron firing frequency, the intravenous administration of tianeptine did not alter the time of recovery of these neurons from microiontophoretic applications of 5-HT, an index of 5-HT uptake activity. In keeping with this observation, the acute administration of tianeptine did not change the effectiveness of the 5-HT reuptake blocker paroxetine (1 mg/kg, i.v.) in prolonging the suppressant effect of microiontophoretically-applied 5-HT. However, in rats that had received tianeptine for 14 days (20 mg/kg/day, s.c.), the recovery time from the suppressant effect of microiontophoretic applications of 5-HT was reduced by 40% and the effectiveness of paroxetine (1 mg/kg, i.v.) was decreased. These effects were no longer observed following a 48 h washout period. In a second series of experiments, the ability of tianeptine to interfere with the uptake blocking capacity of paroxetine was assessed in vitro, using hippocampal slices obtained from rats that had been treated with tianeptine for 14 days (20 mg/kg/day, s.c.; by minipump). The effectiveness of paroxetine to block [³H]5-HT uptake was unchanged in slices obtained from rats still bearing the osmotic minipump at the time of the sacrifice, as well as from those which had undergone a 48 h washout period. To assess whether prolonged administration of tianeptine would induce adaptive changes on 5-HT uptake sites, [³H]cyanoimipramine-binding parameters were measured following a 48 h washout period. Affinity values remained unchanged while density values were significantly increased in cortex (+22%) but not in hippocampus (+12%). It is concluded that, i) the activation of CA₃ pyramidal neurons observed following acute tianeptine administration cannot be attributed to its 5-HT uptake enhancing properties and ii) the prolonged administration of tianeptine induces adaptive changes on cortical but not on hippocampal 5-HT transporters.Deceased 10 May 1994     

Binding of some antidepressants to the 5-hydroxytryptamine transporter in brain and platelets

Antidepressant agents with properties to inhibit 5-hydroxytryptamine (5-HT, serotonin) uptake in brain tissue and platelets bind with high affinities to neuronal and platelet membranes. [³H]Imipramine, [³H]paroxetine and [³H]citalopram label specific binding sites related to the 5-HT transporter. [³H]Paroxetine and [³H]citalopram appear to be better ligands than [³H]imipramine. The former label a homogenous population of binding sites, whereas the displaceable binding of [³H]imipramine is heterogenous. Recent observations in several laboratories, which have taken the heterogeneity of [³H]imipramine binding into account, indicate that the binding of antidepressants to the 5-HT transporter probably occurs to the same site that binds 5-HT for transport and not to a separate site as previously suggested. Additional bonds to subsites in close vicinity to the 5-HT recognition site may contribute to the binding. No convincing evidence has been presented of the existence of an endogenous ligand other than 5-HT itself that binds to the [³H]imipramine binding site. Recent studies also suggest that repeated treatment of rats with antidepressant agents does not produce any alterations of the binding of [³H]imipramine or [³H]paroxetine to membranes of cerebral cortex. It is also doubtful whether the density of the 5-HT uptake site in platelets measured with these ligands is decreased in affective disorders as first reported.     

New Evidence for a Loss of Serotonergic Nerve Terminals in Rats Treated with d,l-Fenfluramine

Abstract: Fenfluramine has been classified as a neurotoxin because animals treated with this anorectic lose 5-HT uptake sites located on serotonergic nerve terminals. However, there are two possible bases for this finding: either uptake sites are lost because the terminals themselves have been destroyed (neurotoxicity); or uptake sites are lost from otherwise intact terminals. To distinguish between these possibilities, we established an animal model in which male Wistar rats were injected (intraperitoneally) with an irreversible 5-HT uptake site antagonist (EEDQ). Since their 5-HT sites were inhibited (blocked) non-competitively, by this agent, such animals had effectively lost 5-HT uptake sites from intact serotonergic terminals. Synaptosomes prepared from such animals showed the predicted reduction in the B _max of [³H]paroxetine binding to the 5-HT uptake site, and a reduction in the V_max of [¹⁴C]5-HT uptake. However, they showed no significant reduction in maximal [¹⁴C]5-HT loading (α) compared with synaptosome from sham-injected controls. In contrast, fenfluramine-treated animals showed reduced [³H]paroxetine binding, reduced maximal [¹⁴C]5-HT uptake and significantly (P<0.02) reduced synaptosomal [¹⁴C]5-HT loading. Therefore, the results suggest that fenfluramine does indeed cause the destruction of serotonergic nerve terminals.     

The activity of 25 paroxetine/femoxetine structure variants in various reactions, assumed to be important for the effect of antidepressants

Structure-activity relationships for 25 structural variants around the 5-hydroxytryptamine (5-HT) uptake inhibitors paroxetine and femoxetine have been investigated. Three parameters related to the 5-HT system were investigated: (i) The inhibition of [3H]5-HT uptake into rat brain synaptosomes, (ii) the inhibition of [3H]paroxetine binding to rat neuronal membranes and (iii) the effect of the compounds on the affinity of [3H]imipramine for the human platelet membrane binding site, measured as the dissociation rate of the [3H]imipramine human platelet membrane binding site complex. A highly significant correlation was found for 5-HT uptake inhibition and inhibition of [3H]paroxetine binding for the different substances, indicating that the two parameters are closely connected. However the slope of the regression line was only 0.6 and not 1.0; this may indicate that [3H]paroxetine binding is necessary, but not sufficient for 5-HT uptake inhibition. No correlation was found between the inhibition of [3H]paroxetine binding and the affinity of the compounds for the [3H]imipramine binding site complex. The two binding sites are therefore probably situated on different parts of the 5-HT transport system, the [3H]paroxetine binding site being part of the 5-HT transport mechanism whereas the [3H]imipramine binding site may represent a site modulating the activity of, and affinity for, 5-HT in the 5-HT transport mechanism. Structure-activity relationships among the substances showed that stereochemical changes from (-)- to (+)-trans changed the activity towards both 5-HT uptake inhibition and [3H]paroxetine displacement for most of the (-)-/(+)-pairs. The substitution of -H with -F or -CH3 also affected the activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

8-Hydroxy-2-(di-n-propylamino) tetralin is devoid of activity at the 5-hydroxytryptamine autoreceptor in rat brain. Implications for the proposed link between the autoreceptor and the [3H] 5-HT recognition site

Summary Experiments have been carried out to provide direct evidence for the proposed presynaptic 5-HT autoreceptor agonist activity of 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) a compound with selectivity for the 5-HT_1A subtype of the 5-HT₁ binding site. Rat brain frontal cortex slices were preincubated with [³H] 5-hydroxytryptamine and continuously stimulated with Krebs solution containing paroxetine and elevated K⁺ ions (25 mmol/l). The elevated efflux of tritium caused by exposure to K⁺ Krebs was inhibited in a dose related manner by 5-hydroxytryptamine and this inhibition was attenuated in the presence of quipazine and methiothepin.In slices of the rat frontal cortex, 8-OH-DPAT was without agonist or antagonist activity at the 5-HT autoreceptor at concentrations up to 1 mol/l. Higher concentrations caused an increase in basal efflux of tritium. 8-OH-DPAT (1 mol/l) was also without inhibitory activity in the piriform cortex, striatum and the hippocampus.These experiments have therefore failed to provide direct evidence for agonist activity of 8-OH-DPAT at the 5-HT autoreceptor and alternative explanations must be sought for its biochemical and behavioural effects in vivo. Moreover, the fact that 8-OH-DPAT is inactive at the autoreceptor at concentrations selective for the 5-HT_1A recognition site suggests that this subtype of the 5-HT_1A binding site may not correspond to the 5-HT autoreceptor.Part of this work was presented at the Joint Meeting of the French and German Pharmacological and Toxicological Societies, Freiburg, September 1983     

Allosteric modulation of 5-HT(1A) receptors by zinc: Binding studies

5-HT_1A receptors were studied via [³H]WAY-100635 and [³H]8-OH-DPAT binding to rat brain cortical membranes. We characterized the effect of zinc (Zn²⁺) on the binding properties of the 5-HT_1A receptor. The allosteric ternary complex model was applied to determine the dissociation constant (K_A) of Zn²⁺ and their cooperativity factors (α) affecting the dissociation constants (K_D, K_i) of [³H]WAY-100635, [³H]8-OH-DPAT, and serotonin (5-HT), the endogenous neurotransmitter. Zn²⁺ (5 μM-1 mM) inhibited the binding of agonist/antagonist to 5-HT1A receptors, mostly by decreasing both the ligands' affinity and the maximal number of sites. In [³⁵S]GTPγS binding assays Zn²⁺ behaved as insourmountable antagonist of 5-HT1A receptors, in agreement with radioligand binding assays. The residues involved in the formation of the inhibitory binding site on the 5-HT1A receptor were assessed by using N-ethyl-maleimide (NEM) or diethylpyrocarbonate (DEPC) which modify preferentially cysteine and histidine residues, respectively. Exposure to both agents did not block the negative allosteric effects of Zn²⁺ on agonist and antagonist binding. Our findings represent the first quantitative analysis of allosteric binding interactions for 5-HT_1A receptors. The physiological significance of Zn²⁺ modulation of 5-HT_1A receptors is unclear, but the colocalization of 5-HT_1A receptors and Zn²⁺ in the nervous system (e.g. in the hippocampus and cerebral cortex) suggests that Zn²⁺ released at nerve terminals may modulate signals generated by the 5-HT_1A receptors in vivo. Finally, these findings suggest that synaptic Zn²⁺ may be a factor influencing the effectiveness of therapies that rely on 5-HT_1A receptor activity.     

A common binding site for tricyclic and nontricyclic 5-hydroxytryptamine uptake inhibitors at the substrate recognition site of the neuronal sodium-dependent 5-hydroxytryptamine transporter

An investigation of the site of interaction of a variety of tricyclic and nontricyclic 5-HT uptake inhibitors with the neuronal sodium-dependent 5-HT transporter was undertaken. The dissociation of [3H]paroxetine binding induced by indalpine (10 microM), SL 81.0385 (10 microM), fluoxetine (10 microM), citalopram (10 microM), paroxetine (0.15 microM), imipramine (10 microM) and 5-HT (50 microM) produced monophasic dissociation curves and gave t1/2 values of dissociation similar to that induced by dilution alone. In inhibition studies of [3H]paroxetine binding with citalopram, imipramine and 5-HT, increases in the concentration of [3H]radioligand used led to parallel rightward shifts of the inhibition curves with no diminution of the maximum degree of inhibition (Imax). "Schild-type" analyses of the data obtained from the inhibition curves with these 3 compounds gave slopes close to unity. In chemical modification studies, treatment of membrane fractions with N-ethylmaleimide led to a pronounded reduction in specific [3H]paroxetine binding. Preincubation of these membranes with SL 81.0385, fluoxetine, imipramine, tryptamine and 5-HT provided significant protection against this NEM-induced inactivation. The above findings are interpreted to provide evidence for a common or at least overlapping binding site for the tricyclic and nontricyclic 5-HT uptake inhibitors with the substrate recognition site of the neuronal sodium-dependent 5-HT transporter.     

Evidence for presynaptic location of inhibitory 5-HT1D\-like autoreceptors in the guinea-pig brain cortex

The effects of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on tritium overflow evoked by high K⁺ were determined in superfused synaptosomes and slices, preincubated with [³H]5-HT, from guinea-pig brain cortex. In addition, we estimated the potencies of 5-HT receptor ligands in inhibiting specific [³H]5-HT binding (in the presence of 8-hydroxy-2(di-n-propylamino)tetralin and mesulergine to prevent binding to 5-HT_1A and 5-HT_2C sites) to guinea-pig cortical synaptosomes and membranes.5-HT receptor agonists inhibited the K⁺-evoked tritium overflow from synaptosomes and slices. In synaptosomes the rank order of potencies was 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indole-3-yl] ethylamine (L-694,247) >5-carboxamidotryptamine (5-CT) > oxymetazoline (in the presence of idazoxan) 5-HT > sumatriptan 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (RU 24969). The potencies of the agonists in inhibiting tritium overflow from slices correlated with those in synaptosomes, suggesting that the same site of action is involved in both preparations. In synaptosomes the nonselective antagonist at cloned human 5-HT_1D, and 5-HT_1D receptors, methiothepin, shifted the concentration-response curve for 5-CT to the right (apparent pA₂: 7.87). In contrast, ketanserin at a concentration which should block the 5-HT_1D, but not the 5-HT_1D\, receptor did not alter the inhibitory effect of 5-CT on tritium overflow. In cortical synaptosomes and membranes, [³H]5-HT bound to a single site with high affinity. In competition experiments, 5-HT receptor agonists and antagonists inhibited specific [³H]5-HT binding. In synaptosomes the rank order was L-694,247 > methiothepin >5-CT >5-methoxytryptamine >5-HT sumatriptan oxymetazoline > RU 24969 > ketanserin > ritanserin. A very similar rank order was obtained in cerebral cortical membranes. The potencies of the 5-HT receptor agonists in inhibiting tritium overflow from synaptosomes and slices correlated with their potencies in inhibiting [³H]5-HT binding to synaptosomes and membranes.In conclusion, the 5-HT receptors mediating inhibition of 5-HT release in the guinea-pig cortex are located on the serotoninergic axon terminals and, hence, represent presynaptic inhibitory autoreceptors. The [³H]5-HT binding sites in cerebral cortical synaptosomes and membranes exhibit the pharmacological properties of 5-HT_1D receptors. The correlation between the functional responses and the binding data confirms the 5-HT_1D character of the presynaptic 5-HT autoreceptors. According to the results of the interaction experiment of ketanserin and methiothepin with 5-CT on 5-HT release, the presynaptic 5-HT autoreceptors can be subclassified as 5-HT_1D\-like.     

5.HT1 receptors in the vertebrate brain

Summary The regional distribution of high affinity [³3H]5-HT recognition sites in the brain of several vertebrates (pigeon, rat, mouse, guinea-pig, cat, dog, monkey and human) was analyzed using in vitro autoradiography. The presence of subtypes of 5-HT₁ binding sites was investigated by selective displacements with 8-OH-DPAT, mesulergine and (±)SDZ 21-009 at appropriate concentrations to block 5-HT_1A, 5-HT_1c and 5-HT_1B sites respectively. In addition, 5-HT_1A, and 5-HT_1c sites were directly visualized with the more selective radioligands [³H]8-OH-DPAT and [³H]mesulergine, respectively. In the pigeon brain, total [³H]5-HT binding sites were enriched in all telencephalic areas. Densely labelled regions were also present in the optic tectum and the brainstem. No binding was observed in the cerebellum. 8-OH-DPAT and mesulergine only displaced a small proportion of [³H]5-HT binding in most of the areas where high concentrations of 5-HT₁ sites were found. (±)SDZ 21-009 did not affect [³H]5-HT binding in the regions examined. Taking into account our pharmacological studies, these results suggest that the majority of 5-HT₁ sites belong to the 5-HT_1D subtype in the pigeon brain. In the mammalian species investigated high levels of [³H]5-HT binding were found in the neo-cortex, hippocampal formation, basal ganglia and related structures (substantia nigra), raphe dorsalis, nucleus superior colliculus and choroid plexus. However, these brain areas were differentially enriched in subtypes of 5-HT₁ recognition sites. 5-HT_1A sites were observed in the neo-cortex, the hippocampal formation and the raphe nucleus, whereas 5-HT_1C sites accounted for all 5-HT₁ binding in the choroid plexus. In the mouse and rat brain, 5-HT_1B binding sites were enriched in the basal ganglia and associated regions (substantia nigra). These areas were enriched in 5-HT_1D sites in the brain of the other mammals studied. In these animals, no site with a 5-HT_1B pharmacological profile were detected.These results indicate that 5-HT_1A 5-HT_1c and 5-HT_1D sites are present already in the lower vertebrate species investigated and that 5-HT_1B appear to be exclusive of the myomorph rodents (mouse, rat). Furthermore, the different subtypes of the 5-HT₁, receptors present a conserved regional distribution with the 5-HT_1D sites being enriched in the basal ganglia and the 5-HT_1A sites predominating in the hippocampal formation.     

Allosteric site in M2 acetylcholine receptors: evidence for a major conformational change upon binding of an orthosteric agonist instead of an antagonist

Muscarinic acetylcholine receptors contain two distinct ligand binding sites, i.e. the orthosteric site for acetylcholine and other conventional ligands, and an allosteric site located at the entrance of the ligand binding pocket. We used a set of allosteric agents to probe whether muscarinic M₂ receptors whose orthosteric site is occupied by an agonist still reveal the common allosteric site that has been identified in M₂ receptors being occupied by an orthosteric antagonist (N-methylscopolamine, NMS). Equilibrium and dissociation binding experiments were carried out in porcine heart homogenates using either the agonist [³H]oxotremorine M ([³H]OxoM) or the antagonist [³H]NMS. The affinities of the allosteric agents were determined for the radioligand-occupied receptor states and, additionally, for the radioligand-free (ground state) M₂ receptor. The archetypal agent W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropyl-ammonium bromide] and its bispyridinio middle chain analogue WDuo3 (1,3-bis[4-(phthalimidomethoxyimino-methyl)-pyridinium-1-yl]propane dibromide) had a clearly lower affinity for [³H]OxoM-liganded receptors compared with [³H]NMS-liganded and ground state receptors. In contrast, a derivative resembling only one half of W84 had equal affinities for both radioligand-occupied receptor states. Also, the agents gallamine and obidoxime did not discriminate between [³H]OxoM- and [³H]NMS-occupied receptors. The allosteric antagonistic tool obidoxime inhibited WDuo3 action in [³H]OxoM-liganded receptors with the same potency as in [³H]NMS-liganded receptors. We conclude that the common allosteric site is still present in OxoM-liganded M₂ receptors, but its spatial conformation is considerably altered compared with NMS-liganded receptors.     

[3H]sumatriptan labels both 5-HT1D and 5-HT1F receptor binding sites in the guinea pig brain: an autoradiographic study

We have used in vitro autoradiography to visualize [³H]sumatriptan binding sites in sections of guinea-pig and rat brain. In saturation studies, this ligand recognized a single saturable population of high affinity binding sites in all regions examined (pK_D = 8.3–9.3). While 5-HT and the sumatriptan derivative CP-122,288 (5-methyl-aminosulfonylmethyl-3-(N-methylpyrrolidin-2R-yl-methyl)-1H-indole) competed for [³H]sumatriptan binding sites with a high affinity and monophasic profile, displacement experiments with 5-carboxamidotryptamine revealed the existence of 2 classes of binding sites. The high affinity component (pKD = 9.2–9.9) probably corresponded to 5-HT_1B (rat) or 5-HT_1D (guinea-pig) receptors. The intermediate affinity (pK_D = 5.7–7.3) of the other component, taken together with their high affinity for [³H]sumatriptan, was similar to that of the cloned 5-HT_1F receptor. The regional distribution of the 5-HT_1B/1D [³H]sumatriptan binding sites was in agreement with previously published studies (striatonigral system, hypothalamus, central gray, superficial layer of the superior colliculus) and corresponded to the pattern of serotonin-5-O-carboxymethyl-glycyl [¹²⁵I]tyrosinamide labeling in consecutive sections. [³H]sumatriptan binding sites with a low affinity for 5-CT predominated in the intermediate neocortical layers, the claustrum (in the guinea-pig only), the mammillary nuclei, most of the thalamic nuclei and the principal oculomotor nucleus (in the guinea-pig only). This distribution is very similar to that of 5-HT_1F mRNA, indicating further the identity of these sites with 5-HT_1F receptors. Very high densities of 5-HT_1F sites were also found in the rat parafascicular nucleus.Some regions, such as the caudate/nucleus, the lateral geniculate nuclei and the spinal trigeminal nucleus appeared to contain both 5-HT_1B/1D and 5-HT_1F binding sites. Ketanserin had a low affinity for [³H]sumatriptan binding sites in all guinea-pig brain regions, compatible with the presence of the 5-HT_1D\ subtype. An exception was the substantia nigra, where a significant proportion of sites displayed an intermediate affinity for this compound, suggesting the presence of 5-HT_1D receptors. [³H]5-HT labeled 5-HT_1F sites in the claustrum and intermediate cortical layers in the guinea-pig. However these data show that [³H]sumatriptan, in the presence of 10 nM 5-carboxamidotryptamine, is a more suitable radioligand to study the distribution of 5-HT_1F binding sites.     

Light-dark differences in [3H]-paroxetine binding to rabbit platelet membranes

Summary [³H]-Paroxetine binding to rabbit blood platelet membranes from samples obtained under light and dark conditions was examined. Animals were kept on a 14 h light (L) — 10 h dark (D) schedule and blood samples were collected at L + 7 and D + 5 h. Significant differences were found for B _max values of [³H]-paroxetine binding, with low B _max values during the light period and high B _max values during the dark period. The K _d values were not significantly different. These results confirm previous observations on light-dark differences of [³H]-imipramine binding in rabbit blood platelets suggesting the existence of a circadian rhythm for the 5-HT transporter complex.Send offprint requests to S. Z. Langer at the above address     

Alterations in hippocampal function following repeated exposure to the amphetamine derivative methylenedioxymethamphetamine (“Ecstasy”)

The effect of the psychomotor stimulant, 3,4-methylenedioxymethamphetamine (MDMA, “Ecstasy”), upon integrated cerebral function was measured in rats using the quantitative [¹⁴C]deoxyglucose autoradiographic technique. Animals were injected with MDMA (20 mg/kg sc) twice daily for 4 days. Fourteen days after the final administration, [³H]-paroxetine binding to 5HT uptake sites was reduced by 89% in membranes prepared from tissue samples of frontal cortex. In the same rats [³H]-paroxetine binding autoradiography revealed heterogeneity in the regional distribution of 5-HT uptake site depletion within neocortex (0–92%) and hippocampus (30–95%). Despite these profound reductions in 5-HT uptake sites no significant alterations were found in glucose utilisation in any area of neocortex examined. However, significant increases in glucose use were found in subregions of the hippocampus, most notably within the pyramidal cell layer of CA2 and CA3 (25–35%). This study provides direct evidence that the loss of 5-HT innervation caused by exposure to MDMA results in lasting functional changes in hippocampus.     

Pharmacological comparison of the sigma recognition site labelled by [3H]haloperidol in human and rat cerebellum

Summary The radioligand binding characteristics of [³H]haloperidol (in the presence of spiperone, 25 nmolL^–1) were investigated in rat and human cerebellar membranes.In both rat and human cerebellar membrane preparations saturation studies with [³H]haloperidol (non-specific binding defined by pentazocine, 10 molL^–1) demonstrated high affinity saturable specific binding to a homogenous population of binding sites (rat, Bmax 6693 ± 1242 fmol mg^–1 protein, pK_D 8.33 ± 0.08; human, Bmax 2550 ± 437 fmol mg^–1 protein, pK_D 8.59 ± 0.11; mean ± SEM, n = 3–6). Competition studies employing a wide range of structurally diverse competing compounds displayed that the [³H]haloperidol binding site was pharmacologically similar in both preparations and comparable to sigma recognition sites previously identified in various tissues originating from different species. In addition, with reference to the potential subtypes of sigma recognition sites, the labelling of these sites by low nanomolar concentrations of [³H]haloperidol provides evidence that they belong to the sigma-1 recognition site subtype.The present findings suggest that the pharmacology of the rat and human cerebellar sigma recognition site are directly comparable and provides further supporting evidence towards the use of [³H]haloperidol radioligand binding studies in the rat to detect sigma receptor ligands with potential therapeutic activity. Send offprint requests to: N.M. Barnes at the above address     

[3H] sertraline binding to rat brain membranes

Tritiated sertraline, a radiolabeled form of a potent and selective inhibitor of serotonin uptake, was found to bind with high affinity to rat whole brain membranes. Characterization studies showed that [³H] sertraline binding occurred at a single site with the following parameters:K _d 0.57 nM,B _max 821 fmol/mg protein,n _h 1.06. This binding was reversible; the dissociation constant calculated from kinetic measurements (K _d 0.81 nM) agreed with that determined by saturation binding experiments. [³H] Sertraline binding in the presence of serotonin, paroxetine, fluoxetine or imipramine suggested competitive inhibition of binding (large increase inK _d with little change inB _max). The rank order of potency of inhibition of [³H] sertraline binding was similar to that of inhibition of serotonin uptake for known uptake inhibitors and the 1-amino-4-phenyltetralin uptake blockers. A marked decrease in ex vivo [³H] sertraline binding in the brain of rats 7 days after treatment withp-chloroamphetamine was consistent with the loss of serotonin uptake sites induced by this agent. The results of our study indicated that [³H] sertraline labels serotonin uptake sites in rat brain.     

Evidence for mediation by 5-HT2 receptors of 5-hydroxytryptamine-induced contraction of canine basilar artery

Summary The agonist potencies of 8 indole derivatives and the potencies of 19 recognized antagonists to inhibit constrictor responses to 5-hydroxytryptamine (5-HT) of canine basilar artery were established. In addition the affinities of the indole derivatives for [³H]5-hydroxytryptamine ([³H]5-HT) binding sites and the affinities of the antagonists for [¹²⁵Iodo]LSD ([¹²⁵I]LSD) binding sites in rat brain cortex membranes were determined. Comparison was also made between the potencies of the antagonists on canine basilar artery and the K _D values published for displacement of [³H]ketanserin binding (Leysen et al. 1982).There was a good correlation between the affinities of the antagonists for 5-HT₂ binding sites labelled by both [¹²⁵I]LSD and [³H]ketanserin and the affinity parameters calculated for inhibition of constrictor responses to 5-HT of canine basilar artery. No correlation could be found between the affinities of the indole derivatives for 5-HT₁ binding sites labelled by [³H]5-HT and their potencies to constrict canine basilar artery.It is concluded that constrictor responses to 5-HT of canine basilar artery are mediated by 5-HT₂-like receptors.     

[3H]ICS 205-930 labels 5-HT3 recognition sites in membranes of cat and rabbit vagus nerve and superior cervical ganglion

Summary The binding characteristics of [³H]ICS 205-930, a 5-hydroxytryptamine 5-HT₃ receptor antagonist, were investigated in membranes prepared from cat and rabbit vagus nerve (VN) and superior cervical ganglion (SCG). The autoradiographic localisation of 5-HT₃ recognition sites was also assessed using [³H]ICS 205-930 in slices from cat medulla oblongata, nodose ganglion and vagus nerve.[³H]ICS 205-930 bound to a homogeneous population of high affinity recognition sites in cat VN: B_max = 201 ± 43 fmol/mg protein, pK_D = 9.26 ± 0.17 and SCG: B_max = 291 ± 40 fmol/mg, pK_D = 9.35 ± 0.80 (n = 3). Competition experiments performed in membranes from cat VN and SCG with agonists and antagonists suggested the presence of a homogeneous population of [³H]ICS 205-930 recognition sites. Competition curves were steep and monophasic and were best fitted by a 1 receptor site model. The following rank order of affinity for [³H]ICS 205-930 binding sites was observed with antagonists: SDZ 206-830 = ICS 205-930 > BRL 43694 > SDZ 206–792 > quipazine > MDL 72222 > metoclopramide > mCPP and agonists: 2-methyl-5-HT = 5-HT > phenylbiguanide. A similar profile was observed for a limited series of compounds in rabbit membranes. Drugs acting at 5-HT₁, 5-HT₂ and dopamine receptors (domperidone, spiperone and metergoline) showed very low affinities for [³H]ICS 205-930 recognition sites. The sites labelled with [³H]ICS 205-930 in vagus nerve and superior cervical ganglion of both species displayed the pharmacological profile of a 5-HT₃ receptor. There was a significant correlation between the rank order of affinity of the tested compounds for [³H]ICS 205-930 recognition sites in cat and rabbit membranes and their rank order of affinity for 5-HT₃ receptors from neuroblastoma-glioma NG 108-15 cells. Autoradiographic studies suggest that [³H]ICS 205-930 binding sites are present over and around the nodose ganglion cell somata, along certain fibers of the vagus nerve and in the terminal areas of this nerve in the medullar nucleus of the vagus.The present data demonstrate that [³H]ICS 205-930 identifies 5-HT₃ receptors in preparations of cat and rabbit vagus nerve and superior cervical ganglion.Send offprint requests to D. Hoyer at the above addressThe present results have been presented in part at the Winter Meeting of the British Pharmacological Society, London, December 20–22, 1988 (Hoyer et al. 1989)     

Inhibition of MAO-B protects against MDMA-induced neurotoxicity in the striatum

The effects of the MAO-B inhibitors, L-deprenyl and MDL-72974 on MDMA-induced serotonergic neurotoxicity in rats were examined. MDMA alone produced a significant decrease in the number of 5-HT uptake sites, measured as a decrease in theB _max for binding of [³H]paroxetine, and in 5-HT and 5-HIAA levels in the striatum.l-Deprenyl and MDL-72974 attenuated this MDMA-induced decrease in serotonergic markers. The data suggest a key role for MAO-B in the expression of the neurotoxicity produced by MDMA in the striatum.