Serotonin Uptake Sites Research Articles

Comparison of the effects of repeated oral versus subcutaneous fenfluramine administration on rat brain monoamine neurons: Pharmacokinetic and dose-response data

The importance of the route of drug administration (oral vs. subcutaneous) on the neurochemical effects and pharmacokinetics of repeated d,1-fenfluramine administration in rats (1-24 mg/kg b.i.d., i.e., 2-48 mg/kg/day for 4 days) was examined. Overall, comparable dose-dependent alterations in brain monoamine markers were observed following repeated oral (PO) and subcutaneous (SC) administration of fenfluramine. Doses of 1 and 2 mg/kg fenfluramine were without significant effects on the density of 3H-paroxetine-labeled serotonin (5-HT) uptake sites. Higher doses of fenfluramine (4, 12 and 24 mg/kg) produced dose-dependent decreases in 5-HT, 5-hydroxyindoleacetic acid and 5-HT uptake sites with maximal decreases (80-90%) occurring at the 12 mg/kg dose. Fenfluramine administration produced dose-dependent and biphasic effects on brain dopamine markers with increases in homovanillic acid (HVA) observed at 2 hours, whereas decreases in the levels of dopamine, HVA and dihydroxyphenylacetic acid were evident at 18 hours posttreatment. Norepinephrine levels were only decreased at the highest dose of fenfluramine. Significantly higher levels of brain fenfluramine were observed following SC than following PO administration of the drug. On the other hand, comparable levels of its active metabolite norfenfluramine were present in the brain following the two routes of fenfluramine administration. These data suggest the importance of norfenfluramine levels in the brain in determining the high-dose neurotoxic effects of fenfluramine on brain 5-HT neurons in rats.

Reversed hemispheric asymmetry of imipramine binding in suicide victims

Changes in indices postulated to reflect central serotonergic mechanisms have been desc~bed in both suicide victims and suicide attempters (Asberg et al 1987; Deakin 1989; Mann et al 1989). Although a considerable body of research has been undertaken in relation to this question, literature to date has been characterized by contradictory findings. For example, in postmortem studies of frontal cortex of suicide victims in which imipramine (IMI) binding was used to label presynaptic serotonin (5HT) uptake sites, reductions have been found by some (Stanley et al 1982; see Mann et al 1989 for review) whereas others report contradictory findings (Meyerson et al 1982; Arora and Meltzer 1989a). Increased postsynaptic 5HT2 binding sites have been described in some investigations (Stanley - ~°~and Mann m~,ao; . . . . . . . . . . rearm ut m-' ~,ou; A~-om and Meltzer 1989b) but not in others (Owen et al 1986; Crow et al 1984; see Mann et al 1989 for review). Though an increasing body of evidence im-

Radiosynthesis and evaluation of N-(3-[ 18F]fluoropropyl)paroxetine as a radiotracer for in vivo labeling of serotonin uptake sites by PET

To visualize serotonin uptake sites by positron emission tomography (PET), N-(3-[ 18F]fluoropropyl)-paroxetine ([ 18F]FPP) (Fig. 1), a derivative of the selective serotonin uptake blocker paroxetine, was synthesized from 3-[ 18F]fluoropropyltosylate and paroxetine via a one-pot procedure. The rate of formation of [ 18F]FPP was a function of the ratio of the initial amount of paroxetine to that of 1,3-propanediol bistosylate with which [ 18F]fluoropropyltosylate was synthesized. When the reaction mixture contained an excess amount of paroxetine over that of the propyl-bistosylate, the radiosynthesis followed by HPLC purification, which took approx. 90 min, gave [ 18F]FPP in a radiochemical yield of approx. 8%, and in high radiochemical and chemical purity. The specific activity was 2640 ± 360 mCi/μmol. The brain biodistribution of [ 18F]FPP showed no distinguishable localization in regions with high density of serotonin uptake sites such as hypothalamus or olfactory tubercles. In vitro binding assays revealed that N-fluoropropylation of paroxetine reduced the affinity for the serotonin uptake site by three orders of magnitude.

Ethanol intake and 3H-serotonin uptake II: A study in alcoholic patients using platelets 3H-paroxetine binding

The kinetic parameters of 3H-paroxetine binding and 3H-serotonin uptake were studied in platelets of alcoholic patients. There was no difference between alcoholic and non alcoholic subjects in 3H-paroxetine binding. When binding and 3H-serotonin uptake were studied, in the same plasma of the same subjects, the Vmax of serotonin uptake was increased in alcoholics. The data confirm the involvement of serotonin uptake system in alcohol dependance and suggest that serotonin uptake and paroxetine binding sites may be regulated independently in this pathology.

Effect of 3,4-methylenedioxymethamphetamine on [ 3H]paroxetine binding in the frontal cortex and blood platelets of rats

The effects of single or repeated administration of the racemic mixture of 3,4-methylenedioxymethamphetamine (MDMA; 20 mg/kg, s.c.) on the number ( B max) of serotonin (5-HT) uptake sites as determined by [ 3H]paroxetine binding and the concentration of 5-HT and its major metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were measured in the frontal cortex and blood platelets of rats 1 and 7 days following its administration. A single injection of MDMA significantly (P < 0.05) decreased the number of [ 3H]paroxetine binding sites as well as the concentrations of 5-HT and 5-HIAA in the frontal cortex but not in platelets 7 days following administration. Repeated injections of MDMA (twice daily for 4 days) significantly (P < 0.05) decreased the number of 5-HT uptake sites and the concentration of 5-HT and 5-HIAA in the frontal cortex but not in platelets 7 days following administration. Pretreatment with the 5-HT 2/5-HT 1C antagonist, ketanserin, inhibited the MDMA-induced decrease in 5-HT and 5-HIAA concentrations and the number of [ 3H]paroxetine binding sites in the frontal cortex 7 days following a single administration. These data are suggestive that blood platelets are less sensitive than brain tissue to the 5-HT-depleting effects of MDMA. The ability of ketanserin pretreatment to block MDMA-induced decreases in [ 3H]paroxetine binding sites in the frontal cortex is suggestive that 5-HT 2/5-HT 1C receptors may be involved in the neurotoxic effects of MDMA.

Behavioral and neurochemical effects of prenatal methylenedioxymethamphetamine (MDMA) exposure in rats

MDMA is a hallucinogenic drug that is used by the general public as a recreational drug of abuse. The neurobehavioral consequences of prenatal MDMA exposure are unknown. Groups of pregnant rats were gavaged with 0, 2.5, or 10 mg/kg MDMA during gestation on alternate gestational days 6–18. Gestational duration, litter size, neonatal birth weights and physical appearance at birth were unaffected by MDMA treatments. Pregnancy weight gain was significantly reduced by MDMA treatment. Progeny growth, maturational parameters (eye opening and incisor eruption times), surface righting reflex, swimming performance, forelimb grip strength, milk-induced behaviors, passive avoidance behavior, figure-8 maze activity over 48 hours, the density of brain serotonin (5-HT) uptake sites, and brain 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) levels were unaffected by MDMA treatments. Olfactory discrimination on postnatal days (PND) 9–11 was enhanced in both male and female MDMA-treated progeny, while negative geotaxis (PND 7–10) was delayed in female pups. In contrast to progeny, MDMA caused dose-dependent decreases in 5-HT and 5-HIAA levels in discrete brain areas of the dam. It is concluded that prenatal exposure to MDMA at the levels used here produces only subtle behavioral alterations in developing rats. The dam is more at risk for MDMA-induced 5-HT depletion than is the conceptus.

3H]Paroxetine binding in human platelets in relation to age and sex

The binding of [ 3H]paroxetine to serotonin uptake sites in human platelets from 47 individuals between 15 and 95 years of age was investigated. There were no significant age-related changes in maximal binding capacity (B max) or apparent binding affinity (K d). There were no significant differences in binding between males (n = 19) and females (n = 28).

Neurotoxic Effects of MDMA on Brain Serotonin Neurons: Evidence from Neurochemical and Radioligand Binding Studiesa

In summary, the data from both neurochemical and neuroanatomical studies demonstrate widespread and long-lasting degeneration of serotonin neurons in brain without any major or consistent effects on catecholamine neurons following in vivo administration of MDMA in both rats and rhesus monkeys. A detailed examination of the parameters involved in the neurotoxic and neurodegenerative effects of MDMA on brain serotonin neurons indicate that the severity of the lesion is dependent on the dose of drug administered with the drug being more potent in rhesus monkeys than in rats. Furthermore, the neurodegenerative effects of the drug are long-lasting (up to one year) with respect to neuronal regeneration (i.e. recovery of serotonin uptake sites) while functional recovery may be permanently impaired since serotonin content remains markedly (40-50%) below levels in age-matched controls for as long as one year after drug administration. The neurochemical and autoradiographic data suggest that there are some regional differences and morphological specificity to the neurodegenerative effects of MDMA as demonstrated by greater reductions in serotonin uptake sites in brain regions containing primarily terminals while regions containing axons of passage and cell bodies are relatively unaffected.

Increased uptake sites for serotonin and dopamine with decreased S2 serotonin receptors in microencephalic rat brain

Methylazoxymethanol (MAM)-induced cerebral hypoplasia resulted in a significant increase in densities of both serotonin uptake sites in frontal cortex and dopamine uptake sites in striatum, suggesting serotonergic and dopaminergic axons terminals were compressed in the smaller brain volumes. The density of S2 serotonin receptors in MAM-lesioned frontal cortex was decreased probably due to down-regulation, while densities of D1 and D2 dopamine receptors in striatum were identical between MAM-lesioned rats and control rats.

Paroxetine as an in vivo indicator of 3,4-methylenedioxymethamphetamine neurotoxicity: a presynaptic serotonergic positron emission tomography ligand?

The present study sought to determine whether [ 3H]paroxetine, a potent and selective inhibitor of serotonin uptake in vitro, could be used to label the serotonin transporter in the rat brain in vivo such that it might be employed to develop a presynaptic serotonergic positron emission tomography ligand. Tritium labeled paroxetine was administered intravenously to rats by means of tail vein injection. Four hours later, specific [ 3H]paroxetine binding was determined by subtracting non-specific binding in the cerebellum from total binding in other brain regions of interest. The distribution of specific [ 3H]paroxetine binding paralleled the distribution of serotonin uptake sites in all brain regions examined. Pretreatment with serotonin re-uptake inhibitors (citalopram or sertraline) reduced in vivo specific [ 3H]paroxetine binding by as much as 99%. Specific in vivo [ 3H]paroxetine binding was further characterized through the use of 5,7-dihydroxytryptamine (5,7-DHT), a known serotonergic neurotoxin. 5,7-DHT (200 μg, i.c.v.) caused a marked reduction in specific [ 3H]paroxetine binding, and induced a prolonged depletion of regional brain serotonin. In a final study, the feasibility of using in vivo [ 3H]paroxetine binding as an indicator of serotonergic damage induced by another neurotoxin (3,4-methylenedioxymethamphetamine, MDMA) was tested. MDMA-treated rats showed a profound reduction in in vivo [ 3H]paroxetine binding, along with a lasting depletion of regional brain serotonin. These results demonstrate that [ 3H]paroxetine can be used to label serotonin uptake sites in rat rat brain in vivo, and that the damage induced by serotonergic neurotoxins can be detected using in vivo [ 3H]paroxetine binding as an indicator. Paroxetine (or one of its derivatives) therefore holds promise as a PET ligand for studying serotonergic neurons in the living human brain in health as well as after neurotoxic injury.

Symmetrical hemispheric distribution of 3H-Paroxetine binding sites in postmortem human brain from controls and suicides

The hemispheres of the human brain are functionally specialized: the dominant hemisphere (usually the left in a right-handed individual) is involved in communicative and logical functions, and the nondominant (usually right) hemisphere is involved in emotional, aggressive, and sexual behavior. This specialization suggests a neurochemical asymmetry. Indeed, there have been a number of reports of asymmetrical distribution of various neurotransmitters and related enzymes in human brain (Amaducci et al. 1981; Glick et ai. 1982). 3H-Imipramine has been widely used to label serotonin (5-HT) uptake sites. Studies of 3H-imipramine binding in postmortem human frontal cortex from suicide victims has produced inconsistent findings, with decreased, increased, and unaltered binding compared with controls (Stanley et al. 1982; Meyerson eta!. 1982; Owen et al. 1986). These discrepant findings may be partly related to the recently reported marked asym-

Brain 5-HT uptake sites, labelled with [ 3H]paroxetine, in antidepressant-free depressed suicides

Brain serotonin (5-HT) uptake sites were quantitated, by saturation binding of [ 3H]paroxetine, in 10 brain regions from 22 suicide victims and 20 control subjects. Suicide victims were restricted to those subjects in whom a firm retrospective diagnosis of depression was established and who had not recently been prescribed antidepressant drugs. The K d and B max of [ 3H]paroxetine did not differ significantly between controls and depressed suicides in any of the brain regions. In putamen, B max values of suicides who died non-violently were lower than controls, whereas those who died by violent methods did not differ from controls. No significant differences between violent or non-violent suicides and their matched controls were found in other brain areas. These results offer little support for the view that suicide/depression is associated with an abnormality in 5-HT uptake.

Olfactory bulbectomy alters alpha-1 acid glycoprotein levels in rat plasma

Olfactory bulbectomy in rats causes neurochemical, behavioral, as well as physiological alterations. These alterations make this surgical procedure a useful animal model for depression. In humans, depression was shown to be accompanied by increases in plasma cortisol, inability to decrease cortisol in the dexamethasone suppression test and increases in plasma alpha-1 acid glycoprotein (AGP), an endogenous modulator for the serotonin uptake site. Utilizing a recently developed radial immunodiffusion assay for rat AGP we were able to confirm the increases in plasma AGP in the rat. However, we did not observe increased corticosterone in the rat. We also observed the aggressive behavior of muricide in olfactory bulbectomized rats. These results seem to indicate that olfactory bulbectomy is a good model for depression in the human condition and that AGP may be a putative marker for this condition.

Lack of asymmetry in serotonin uptake sites in human brain : Bruce Parsons, M.D., Ph.D., Yung-Yu Huang, M.S., Narendra Budhakar, M.D., Shaoning Wang, M.D., Michael Stanley, Ph.D. New York State Psychiatric Institute, New York, NY 10032

Lack of asymmetry in serotonin uptake sites in human brain : Bruce Parsons, M.D., Ph.D., Yung-Yu Huang, M.S., Narendra Budhakar, M.D., Shaoning Wang, M.D., Michael Stanley, Ph.D. New York State Psychiatric Institute, New York, NY 10032

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: KD 0.57 nM, Bmax 821 fmol/mg protein, nH 1.06. This binding was reversible; the dissociation constant calculated from kinetic measurements (KD 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 in KD with little change in Bmax). 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 with p-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.

Laminar distributions of muscarinic acetylcholine, serotonin, GABA and opioid receptors in human posterior cingulate cortex

Experimental animal studies have demonstrated a number of receptor localizations on specific cortical afferents and neurons. The present study of human posterior cingulate cortex evaluates the laminar distributions of particular receptors and their likely association with components of the neuropil. Coverslip autoradiographic and single grain counting techniques were used followed by heterogeneity analysis in which the layer of peak binding and an index of heterogeneity were determined for each ligand. The index was calculated by determining specific binding by layer as a percentage of binding in all layers. The differences from an absolutely homogeneous distribution, i.e. 11.1% for each of nine layers, were subtracted and the absolute laminar differences summed to form the index. High indices of over 15 reflected heterogeneous binding patterns in neocortex. The binding of ligands for muscarinic acetylcholine, serotonin, opioid, GABA and beta adrenoceptors was evaluated. Pirenzepine binding peaked in layer II of area 23a but was extremely homogeneous with an index of heterogeneity of 8.9. In contrast, oxotremorine-M binding had a peak in layer IIIc and an index of 16.4, while AF-DX 116 binding peaked in layer IIIa-b and had an index of 30.6. Of the ligands for serotonin uptake and receptor binding paroxetine binding was evenly distributed in layers I–III and had a low index of heterogeneity of 9.8. Ketanserin binding was also homogeneous and, since it had an index of 8.9, this pattern was virtually the same as that for paroxetine. In contrast, serotonin and 8-hydroxy-2-(di- n-propylamino)tetralin binding peaked in layer II and had very high indices of 20.8 and 50.3, respectively, suggesting only a limited association with that of the paroxetine distribution. Finally, there were three layers which contained peaks in binding for ligands for opioid, GABA and beta adrenoceptors. Firstly, layer Ia had peak dynorphin-A binding, the latter of which had an index of 22.6. Secondly, Tyr- d-Ala-Gly-MePhe-Gly-ol and 2- d-penicillamine-5- d-penicillamine-enkephalin binding peaked in layer II and had indices of 8.6 and 17.4, respectively. Thirdly, muscimol and (−)-cyanopindolol binding peaked in layer IIIa-b and had indices of 29.6 and 11.1, respectively. When viewed in the context of experimental animal studies, it is likely that heterogeneities in oxotremorine-M and paroxetine binding are associated with the termination of the thalamic and raphe nuclei, respectively. While serotonin, receptors are co-distributed with serotonin uptake sites, serotonin 1A receptors have a significant mismatch with these sites. Finally, postsynaptic receptors preferentially peak in different layers including kappa opioid receptors in layer Ia, M 1 acetylcholine, serotonin 1A, mu and delta opioid receptors in layer II, GABA A and beta adrenoceptors in layer IIIa-b and serotonin 2 receptors in layer IIIc. Thus, ligand binding analyses provide new insights into the structure and connections of human cerebral cortex.

Synthesis of a selective serotonin uptake inhibitor: [ 11C]citalopram

Citalopram (Fig. 1), a selective serotonin uptake inhibitor, was labeled with 11C for non-invasive in vivo studies of serotonin uptake sites in the human brain using positron emission tomography. The synthesis was completed in approximately 17 min using [ 11C]methyl iodide as the precursor. The synthesis, purification, characterization, and determination of specific activity are described.

Regional distribution of the serotonin transport complex in human brain, identified with 3H-Paroxetine, 3H-Citalopram and 3H-Imipramine

1. 1. Regional distribution of the serotonin transport complex was studied in 12 different brain areas from human brains. The serotonin uptake complex was measured with 3H-paroxetine, and 3H-imipramine. The binding site density was highest in the nucleus of raphe, medium in the basal ganglia, and lowest in cortical areas. The specific binding measured with 3H-paroxetine and 3H-citalopram was compared with the high affinity 3H-imipramine binding determined with either 100 μM 5HT or 1 μM imipramine as non specific displacers. 3H-paroxetine and 3H-citalopram allowed a more precise determination of B max, and are both good ligands for the serotonin uptake site, but the determinations with 3H-imipramine were within the same range. 2. 2. Protease digestion of brain membranes showed that the binding site measured with all three ligands disappeared with the same rate as other membrane proteins, and not faster as might be expected from the literature. 3. 3. Left/right hemisphere distribution was measured in cortical tissue from 6 brains using 3H-paroxetine. No difference between the two hemispheres was found. In one brain from a lithium treated patient a very low binding was measured, possibly indicating that the lithium treatment had decreased the serotonin uptake mechanism.

Effects of high-dose methamphetamine administration on serotonin uptake sites in rat brain measured using [ 3H]cyanoimipramine autoradiography

Repeated administration of high doses of methamphetamine (15 mg/kg given for 5 doses over 24 h) resulted in long-term decreases in the binding of [ 3H]cyanoimipramine ([ 3H]CN-IMI) to serotonin uptake sites measured using quantitative autoradiography. Seven days after termination of drug administration decreases were seen in 23 of 28 regions examined. This is consistent with previous studies indicating that methamphetamine and related amphetamines are neurotoxic to serotonin neurons. Significant decreases were still present in many brain areas on the same dosage schedule 30 days after drug administration. However, the number of areas affected was considerably less, consistent with some regrowth of serotonin neurons. At a lower dosage (7.5 mg/kg on the same schedule) no effects on [ 3H]CN-IMI binding were seen. The results of this study provide support for the serotonergic neurotoxicity of repeated methamphetamine administration in rats. They also show that the neurotoxicity is highly regional and dose dependent.

In Vivo Labeling of Serotonin Uptake Sites with [3H]Paroxetine

Previous work has shown that [3H]paroxetine is a potent and selective in vitro label for serotonin uptake sites in the mammalian brain. In the present study, [3H]paroxetine was tested in mice as an in vivo label for serotonin uptake sites. Maximum tritium concentration in the whole brain (1.4% of the intravenous dose) was reached 1 h after injection into a tail vein. Distribution of the tracer at 3 h after injection followed the distribution of serotonin uptake sites known from previous in vitro binding studies (r = 0.85). The areas of highest [3H]paroxetine concentration, in decreasing order, were: hypothalamus greater than frontal cortex greater than olfactory tubercles greater than thalamus greater than upper colliculi greater than brainstem greater than hippocampus greater than striatum greater than cerebellum. Preinjection of carrier paroxetine (1 mg/kg) significantly decreased [3H]paroxetine concentration in all areas except in the cerebellum, which is known to contain a relatively low number of specific binding sites. Kinetic studies showed highest specific [3H]paroxetine binding (tissue minus cerebellum) at 2 h after injection and slow clearance of activity thereafter (half-time of dissociation from the hypothalamus, 215 min). The specificity of in vivo [3H]paroxetine binding was studied by preinjecting monoamine uptake blockers or receptor antagonists 5 min before administration of [3H]paroxetine. Serotonergic or muscarinic cholinergic receptor antagonists and dopamine or norepinephrine uptake blockers did not reduce the in vivo binding of [3H]paroxetine. In contrast, there was an excellent correlation (r = 0.99) between the in vivo inhibitory potencies of serotonin uptake blockers in this study and previously published in vitro data on inhibition of [3H] serotonin uptake in brain synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)