Citalopram Binding Sites Research Articles

Neurochemical Changes in the Mouse Hippocampus Underlying the Antidepressant Effect of Genetic Deletion of P2X7 Receptors

Recent investigations have revealed that the genetic deletion of P2X7 receptors (P2rx7) results in an antidepressant phenotype in mice. However, the link between the deficiency of P2rx7 and changes in behavior has not yet been explored. In the present study, we studied the effect of genetic deletion of P2rx7 on neurochemical changes in the hippocampus that might underlie the antidepressant phenotype. P2X7 receptor deficient mice (P2rx7−/−) displayed decreased immobility in the tail suspension test (TST) and an attenuated anhedonia response in the sucrose preference test (SPT) following bacterial endotoxin (LPS) challenge. The attenuated anhedonia was reproduced through systemic treatments with P2rx7 antagonists. The activation of P2rx7 resulted in the concentration-dependent release of [3H]glutamate in P2rx7+/+ but not P2rx7−/− mice, and the NR2B subunit mRNA and protein was upregulated in the hippocampus of P2rx7−/− mice. The brain-derived neurotrophic factor (BDNF) expression was higher in saline but not LPS-treated P2rx7−/− mice; the P2rx7 antagonist Brilliant blue G elevated and the P2rx7 agonist benzoylbenzoyl ATP (BzATP) reduced BDNF level. This effect was dependent on the activation of NMDA and non-NMDA receptors but not on Group I metabotropic glutamate receptors (mGluR1,5). An increased 5-bromo-2-deoxyuridine (BrdU) incorporation was also observed in the dentate gyrus derived from P2rx7−/− mice. Basal level of 5-HT was increased, whereas the 5HIAA/5-HT ratio was lower in the hippocampus of P2rx7−/− mice, which accompanied the increased uptake of [3H]5-HT and an elevated number of [3H]citalopram binding sites. The LPS-induced elevation of 5-HT level was absent in P2rx7−/− mice. In conclusion there are several potential mechanisms for the antidepressant phenotype of P2rx7−/− mice, such as the absence of P2rx7-mediated glutamate release, elevated basal BDNF production, enhanced neurogenesis and increased 5-HT bioavailability in the hippocampus.

Increases in serotonergic neuronal activity following intracerebroventricular administration of AF64A in rats

Changes in the serotonergic nervous system after the intracerebroventricular (i.c.v.) administration of ethylcholine aziridinium (AF64A, 3 nmol/each ventricle) were studied in rats. Two weeks after the infusion of AF64A, the levels of 5-HT and 5-HIAA in microdialysed cerebrospinal fluid (CSF), the levels of total 5-HT and 5-HIAA, the density of serotonin uptake sites and the activities of tryptophan hydroxylase (TPH) and monoamine oxidase (MAO) in various brain regions were determined. After AF64A administration, the concentrations of 5-HT in lateral ventricle were increased and the levels of 5-HIAA were decreased. However, the hippocampal levels of total 5-HT were decreased without changes in the levels of 5-HIAA and the hippocampal turnover rates of 5-HT increased. Also, the density of uptake sites of serotonin ([(3)H]citalopram binding sites) was decreased in the various brain. The activities of TPH were increased in striatum and frontal cortex and the activity of MAO was also increased in striatum. These results indicate that AF64A induces an increase in serotonergic neuronal activity and decreased densities of 5-HT uptake sites which may affect the change in the other parameters of serotonergic neuronal activities. Furthermore, these results suggest that the impaired cholinergic neuronal activity induces the alteration in the serotonergic nervous activities.

A single nucleotide polymorphism in the human serotonin transporter introduces a new site for N-linked glycosylation

The human serotonin transporter (hSERT) is responsible for reuptake of serotonin (5-HT) from the synaptic cleft and is target for antidepressant medicine. Differential hSERT activity caused by genetic polymorphisms is believed to affect the risk of developing depression and, moreover, to affect the response to antidepressant therapy. The hSERT contains in the second extracellular loop (EL2) two sites for N-linked glycosylation that are critical for functional transporter expression. Here we examine a non-synonymous single nucleotide polymorphism (SNP) in EL2 that gives rise to a potential third glycosylation site due to substitution of a lysine at position 201 with an asparagine (K201N). In agreement with introduction of an additional glycosylation site, western blot analysis showed migration of hSERT K201N corresponding to a higher molecular weight than wild type hSERT upon expression in both HEK293 cells and primary cultures of cortical neurons. An increase in molecular weight was not observed after removal of glycans with peptide N-glycosidase F (PNGase F). Quantitative analysis of western blots indicated significantly increased total transporter expression (∼30%) for hSERT K201N as compared to hSERT in both cell systems. The increase in expression was accompanied by corresponding significant increases in the number of [ 3H]citalopram binding sites and in the V max for [ 3H]5-HT uptake. Characterization of mutants carrying all possible combinations of glycosylation sites demonstrated clear correlation between the number of glycosylation sites and the level of transporter activity, and showed that K201N could substitute for either one of the two original glycosylation sites.

Serotonin transporter characteristics in lymphocytes and platelets of male aggressive schizophrenia patients compared to non-aggressive schizophrenia patients

A large body of literature indicates that disturbances of central serotonin (5-HT) function play an important role in aggressive behavior. Results from open-label and placebo-controlled trials as well as the reported inverse relationship between 5-HT function and aggression in human subjects, suggest that reduced 5-HT activity is associated with aggressive behavior. The activity of the 5-HT transporter (5-HTT), as determined by [3H]5-HT uptake to blood lymphocytes, was measured in 20 currently aggressive and 20 non-aggressive male schizophrenia patients. In addition, the pharmacodynamic characteristics of platelet 5-HTT were assessed by [3H]citalopram binding.There were no significant differences in the density (Bmax) of platelet [3H]citalopram binding sites between the two groups. Similarly, the dissociation constant (Kd) values were indistinguishable. The maximum uptake velocity (Vmax) of [3H]5-HT to fresh lymphocytes and the Km values of the 5-HT to the transporter were significantly higher in currently aggressive compared to the non-aggressive schizophrenia patients. The association of high Vmax values with current aggressive behavior provides further support to the involvement of the 5-HTT in aggressive behavior as well as to the efficacy of 5-HTT blockers in the control of aggression. The role of the various components of the serotonergic system in the pathophysiology and treatment of aggressive behavior in schizophrenia needs to be further evaluated.

The serotonin transporter in rhesus monkey brain: comparison of DASB and citalopram binding sites

We have characterized the interaction of the serotonin transporter ligand [3H]-N,N-dimethyl-2-(2-amino-4-cyanophenylthio)-benzylamine (DASB) with rhesus monkey brain in vitro using tissue homogenate binding and autoradiographic mapping. [3H]-DASB, a tritiated version of the widely used [11C] positron emission tomography tracer, was found to selectively bind to a single population of sites with high affinity (K(d)=0.20+/-0.04 nM). The serotonin transporter density (B(max)) obtained for rhesus frontal cortex was found to be 66+/-8 fmol/mg protein using [3H]-DASB, similar to the B(max) value obtained using the reference radioligand [3H]-citalopram, a well-characterized and highly selective serotonin reuptake inhibitor (83+/-22 fmol/mg protein). Specific binding sites of both [3H]-DASB and [3H]-citalopram were similarly and nonuniformly distributed throughout the rhesus central nervous system, in a pattern consistent with serotonin transporter localization reported for human brain. Regional serotonin transporter densities, estimated from optical densities of the autoradiographic images, were well correlated between the two radioligands. Finally, DASB and fluoxetine showed dose-dependent full inhibition of [3H]-citalopram binding in a competition autoradiographic study, with K(i) values in close agreement with those obtained from rhesus brain homogenates. This side-by-side comparison of [3H]-DASB and [3H]-citalopram binding sites in rhesus tissue homogenates and in adjacent rhesus brain slices provides additional support for the use of [11C]-DASB to assess the availability and distribution of serotonin transporters in nonhuman primates.

Characterization of the serotonin transporter in lymphocytes and platelets of schizophrenia patients treated with atypical or typical antipsychotics compared to healthy individuals

A rapidly growing body of data suggests that abnormalities in serotonergic function might be involved in the pathophysiology of schizophrenia and that serotonergic mechanisms play a role in the therapeutic effects of antipsychotics. The activity of the serotonin transporter (5-HTT), as determined by [ 3H]5-HT uptake to blood lymphocytes, was measured in 38 medicated schizophrenia patients (15 of them treated with typical antipsychotics and 23 treated with atypical antipsychotics) and 15 healthy control subjects. In addition, the pharmacodynamic characteristics of platelet 5-HTT were assessed by [ 3H]citalopram binding. There were no significant differences in the density ( B max) of platelet [ 3H]citalopram binding sites between the three groups. Similarly, the dissociation constant ( K d) values were indistinguishable. There were no significant differences in the maximal uptake velocity ( V max) of [ 3H]5-HT to fresh lymphocytes between the three groups. The affinity ( K m) values of 5-HT to the 5-HTT were significantly higher in schizophrenia patients treated with typical antipsychotics compared with control subjects. The K m values in schizophrenia patients treated with atypical antipsychotics were significantly lower compared with those observed in the group of schizophrenia patients treated with typical antipsychotics; however, they were comparable to values in the control group. The high values of K m associated with typical antipsychotic treatment may be relevant to the high risk of developing extrapyramidal side effects (EPS). The role of the various components of the serotonergic system in the etiopathology of schizophrenia and the mechanisms by which antipsychotics achieve their therapeutic effects need to be further evaluated.

Reduced serotonin release and serotonin uptake sites in the rat nucleus accumbens and striatum after prenatal cocaine exposure

This study was designed to assess the effects of prenatal cocaine exposure on the development of the serotonergic system. Pregnant Sprague–Dawley rats received daily sc injections of either cocaine (30 mg/kg) or saline from gestation day 7 (GD 7) to GD 20. At 1 week postnatal, all pups were killed and tissues containing the striatum and nucleus accumbens dissected out. In superfusion experiments, tissue slices were incubated with [ 3H]serotonin ([ 3H]5-HT) for 30 min and then superfused. The [ 3H]5-HT release was induced by exposures to the following treatments: electrical stimulations (20 mA or 40 mA, 0.5 Hz, 4 min), the medium containing 15 or 30 mM potassium (2 min), fenfluramine (1 or 2 μM for 2 min), para-chloroamphetamine (1 or 2 μM for 2 min), methiothepin (1 or 2 μM for 2 min), and fluoxetine (1 or 2 μM for 2 min). The results showed that the treatment-induced [ 3H]5-HT releases were all significantly less pronounced in the pups prenatally exposed to cocaine than in those prenatally exposed to saline regardless of the mechanisms by which the treatment increases extracellular 5-HT. Saturation analysis showed that the B max of [ 3H]citalopram binding sites was also significantly lower in the pups prenatally treated with cocaine than in those prenatally treated with saline. The results are consistent with the concept that less serotonergic innervation may exist in the examined brain areas of cocaine-treated offspring at 1 week postnatal, and support the hypothesis that prenatal cocaine exposure affects the postnatal development of the serotonergic system.

Non-amines, drugs without an amine nitrogen, potently block serotonin transport: novel antidepressant candidates?

The serotonin transporter (SERT) is a principal site of action of therapeutic antidepressants in the brain. Without exception, these inhibitors of serotonin transport contain an amine nitrogen in their structure. We previously demonstrated that novel compounds without an amine nitrogen in their structure (non-amines), blocked dopamine transport in cells transfected with the human dopamine transporter. The present study investigated whether, in the absence of an amine nitrogen, certain non-amines bind selectively to the SERT and block the transport of serotonin. At 10 microM concentration, select non-amines displayed no, or little, affinity for 9 serotonin, 5 dopamine, 7 adrenergic, 5 muscarinic cholinergic, 3 opiate and histamine receptors. The affinities of non-amines for [(3)H]citalopram binding sites on the SERT and their potencies for blocking [(3)H]serotonin transport were measured in cloned human SERT stably or transiently expressed in HEK-293. Whether oxa- or carba-based, non-amines bound to [(3)H]citalopram-labeled sites and blocked [(3)H]serotonin transport in the low nanomolar range, at values equal to or higher than those of some conventional antidepressants. A non-amine, O-1809, was 99-fold more selective for the serotonin over the dopamine transporter. As substituents on the aromatic ring of non-amines confer high affinity for the SERT, we investigated the hypothesis that aromatic-aromatic interactions may contribute significantly to non-amine/transporter association. A SERT mutant was produced in which a highly conserved aromatic amino acid, phenylalanine 548, was replaced by an alanine (F548A). Although the affinities of several non-amines were unchanged in the mutant SERT, the affinity of imipramine was decreased, revealing possible differences in amine and non-amine binding domains on the SERT. The similar affinities of non-amines and conventional antidepressant drugs for the SERT support the view that an amine nitrogen is not essential for drugs to block serotonin transport with high affinity. Non-amines open avenues for developing a new generation of antidepressants.

Distribution of [3H]citalopram binding sites in the nonhuman primate brain.

Annals of the New York Academy of SciencesVolume 877, Issue 1 p. 700-702 Distribution of [3H]Citalopram Binding Sites in the Nonhuman Primate Brain HILARY R. SMITH, HILARY R. SMITH Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USASearch for more papers by this authorJAMES B. DAUNAIS, JAMES B. DAUNAIS Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USASearch for more papers by this authorMICHAEL A. NADER, MICHAEL A. NADER Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USASearch for more papers by this authorLINDA J. PORRINO, Corresponding Author LINDA J. PORRINO Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA Corresponding author: Linda J. Porrino, Ph.D., Dept. of Physiology and Pharmacology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. Voice: 336-716-8575; fax: 336-716-8501; [email protected]Search for more papers by this author HILARY R. SMITH, HILARY R. SMITH Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USASearch for more papers by this authorJAMES B. DAUNAIS, JAMES B. DAUNAIS Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USASearch for more papers by this authorMICHAEL A. NADER, MICHAEL A. NADER Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USASearch for more papers by this authorLINDA J. PORRINO, Corresponding Author LINDA J. PORRINO Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA Corresponding author: Linda J. Porrino, Ph.D., Dept. of Physiology and Pharmacology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. Voice: 336-716-8575; fax: 336-716-8501; [email protected]Search for more papers by this author First published: 06 February 2006 https://doi.org/10.1111/j.1749-6632.1999.tb09305.xCitations: 24Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume877, Issue1ADVANCING FROM THE VENTRAL STRIATUM TO THE EXTENDED AMYGDALA: IMPLICATIONS FOR NEUROPSYCHIATRY AND DRUG ABUSEJune 1999Pages 700-702 RelatedInformation

Thalamocortical afferents in rat transiently express high-affinity serotonin uptake sites

Autoradiographic techniques using [ 3H]citalopram were employed in 8-day-old (P-8) and adult rats to delineate the distribution of high-affinity serotonin (5-HT) uptake sites in the cerebral cortex. In the postnatal rats, [ 3H]citalopram binding sites were densely distributed in the lower portion of layer III, lamina IV, and upper layer V in the primary visual, somatosensory, and auditory cortices. In the primary somatosensory cortex, these binding sites were arrayed in a manner exactly matching the representation of the body surface as demonstrated by other methods such as staining for cytochrome oxidase (CO) or acetylcholinesterase (AChE). In adult rats, there was no differential distribution of [ 3H]citalopram binding sites in the cerebral cortex. Neonatal administration of the 5-HT neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), resulted in a nearly complete destruction of the 5-HT innervation of the cortex on P-8, but the patterned distribution of [ 3H]citalopram binding sites remained visible. In contrast, thalamic lesions carried out on P-4 caused a complete loss of the patterned distribution of [ 3H]citalopram binding sites in rats killed on either P-5 or P-8. These results are consistent with the conclusion that thalamocortical afferents in postnatal rats transiently express high-affinity uptake sites for 5-HT and thus may accumulate this amine.

Serotoninergic terminal transporters are differentially affected in Parkinson's disease and progressive supranuclear palsy: An autoradiographic study with [ 3H]citalopram

Receptor autoradiography with [ 3H]citalopram as ligand was used to study the distribution of serotonin uptake binding sites in post mortem brain tissues from patients with Parkinson's disease, progressive supranuclear palsy and from age-matched controls. Significant decreases in [ 3H]citalopram binding sites were found in the cerebral cortex of patients with Parkinson's disease and progressive supranuclear palsy. Densities of [ 3H]citalopram binding sites were significantly reduced in all components of the basal ganglia of Parkinson's disease but only in the head of caudate nucleus of progressive supranuclear palsy patients. The density of [ 3H]citalopram binding sites in the raphe nuclei of Parkinson's disease was comparable to control values. Our results suggest that serotoninergic terminals are differentially affected in Parkinson's disease and in progressive supranuclear palsy.

Binding of 5-HT 1A receptor and 5-HT transporter ligands in rat cortex and hippocampus following cholinergic and serotonergic lesions

The cellular localisation of 5-HT 1A receptor and 5-HT transporter binding sites in the rat cortex and hippocampus has been examined. Lesions of either basal forebrain neurones or serotonergic neurones did not affect [ 3H]8-OH-DPAT binding, suggesting that 5-HT 1A binding sites are not localised on cholinergic or serotonergic nerve terminals. The binding of the 5-HT transporter ligand, [ 3H]citalopram was unaffected by the cholinergic lesion whereas binding was reduced in both the hippocampus and cortex following serotonergic lesions. A reduction in binding site density rather than an alteration in affinity was responsible for this effect. While these data suggest that [ 3H]citalopram binding sites are located on serotonergic nerve terminals, the abolition of hippocampal binding sites contrasted with a 50% loss in cortical tissue.

Autoradiographic characterization of [ 3H]imipramine and [ 3H]citalopram binding in rat and human brain: species differences and relationships to serotonin innervation patterns

The neuroanatomical distribution of binding sites for [ 3H]imipramine and [ 3H]citalopram was assessed by in vitro autoradiography in select regions of the rat and human forebrain. To determine involvement of serotonin-containing terminals in the binding of [ 3H]imipramine and [ 3H]citalopram, binding of these compounds was measured in rats after destroying serotonin-containing neurons with 5,7-dihydroxytryptamine (5,7-DHT). Treatment with this neurotoxin decreased serotonin content by 90% and reduced [ 3H]citalopram binding to a similar extent. These results demonstrate that [ 3H]citalopram binding is a reliable marker for serotonin-containing terminals. Binding of [ 3H]imipramine was reduced by only 15–35% after 5,7-DHT treatment. These latter results suggest that only a small fraction of [ 3H]imipramine binding to brain sections is associated with serotonergic terminals under standard conditions used in autoradiographic studies with the ligand. Dose-response effects of fluoxetine and desipramine on displacement of [ 3H]imipramine binding in forebrain regions indicate that the ligand labels predominantly high capacity, low affinity binding sites. To determine the utility of the rat brain as a model for [ 3H]imipramine and [ 3H]citalopram binding in the human brain, binding of the ligands was compared in human and rat hypothalamus, amygdala, and hippocampus. The pharmacological characteristics of [ 3H]imipramine and [ 3H]citalopram binding were similar in the rat and human brain. However, substantial species differences were observed in topographic patterns of [ 3H]imipramine binding within the hippocampus and hypothalamus. The distribution of [ 3H]citalopram binding sites within the amygdala and hypothalamus were also strikingly different in rats compared to humans. This work provides the first demonstration that marked species differences exist in the topography of serotonergic innervation and in the distribution of [ 3H]imipramine binding sites within the rat and human brain regions examined.