A requirement of serotonergic p38α mitogen-activated protein kinase for peripheral immune system activation of CNS serotonin uptake and serotonin-linked behaviors.

Alterations in central serotonin (5-hydroxytryptamine, 5-HT) neurotransmission and peripheral immune activation have been linked to multiple neuropsychiatric disorders, including depression, schizophrenia and autism. The antidepressant-sensitive 5-HT transporter (SERT, SLC6A4), a critical determinant of synaptic 5-HT inactivation, can be regulated by pro-inflammatory cytokine signaling. Systemic innate immune system activation via intraperitoneal lipopolysaccharide (LPS) injection rapidly elevates brain SERT activity and 5-HT clearance. Moreover, the pro-inflammatory cytokine interleukin (IL)-1ß rapidly stimulates SERT activity in raphe nerve terminal preparations ex vivo, effects that are attenuated by pharmacological p38 MAPK inhibition. To establish a role of serotonergic p38α MAPK signaling in LPS/IL-1ß-induced SERT regulation and attendant behavioral responses, we pursued studies in mice that afford conditional elimination of p38α MAPK in 5-HT neurons (p38α(5HT-)). We found p38α(5HT-) and control (p38α(5HT+)) littermates to be indistinguishable in viability and growth and to express equivalent levels of SERT protein and synaptosomal 5-HT transport activity. Consistent with pharmacological studies, however, IL-1ß fails to increase SERT activity in midbrain synaptosomes prepared from p38α(5HT-) animals. Moreover, although LPS elevated plasma corticosterone and central/peripheral pro-inflammatory cytokines in p38α(5HT-) animals, elevations in midbrain SERT activity were absent nor were changes in depressive and anxiety-like behaviors observed. Our studies support an obligate role of p38α MAPK signaling in 5-HT neurons for the translation of immune activation to SERT regulation and 5-HT-modulated behaviors.

Expression mapping of 5-HT1 serotonin receptor subtypes during fetal and early postnatal mouse forebrain development.

Serotonin (5-HT) is implicated in several aspects of brain development, yet the ontogenetic expression patterns of 5-HT receptors responsible for transducing specific effects have largely not been characterized. Fifteen different 5-HT receptor genes have been cloned; therefore any spatial and/or temporal combination of their developmental expression could mediate a wide array of 5-HT effects. We undertook a detailed analysis of expression mapping of the Gi/o-coupled 5-HT1 (5-HT1A, 1B, 1D and 1F) receptor subtypes in the fetal and early postnatal mouse forebrain. Using receptor subtype-specific riboprobes and in situ hybridization, we observed that all 5-HT1 receptor subtypes are expressed as early as embryonic day (E) 14.5 in the forebrain, typically in gradients within specific structures. Among 5-HT1 receptors, the 5-HT1A receptor transcript is expressed densely in E14.5-16.5 thalamus, in hippocampus, and in a medial to lateral gradient in cortex, whereas the 5-HT1B receptor mRNA is expressed in more lateral parts of the dorsal thalamus and in the striatum at these ages. The 5-HT1D receptor transcript, which also is expressed heavily in E14.5-E16.5 thalamus, appears to be down-regulated at birth. The 5-HT1F receptor transcript is present in proliferative regions such as the cortical ventricular zone, ganglionic eminences, and medial aspects of the thalamus at E14.5-16.5, and otherwise presents similarities to the expression patterns of 5-HT1B and 1D receptor transcripts. Overall, the 5-HT1 subfamily of Gi/o-coupled 5-HT receptors displays specific and dynamic expression patterns during embryonic forebrain development. Moreover, all members of the 5-HT1 receptor class are strongly and transiently expressed in the embryonic dorsal thalamus, which suggests a potential role for serotonin in early thalamic development.

Anxiolytic-like effects of DAIZAC, a selective high-affinity 5-HT(3) receptor antagonist, in the mouse elevated plus-maze.

Behavioral effects of desamino-3-iodozacopride (DAIZAC) [(S)-5-chloro-3-iodo-2-methoxy-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide], a selective high-affinity 5-HT(3) receptor antagonist (K(D) 0.14 nM), were evaluated in the mouse elevated plus-maze using the anxiolytic benzodiazepine, diazepam, as a positive control. DAIZAC treatment produced a significant dose-related increase in the time spent in the open arm. The increased total time in the open arm resulted from a significant dose-dependent increase in the number of entries into that arm. The minimum dose of DAIZAC associated with a statistically significant increase in entries and time spent in the open arm was 0.05 mg/kg ip, consistent with its high affinity for the 5-HT(3) receptor. DAIZAC did not affect the amount of time spent in the open arm after each entry. Thus, DAIZAC reduced apparent avoidance of the open arm when the animal was in the central compartment, without affecting active avoidance of that arm when the animal was in the exposed condition. The increase in the open-arm entries was accompanied by a corresponding reduction in the number of entries into the closed arm with a consequent reduction in the time spent in the closed arm. The time spent in the closed arm after each entry was not altered by DAIZAC administration. As such, the sole apparent effect of DAIZAC was to alter the choice of arm to enter when the animal was in the central compartment. Diazepam also significantly increased total time in the open arm; however, the increase was not attributable to a single behavioral factor. The anxiolytic-like effects of DAIZAC reached maximum by 20-30 min and returned to baseline levels by 90 min. Ex vivo binding studies found that levels of DAIZAC-like activity assayed in brains of mice 25 min after DAIZAC injection were significantly correlated with the behavioral parameters associated with anxiolysis. These results indicate that DAIZAC produces dose-dependent anxiolytic-like behavioral changes in the mouse elevated plus-maze that are correlated with brain DAIZAC-like activity.

Characterization of (S)-des-4-amino-3-[125I]iodozacopride ([125I]DAIZAC), a selective high-affinity radioligand for 5-hydroxytryptamine3 receptors.

The 5-hydroxytryptamine(HT)3 receptor subtype is present in the central nervous system (CNS) in low abundance, and few selective radiolabeled antagonists with high specific activity are available to study these sites. DAIZAC [desamino-3-iodo-(S)-zacopride; (S)-5-chloro-3-iodo-2-methoxy-N-(1-azobicyclo-[2.2. 2]oct-3-yl)benzamide] is a compound with high affinity and selectivity for the 5-HT3 receptor. Scatchard analysis of specific binding to NCB-20 cell membranes gave a Bmax of 340 +/- 58 fmol/mg protein and a KD of 0.14 +/- 0.03 nM, which is in agreement with the value previously reported in rat brain (KD = 0.15 nM). Nonspecific binding of [125I]DAIZAC in NCB-20 cells was <1% of total binding at the KD for DAIZAC compared with 17% in the rat brain preparation. Unlabeled DAIZAC (10 microM) showed minimal ability to displace binding of radiolabeled ligands selected for their affinities for other CNS receptor and uptake carrier binding sites. The discrimination ratio of DAIZAC for the 5-HT3 receptor over the M1 muscarinic binding site, the non-5-HT3 site at which it was most potent, was >2800. Serotonergic antagonists at every other known CNS serotonergic binding sites (3-30 microM) were ineffective in displacing [125I]DAIZAC binding in rat brain membranes. Similarly, antagonists (3-30 microM) for other nonserotonergic receptors and uptake sites were ineffective in displacing [125I]DAIZAC binding. Autoradiographic studies showed highest specific binding in area postrema and nucleus solitarius, with intermediate levels of binding in entorhinal cortex and hippocampus. DAIZAC inhibited 5-HT3 receptor-mediated inward cation current in NCB-20 cells with an IC50 of 0.24 nM. [125I]DAIZAC is a potent and highly selective ligand for in vitro studies of the 5-HT3 receptor.

Characterization of desamino-5-[125I]iodo-3-methoxy-zacopride ([125I]MIZAC) binding to 5-HT3 receptors in the rat brain.

1. Antagonists at 5-HT3 receptors have shown activity in animal models of mental illness, however, few radiolabeled 5-HT3 ligands are available for preclinical studies. MIZAC, an analogue of the selective 5-HT3 antagonist, zacopride, binds with high affinity (1.3-1.5 nM) to CNS 5-HT3 sites. The authors report here the selectivity of MIZAC for these sites in rat brain homogenates. 2. Ninety-seven percent of total specific binding of [125I]MIZAC (0.1 nM) of was displaced by bemesetron (3 microM), a selective 5-HT3 antagonist. Competition studies using ligands with known affinities for 5-HT3 sites give a high correlation with reported pKi values (r2 0.98). Bemesetron displaceable binding has a regional distribution consistent with that of the 5-HT3 receptor, i.e. highest in cortex and hippocampus, and lowest in striatum and cerebellum. 3. Potent antagonists present at concentrations sufficient to occupy 95% of other 5-HT receptor populations (1A, 1B, 1D, 2A, 2B, 2C, 5A, 5B, 6, and 7) showed minimal ability to displace [125I]MIZAC binding (3 nM). Specificity studies using radioligand binding assays selective for 5-HT4, 5-HT6, and 5-HT7 receptors, and for binding sites of other neurotransmitters indicate a high degree of selectivity of [125I]MIZAC for the 5-HT3 receptor. 4. [125I]MIZAC binds to an apparent low affinity (benzac) site having a unique pharmacology. Low affinity binding was displaceable by benztropine, but not by other muscarinic agents nor inhibitors of dopamine uptake. The regional distribution of the low affinity site differed markedly from that of the high affinity site. The apparent affinity of [125I]MIZAC for the benzac site is two orders of magnitude lower than for the 5-HT3 receptor. Given its high selectivity for 5-HT3 binding sites, [125I]MIZAC appears to be a promising ligand for labeling 5-HT3 receptors in vitro and in vivo.

Synthesis and 5-HT-3 receptor binding of 2- and 3-(halo)alkoxyl substituted (S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-5-chlorobenzamides as potential radioligands.

In an effort to develop selective, high-affinity radioligands for the 5-HT-3 receptor, a series of homologues of 5-chloro-2,3-dimethoxy-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide (2b) was prepared in which individual methoxy groups were replaced by ethoxyl, (2-fluoroethoxyl), allyloxyl, propargyloxyl, or (3-iodoallyl)oxyl groups. Affinities for the 5-HT-3 receptor were determined by displacement of the binding of [125I]MIZAC (2a), a selective 5-HT-3 receptor antagonist radioligand, in rat brain homogenates. The 3-substituted homologues were more potent than the lead compound, 2b. The homologue having the largest 3-substituent, i.e., E-(S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-5-chloro-3-(3-iodo-2-propenyl)oxy- 2-methoxybenzamide (3b, THIZAC), had one of the highest affinities, Ki 0.08 nM. The 2-substituted homologues were equipotent with 2b, having Ki 0.2-0.3 nM, regardless of the size of the substituent. The corresponding iodoallyl derivative, E-(S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-5-chloro-2-(3-iodo-2-propenyl)oxy- 3-methoxybenzamide (4, LIZAC), displayed a Ki of 0.29 nM. Saturation binding of [125I]-4 gave a KD of 0.31 +/- 0.04 nM and a Bmax of 2.36 +/- 0.10 fmol/mg of entorhinal cortex. In vivo biodistribution of [125I]-4 in the rat brain showed increased accumulation in hippocampus relative to that in cerebellum. Both the high-affinity ligands [125I]-3b and [125I]-4 are potentially useful radioligands for studying the 5-HT-3 receptor.

Synthesis and radiolabeling of (S)-4-amino-5-iodo-2-methoxy-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide, the active enantiomer of [125I]iodozacopride, and re-evaluation of its 5-HT3 receptor affinity.

We report an improved synthesis of unlabeled (S)-iodozacopride, the radiolabeling of (S)-[125I]iodozacopride via deschloro-(S)-zacopride, and a re-evaluation of its affinity for the 5-HT3 receptor. Unlabeled (S)-iodozacopride was prepared in seven steps from 4-aminosalicylic acid via alkaline hydrolysis of its 4-acetamide derivative. Catalytic hydrogenation of (S)-iodozacopride gave deschloro-(S)-zacopride, identical to that obtained from (S)-3-amino-quinuclidine and 4-amino-2-methoxybenzoic acid via its corresponding 1-imidazole derivative. Radioiodination to produce (S)-[125I]iodozacopride was accomplished by treatment of deschloro-(S)-zacopride with 5 mCi sodium 125iodide and chloramine-T in hydrochloric acid. Purification of the reaction products using an HPLC system capable of detecting chlorinated side-products revealed a mixture of 2.1 mCi (1.3 nmol) (S)-[125I]iodozacopride and (S)-zacopride (1.5 nmol). Saturation analysis of the binding of the purified (S)-[125I]iodozacopride to whole rat brain homogenates gave an estimated KD of 1.10 +/- 0.07 nM. As anticipated, this is approximately half the KD reported for binding of racemic [125I]iodozacopride, and differs from the previously reported value by an order of magnitude. Analysis of the apparent binding affinity of a 1:1 mixture of (S)-[125I]iodozacopride and (S)-zacopride suggests that the previous result may have been confounded by contamination of the product with unlabeled (S)-zacopride. Competition analysis of the displacement of (S)-[125I]iodozacopride binding by unlabeled (S)-iodozacopride and (S)-zacopride gave Ki values of 0.95 and 0.21 nM, respectively.

Clomipramine, clonazepam, and clonidine treatment of obsessive-compulsive disorder.

Serotonergic reuptake inhibitors have been the primary medications for treatment of obsessive-compulsive disorder (OCD); however, other serotonergic and alpha 2-adrenergic medications also have been reported to reduce obsessive-compulsive symptoms. In this study, we compare three medications with reported efficacy in OCD to a control medication, diphenhydramine, a medication without theoretical or demonstrated treatment benefit. The three active medications were clomipramine, a serotonergic reuptake inhibitor; clonazepam, a benezodiazepine with putative serotonergic properties; and clonidine, an alpha 2-adrenergic agonist. Twenty-eight subjects with DSM-III-R diagnosis of OCD rotated through 6-week trials of each of the four medications in a randomized, double-blind, multiple crossover protocol. Clomipramine and clonazepam were both effective relative to the control medication in reducing OCD symptoms. There was a significant cross-response between these two medications; however 40% of subjects failing clomipramine trials had a clinically significant response to clonazepam treatment. The control medication, diphenhydramine, itself produced a significant decrement in symptoms, whereas clonidine was ineffective in reducing OCD symptoms. Clonazepam improvement was unrelated to changes in anxiety and occurred early in treatment. Clonazepam was significantly more effective than the other medications during the first 3 weeks of treatment. The results confirm the efficacy of clomipramine in the treatment of OCD and suggest that clonazepam might be a useful alternative treatment for patients with this disorder.

Fenfluramine stimulation of prolactin in obsessive-compulsive disorder.

The success of serotonergic reuptake inhibitors in the treatment of obsessive-compulsive disorder (OCD) has suggested that serotonergic neurotransmission may play a role in the pathogenisis of this disorder. Prolactin responses to a 60-mg oral dose of fenfluramine in 26 medication-free patients with a DSM-III-R diagnosis of OCD were compared with those of 20 controls subjects. Fenfluramine produced a significant elevation of prolactin levels in both OCD patients and controls. Prolactin responses were significantly blunted in OCD patients compared with responses in control subjects. Female subjects in both groups showed greater prolactin responses to fenfluramine than did their male counterparts. There was a significant interaction between sex and the presence of OCD such that female patients had lower prolactin responses than their controls, while the difference between male patients and controls was not significant. Prolactin responses were not correlated with age, weight, drug level, depression, anxiety, or degree of OCD symptoms. The results are consistent with a relative reduction in serotonergic efficacy in the setting of OCD.

Fluoxetine for trichotillomania: an open clinical trial.

Of 17 adult patients with long-standing trichotillomania, 13 completed an 8- to 12-week open trial of fluoxetine, up to 80 mg per day. No patient had obsessive-compulsive disorder or major depression. We used the compulsions subscale of the Yale-Brown Obsessive-Compulsive Scale (YBOCS) to rate patients' hair-pulling behavior. The 13 completing patients' mean YBOCS score decreased significantly from 10.15 at baseline to 5.92 at the completion visit (Student's paired t = 4.82, df = 12, two-tailed p less than .001). Of these 13 patients, 5 experienced a 50 percent or greater decrease in their pulling behavior as measured by the YBOCS; 4 experienced between a 25 percent and 50 percent decrease. Three of the patients stopped pulling entirely, as did 2 of the 4 noncompleting patients. Three noncompleting patients discontinued treatment because of side effects, and 1 insisted on early use of behavior therapy. Comparative treatment trials elucidating the indications, risks, and expectable benefits of psychotherapeutic and pharmacological treatments are needed.

Clonazepam treatment of obsessions and compulsions.

Obsessive compulsive disorder has recently been successfully treated with antidepressants that selectively inhibit serotonin reuptake, and a serotonergic hypothesis related to the etiology and treatment of obsessive compulsive disorder has been proposed. Clonazepam, a novel benzodiazepine, uniquely affects serotonergic neurotransmission. It has been employed in the treatment of other neuropsychiatric syndromes that respond to serotonergic medications. Three patients with obsessive compulsive disorder who were treated with clonazepam for periods of up to 1 year experienced substantial improvement in their symptoms. Clonazepam had a rapid onset of antiobsessive action with accompanying decreases in both depression and anxiety. One patient showed reductions in obsessions and compulsions that were equivalent to or greater than those seen during previous treatment with clomipramine: Clonazepam may be a useful alternative to serotonergic antidepressants in patients who cannot tolerate the toxic effects of these medications.

Isolation of enzyme cDNA clones by enzyme immunodetection assay: isolation of a peptide acetyltransferase.

The biological activity of many proteins and peptides can be profoundly affected by enzyme-catalyzed covalent modifications such as acetylation, sulfation, glycosylation, or amidation. This article describes the cloning of such an enzyme, a peptide acetyltransferase from rat brain that catalyzes the amino-terminal acetylation of endorphins and perhaps other substrates in vivo. Blot-hybridization analysis suggests that the mRNA encoding the acetyltransferase is approximately 2.0 kilobases, is present in whole rat brain and rat hypothalamus, and is slightly larger in mouse AtT20 tumor cells. The acetyltransferase was cloned by using a strategy whereby a cDNA expression library was screened with a solid-phase enzyme-activity assay; this technique combines the use of the substrate coupled to a solid support and subsequent recognition of the product by using a specific antiserum. We have called this method the enzyme immunodetection assay (EIDA). The EIDA should prove useful in the isolation of other clones for proteins that possess enzymatic activity upon expression in bacterial hosts.

Regional interactions of opioid peptides at mu and delta sites in rat brain.

Opiate binding sites in five brain regions were labeled with the mu and delta markers, 3H-morphine and 3H-[D-Ala2,D-leu5]enkephalin, respectively. The highest densities of both 3H-morphine and 3H-DADLE labeled sites are found in striatum and frontal cortex. Hypothalamus and midbrain contain predominantly 3H-morphine labeled sites. The selectivity of the opioid peptides [D-Ala2,D-leu5]enkephalin, beta-endorphin and dynorphin(1-13) for the two opiate sites was investigated by comparing the potency of these unlabeled compounds against the mu and delta markers in different brain regions. This determination has the effect of controlling for the breakdown of peptides within each region. While the enkephalin analogue shows a preference for the delta binding site and beta-endorphin is more nearly equipotent towards the two binding sites, dynorphin(1-13) shows a high affinity and selective preference for the mu binding site over the delta site. The potency of the opioid peptides in displacing the mu and delta markers varies from region to region according to the relative densities of the two opiate binding site populations.

Binding of 3H-beta-endorphin to rat brain membranes: characterization of opiate properties and interaction with ACTH.

The binding of tritiated beta-endorphin (3H-beta-EP) to brain homogenates is described. This has been difficult to achieve due to the lack of availability of 3H-beta-EP and to technical difficulties associated with high non-specific binding of beta-EP. We now report that 3H-beta-EP binding is saturable, stereospecific, has high affinity and is inhibited by sodium. Its dissociation rate is ten-fold longer than that of naloxone. Its regional distribution exhibits interesting differences from naloxone and enkephalin binding. ACTH1-24 appears to displace it more effectively than it displaces 3H-naloxone. The results are discussed in terms of multiple transmitter systems and the multiple opiate receptor hypothesis.