Structure-toxicity relationships of saturated and unsaturated free fatty acids for elucidating the lipotoxic effects in human EndoC-ßH1 beta-cells.

Lipotoxicity has been considered a major cause for beta-cell dysfunction in type 2 diabetes mellitus. However, the underlying mechanisms are still unclear. To achieve a better understanding of the toxicity a wide range of structurally different free fatty acids (FFAs) has been analyzed in human EndoC-ßH1 beta-cells. Exposure of human EndoC-ßH1 beta-cells to physiological saturated and monounsaturated long-chain FFAs induced apoptosis. Particularly noteworthy was that the toxicity increased more rapidly with increasing chain length of saturated than of unsaturated FFAs. The highest toxicity was observed in the presence of very long-chain FFAs (C20-C22), whereas polyunsaturated FFAs were not toxic. Long-chain FFAs increased peroxisomal hydrogen peroxide generation slightly, while very long-chain FFAs increased hydrogen peroxide generation more potently in both peroxisomes and mitochondria. The greater toxicity of very long-chain FFAs was accompanied by hydroxyl radical formation, along with cardiolipin peroxidation and ATP depletion. Intriguingly, only saturated very long-chain FFAs activated ER stress. On the other hand saturated very long-chain FFAs did not induce lipid droplet formation in contrast to long-chain FFAs and unsaturated very long-chain FFAs. The present data highlight the importance of structure-activity relationship analyses for the understanding of the mechanisms of lipotoxicity. Chain length and degree of saturation of FFAs are crucial factors for the toxicity of FFAs, with peroxisomal, mitochondrial, and ER stress representing the major pathogenic factors for induction of lipotoxicity. The results might provide a guide for the composition of a healthy beta-cell protective diet.

Safety and efficacy of home intravenous antimicrobial infusion therapy in older patients: a comparative study with younger patients.

BACKGROUND: Home intravenous antimicrobial infusion therapy has proved its safety and efficacy in a great number of infections. Despite this there are few published studies about this way of managing in the elderly patient. OBJECTIVE: To study the safety and efficacy of home intravenous antimicrobial infusion therapy in elderly patients. STUDY DESIGN: A prospective and comparative study of an elderly group of patients > or =70 years old vs. a cohort of younger adult patients as a control group. All patients were followed until 3 months after discharge. SETTING: Hospital at Home Programme (HHP) as part of the Internal Medicine Department at Valle de Hebrón Hospital, Barcelona, Spain. PATIENTS: All patients admitted to HHP diagnosed of infections requiring intravenous antibiotic therapy between March 2006 and March 2007. RESULTS: We included 145 patients, 90 of whom were 70 years or older. Diabetes mellitus, heart failure and respiratory tract infection were more frequent in these elderly patients. In this group 14 (12%) developed some type of adverse event during treatment, phlebitis being the most common. The majority of those in the elderly patients group were discharged because of satisfactory clinical evolution and only 7 (7%) were re-admitted to hospital. Another 13 (14%) were re-admitted to hospital 3 months after discharge from HHP, mostly for chronic diseases. There were no significant differences between these results and those obtained from the control group. CONCLUSION: Home intravenous antimicrobial infusion therapy in elderly patients is safe and effective.

Processing of urushiol (poison ivy) hapten by both endogenous and exogenous pathways for presentation to T cells in vitro.

The antigen processing requirements for urushiol, the immunogen of poison ivy (Toxicodendron radicans), were tested by presentation of urushiol to cultured human urushiol-responsive T cells. Urushiol was added to antigen-presenting cells (APC) either before or after fixation with paraformaldehyde. Three distinct routes of antigen processing were detected. CD8+ and CD4+ T cells, which were dependent upon processing, proliferated if urushiol was added to APC before fixation, but did not proliferate when urushiol was added to APC after fixation. Processing of urushiol for presentation to CD8+ T cells was inhibited by azide, monensin, and brefeldin A. This suggests that urushiol was processed by the endogenous pathway. In contrast, presentation of urushiol to CD4+ T cells was inhibited by monensin but not by brefeldin A. This was compatible with antigen processing by the endosomal (exogenous) pathway. Finally, certain CD8+ T cells recognized urushiol in the absence of processing. These cells proliferated in response to APC incubated with urushiol after fixation. Classification of contact allergens by antigen processing pathway may predict the relative roles of CD4+ and CD8+ cells in the immunopathogensis of allergic contact dermatitis.

The monounsaturated fatty acid oleate is the major physiological toxic free fatty acid for human beta cells.

Free fatty acids (FFAs) can cause glucose intolerance and diabetes. Lipotoxicity to the pancreatic beta cells is considered to be a major underlying cause for this phenomenon. The aim of this study was to analyse the toxicity profile of FFAs in the human EndoC-ßH1 beta-cell line and to compare the results with isolated rat and human islets with special reference to the physiologically most prevalent FFAs palmitic acid (PA) and oleic acid (OA). Toxicity after a 2-day incubation with the different FFAs was analysed by the caspase-3 assay and confirmed by the propidium iodide and annexin V staining tests. The long-chain saturated PA (C16:0) and the monounsaturated OA (C18:1) were both toxic to human EndoC-ßH1 beta cells and pseudoislets, as well as to rat islets, and, as confirmed in a pilot experiment, also to human islets. Furthermore, OA provided no protection against the toxicity of PA. Likewise, elaidic acid (EA, the trans isomer of OA; trans-OA) was significantly toxic, in contrast to the non-metabolisable analogues methylated PA (MePA) and methylated OA (MeOA). Fatty acids with a chain length < C16 were not toxic in EndoC-ßH1 beta cells. Caspase-3 was also activated by linoleic acid (LA)(C18:2) but not by γ-linolenic acid (γ-LNA)(C18:3). Overall, only long-chain FFAs with chain lengths > C14, which generate hydrogen peroxide in the peroxisomal beta-oxidation, were toxic. This conclusion is also supported by the toxicity of the branched-chain FFA pristanic acid, which is exclusively metabolised in the peroxisomal beta-oxidation. The lack of a protective effect of the monounsaturated fatty acid OA has important consequences for a beta-cell protective lipid composition of a diet. A cardioprotective diet with a high OA content does not fulfil this requirement.

Factors associated with tuberculosis at AIDS diagnosis in France.

OBJECTIVE: To identify factors associated with tuberculosis (TB) at AIDS diagnosis in France. DESIGN: Analysis of surveillance data. METHODS: Among all adult AIDS cases diagnosed since January 1988 and reported by December 1993 in France, the proportion diagnosed with AIDS-defining TB (extrapulmonary TB among cases diagnosed between 1988 and 1992, all forms of TB among cases diagnosed in 1993) was analysed by year of diagnosis, sex, age, nationality, profession, HIV transmission group and region of residence by multiple logistic regression. RESULTS: Between 1988 and 1992, 5.7% (1134 out of 19,968) of AIDS patients were diagnosed with AIDS-defining extrapulmonary TB. Presence of extrapulmonary TB was associated with male sex [adjusted odds ratio (AOR), 1.7], nationality from a sub-Saharan country (AOR, 4.8), heterosexual contact or injecting drug use (AOR, 2.4 and 2.7, respectively), residence in the Paris area (AOR, 1.7), and unemployment or factory work (AOR, 2.5 and 2.4, respectively). In 1993, 10.6% (393 out of 3721) of AIDS patients were diagnosed with TB (all forms). In multivariate analysis, three factors were independently associated with the risk of presenting TB at AIDS diagnosis: transmission category, nationality, and region of residence. CONCLUSIONS: Some factors associated with TB at AIDS diagnosis in France are known to be related to a high incidence of TB in industrialized countries (nationality, from a developing country, male sex, low socioeconomic status). The independent association with injecting drug use or residence in Paris suggests a contribution of recent TB infection in specific groups of HIV-infected persons. This contribution should be evaluated to implement appropriate preventive measures.

Differential effects of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) on various 5-HT receptor binding sites in the rat brain.

The effects of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), an alkylating agent producing irreversible blockade of various membrane bound receptors in brain, were investigated on four different types of serotonin receptors, 5-HT1A, 5-HT1B, 5-HT2A and 5-HT3, in various brain regions in the rat. In addition, the fate of central benzodiazepine- and "R"-zacopride-specific binding sites was also examined in rats treated with EEDQ. Membrane binding assays and/or quantitative autoradiography with appropriate radioligands indicated that EEDQ inactivated 5-HT1A, 5-HT1B and 5-HT2A sites, but was poorly active on 5-HT3, benzodiazepine and "R" sites. Among the receptors affected by EEDQ, hippocampal 5-HT1A sites were the most sensitive to the alkylating agent (ID50 approximately 1 mg/kg i.p.), followed by the cortical 5-HT2A (ID50 approximately 3 mg/kg i.p.) and the striatal 5-HT1B (ID50 approximately 6 mg/kg i.p.) sites. Pretreatment by selective ligands partially protected hippocampal 5-HT1A sites from irreversible inactivation by EEDQ (10 mg/kg i.p.) with the following order of efficacy: WAY 100635 > spiperone > BMY 7378 > ipsapirone. Similarly, pretreatment by spiperone (5 mg/kg i.p.) also reduced the ability of EEDQ to inactivated cortical 5-HT2A receptors. Analyses of the time-course recovery of respective binding sites after EEDQ administration showed that the turnover rate of 5-HT1A sites did not significantly differ in the dorsal raphe nucleus and in various forebrain areas (hippocampus, septum, cerebral cortex; half-life: approximately 4 days), but was lower than that of cortical 5-HT2A sites (half-life: 2.9 days).

Differential adaptation of brain 5-HT1A and 5-HT1B receptors and 5-HT transporter in rats treated chronically with fluoxetine.

Quantification of receptor binding sites and their encoding mRNAs, and electrophysiological recordings, were used to assess central serotonin (5-HT) neurotransmission in rats 24 h after a 2-3 week treatment with the selective 5-HT reuptake inhibitor fluoxetine (8 mg/kg i.p., daily). Binding studies showed that this treatment affected neither 5-HT1A nor 5-HT1B binding sites in all brain areas examined. However, a significant decrease (-38%) in 5-HT1A mRNA levels in the anterior raphe area (but not forebrain regions) and increases in 5-HT1B mRNA levels in the striatum (+127%) and the cerebral cortex (+34%) were noted in fluoxetine-treated rats. Electrophysiological recordings in brain slices showed that chronic fluoxetine treatment reduced the potency of the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin to inhibit neuronal activity in the dorsal raphe nucleus, but did not affect 5-HT1A-evoked responses of CA1 pyramidal cells in the hippocampus. These data further demonstrate that fluoxetine-induced adaptive changes in 5-HT neurotransmission exhibit marked regional differences. The decrease in 5-HT1A mRNA levels in the anterior raphe suggests that fluoxetine-induced desensitization of 5-HT1A autoreceptors involves changes at the transcription level.

Rapid eye movement sleep induction by vasoactive intestinal peptide infused into the oral pontine tegmentum of the rat may involve muscarinic receptors.

In rats, rapid eye movement sleep can be induced by microinjection of either the cholinergic agonist carbachol or the neuropeptide vasoactive intestinal peptide into the oral pontine reticular nucleus. Possible involvement of cholinergic mechanisms in the effect of vasoactive intestinal peptide was investigated using muscarinic receptor ligands. Sleep-waking cycles were analysed after infusion into the oral pontine reticular nucleus of vasoactive intestinal peptide (10 ng in 0.1 microl), carbachol (20 ng), atropine (200 ng) and pirenzepine (50, 100 ng), performed separately or in combination at 15-min intervals. The increase in rapid eye movement sleep due to the combined infusion of vasoactive intestinal peptide and carbachol (+58.7+/-4.6% for 8 h, P<0.05) was not significantly different from that induced by each compound separately. The enhancement of rapid eye movement sleep by vasoactive intestinal peptide was totally prevented by infusion of atropine, but not pirenzepine, a relatively selective M1 antagonist. On their own, none of the latter two compounds affected the sleep-waking cycle. Quantitative autoradiographic studies using [3H]quinuclidinyl benzylate (1 nM) and pirenzepine (0.5 microM) indicated that muscarinic receptors correspond to pirenzepine-insensitive binding sites in the oral pontine reticular nucleus. In vitro, vasoactive intestinal peptide (1-100 nM) significantly increased (+30-40%) the specific binding of [3H]quinuclidinyl benzylate to the oral pontine reticular nucleus in rat brain sections. This effect appeared to be due to an increased density, with no change in affinity, of pirenzepine-insensitive binding sites in this area. These data suggest that pirenzepine-insensitive muscarinic binding sites are involved in the induction of rapid eye movement sleep by vasoactive intestinal peptide at the pontine level in the rat.

Differential effects of stress on presynaptic and postsynaptic 5-hydroxytryptamine-1A receptors in the rat brain: an in vitro electrophysiological study.

Extracellular and intracellular recording techniques were used to assess possible changes in the functional properties of 5-hydroxytryptamine-1A receptors in brain slices prepared from rats subjected to different stress paradigms. Whereas a 30-min restraint stress did not alter the inhibitory influence of ipsapirone on the firing of serotoninergic neurons in the dorsal raphe nucleus, the same session followed by a 24-h isolation produced a significant decrease in the potency of the 5-hydroxytryptamine-1A agonist to inhibit the electrical activity of these cells. Similarly, exposure of the animals to novel uncontrolled environmental conditions for 16 h significantly reduced the potency of ipsapirone to decrease the firing rate of serotoninergic neurons in brain stem slices. The effects of the latter two stressful paradigms were observed in slices from intact rats, but not in those from adrenalectomized animals. Intracellular recording showed that exposure of the animals to novel uncontrolled environmental conditions markedly reduced the potency of 5-carboxamidotryptamine to hyperpolarize serotoninergic neurons in the dorsal raphe nucleus and to decrease the input resistance of their plasma membrane. In contrast, the same stressful paradigm exerted no significant influence on the membrane effects of this 5-hydroxytryptamine-1A agonist on pyramidal cells in the CA1 hippocampal area. These data show that, like the direct application of corticosterone on to brain slices [Laaris N. et al. (1995) Neuropharmacology 34, 1201-1210], the stress-induced in vivo elevation of serum levels of endogenous corticosterone is associated with desensitization of somatodendritic 5-hydroxytryptamine-1A receptors in the dorsal raphe nucleus. The differential changes in 5-hydroxytryptamine-1A receptor sensitivity due to stress in the latter area versus the hippocampus further support the idea that somatodendritic and postsynaptic 5-hydroxytryptamine-1A receptors are regulated differently in the rat brain.

Baroreceptor reflex inhibition induced by the stimulation of serotonin3 receptors in the nucleus tractus solitarius of the rat.

Previous studies suggested that in the nucleus tractus solitarius, cardiovascular responses to serotonin may involve the simultaneous activation of more than one receptor subtype. In the present study, the cardiovascular effects of the local application of serotonin and different serotonin3 agonists and antagonists into the nucleus tractus solitarius were analysed in intact and unilaterally ganglionectomized rats. Unilateral injections of serotonin (5-15 nmol) produced a dose-dependent increase in blood pressure and partially antagonized the arterial baroreflex responses evoked by an i.v. injection of phenylephrine. Similar blood pressures response were obtained after unilateral microinjections of phenylbiguanide (5 nmol) and 2-methyl-serotonin (5 nmol), two serotonin3 receptor agonists. Bilateral microinjections of serotonin or phenylbiguanide produced more pronounced blood pressure effects and antagonized completely the baroreflex responses. Both blood pressure and baroreflex effects were antagonized by prior injections of specific serotonin3 antagonists such as zacopride (100 pmol) and ondansetron (100 pmol). Concomitant autoradiographic studies performed in intact and ganglionectomized rats, using [125I]iodozacopride, confirmed that serotonin3 receptors in the nucleus tractus solitarius are mainly located on vagal afferent fibers. In addition, serotonin microinjections made in the nucleus tractus solitarius ipsilateral to the ganglionectomy revealed a significant reduction in cardiovascular responses compared to intact animals. These results suggest that in the nucleus tractus solitarius of the rat, serotonin is involved in the reflex regulation of blood pressure through the stimulation of serotonin3 receptors presumably located on vagal afferent fibers. Since bicuculline antagonized the serotonin-mediated pressor responses, a serotonin3-dependent activation of an inhibitory GABAergic system within the nucleus tractus solitarius might be involved in blood pressure regulatory mechanisms.

Antagonist properties of (-)-pindolol and WAY 100635 at somatodendritic and postsynaptic 5-HT1A receptors in the rat brain.

1. The aim of the present work was to characterize the 5-hydroxytryptamine1A (5-HT1A) antagonistic actions of (-)-pindolol and WAY 100635 (N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexane carboxamide). Studies were performed on 5-HT1A receptors located on 5-hydroxytryptaminergic neurones in the dorsal raphe nucleus (DRN) and on pyramidal cells in the CA1 and CA3 regions of the hippocampus in rat brain slices. 2. Intracellular electrophysiological recording of CA1 pyramidal cells and 5-hydroxytryptaminergic DRN neurones showed that the 5-HT1A receptor agonist 5-carboxamidotryptamine (5-CT) evoked in both cell types a concentration-dependent cell membrane hyperpolarization and a decrease in cell input resistance. On its own, (-)-pindolol did not modify the cell membrane potential and resistance at concentrations up to 10 microM, but it antagonized the 5-CT effects in a concentration-dependent manner. Similar antagonism of 5-CT effects was observed in the CA3 hippocampal region. (-)-Pindolol also prevented the 5-HT1A receptor-mediated hyperpolarization of CA1 pyramidal cells due to 5-HT (15 microM). In contrast, the 5-HT-induced depolarization mediated by presumed 5-HT4 receptors persisted in the presence of 3 microM (-)-pindolol. 3. In the hippocampus, (-)-pindolol completely prevented the hyperpolarization of CA1 pyramidal cells by 100 nM 5-CT (IC50=92 nM; apparent KB=20.1 nM), and of CA3 neurones by 300 nM 5-CT (IC50=522 nM; apparent KB= 115.1 nM). The block by (-)-pindolol was surmounted by increasing the concentration of 5-CT, indicating a reversible and competitive antagonistic action. 4. Extracellular recording of the firing rate of 5-hydroxytryptaminergic neurones in the DRN showed that (-)-pindolol blocked, in a concentration-dependent manner, the decrease in firing elicited by 100 nM 5-CT (IC50=598 nM; apparent KB= 131.7 nM) or 100 nM ipsapirone (IC50= 132.5 nM; apparent KB= 124.9 nM). The effect of (-)-pindolol was surmountable by increasing the concentration of the agonist. Intracellular recording experiments showed that 10 microM (-)-pindolol were required to antagonize completely the hyperpolarizing effect of 100 nM 5-CT. 5. In vivo labelling of brain 5-HT1A receptors by i.v. administration of [3H]-WAY 100635 ([O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1 -piperazinyl)ethyl-N-(2-pyridyl)cyclo-hexane-carboxamide) was used to assess their occupancy following in vivo treatment with (-)-pindolol. (-)-Pindolol (15 mg kg[-1]) injected i.p. either subchronically (2 day-treatment before i.v. injection of [3H]-WAY 100635) or acutely (20 min before i.v. injection of [3H]-WAY 100635) markedly reduced [3H]-WAY 100635 accumulation in all 5-HT1A receptor-containing brain areas. In particular, no differences were observed in the capacity of (-)-pindolol to prevent [3H]-WAY 100635 accumulation in the DRN and the CAI and CA3 hippocampal areas. 6. Intracellular electrophysiological recording of 5-hydroxytryptaminergic DRN neurones showed that WAY 100635 prevented the hyperpolarizing effect of 100 nM 5-CT in a concentration-dependent manner (IC50=4.9 nM, apparent KB=0.25 nM). In CA1 pyramidal cells, hyperpolarization induced by 50 nM 5-CT was also antagonized by WAY 100635 (IC50 = 0.80 nM, apparent KB= 0.28 nM).

Autoradiographic mapping of serotonin 5-HT1A, 5-HT1D, 5-HT2A and 5-HT3 receptors in the aged human spinal cord.

Quantitative autoradiography with selective radioligands was used to establish the respective distribution of serotonin 5-HT1A, 5-HT1D, 5-HT2A and 5-HT3 receptors at the cervical, thoracic and lumbar levels of the spinal cord from subjects who died at 81-94 years. A high density of 5-HT1A receptors, labeled by [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT), was found in the superficial layers of the dorsal horn, with a significant enrichment ( approximately 20%) in the lumbar vs. the thoracic and cervical segments. In contrast, only very low specific labeling by [3H]8-OH-DPAT (i.e. less than 10% of that measured in the dorsal horn), was detected in the ventral horn. 5-HT1D sites labeled by [125I]serotonin-O-carboxymethyl-glycyl-iodo-tyrosinamide ([125I]GTI) were also mainly located within the superficial layers of the dorsal horn, but no difference in their relative density was noted at the three levels of the spinal cord examined. 5-HT2A sites labeled by [3H]ketanserin were found in the dorsal horn of the cervical segments but no specific binding of this radioligand could be detected at any other level of the spinal cord of such aged subjects. Finally, a high density of [3H]S-zacopride-labeled 5-HT3 receptors was noted especially in the most superficial layer (lamina I) of the dorsal horn at all segments examined. These data provide anatomical support for a role of spinal serotonin especially in nociception processing.

Record-linkage between two anonymous databases for a capture-recapture estimation of underreporting of AIDS cases: France 1990-1993. The Clinical Epidemiology Group from Centres d'Information et de Soins de l'Immunodéficience Humaine.

OBJECTIVE: To estimate the completeness of the French mandatory AIDS surveillance system (Declaration Obligatoire DO) over the 1990-1993 period using a capture-recapture approach, by matching the mandatory reports with the AIDS cases present in the French Hospital Database on HIV infection (FHDH). METHODS: An anonymous record-linkage algorithm was developed to identify those cases common to both anonymous surveillance systems. The linkage was based on sex, date of birth, and infection risk group, all strictly matched, and on the dates of AIDS diagnosis and of death, the places of diagnosis and residence, and the AIDS-defining diseases at diagnosis. The total number of AIDS cases and completeness of both surveillance systems were estimated using a capture-recapture approach, assuming independence of the ascertainment sources. RESULTS: The completeness of the mandatory reporting was estimated at 83.6% (95% CI: 82.9-84.3), and that of the FHDH at 47.6% (95% CI: 46.9-48.3) for the surveillance of AIDS cases diagnosed among adults in France between 1990 and 1993. The completeness of the system based on FHDH increased over the study period as more hospitals joined the project, while the completeness of the DO surveillance system remained stable. CONCLUSION: This approach was useful in estimating the underreporting of AIDS cases in France. Regularly performed, it will allow the impact of underreporting to be monitored over time.

Microgram doses of diazepam do not induce proconvulsant or proconflict effects in rodents.

Cooper (1983, 1985) observed an inhibition of ambulation and fluid intake in rats following diazepam in the microgram dose range. The present study aimed at investigating the effects of microgram doses of this benzodiazepine on i) the threshold doses of either pentylenetetrazole, picrotoxin or bicuculline required to induce seizures in mice and ii) the suppression of lever pressing for food induced by the delivery of one electric footshock every ten presses in rats pretreated or not with isoniazid (64 mg/kg IP). Diazepam 4-32 micrograms/kg IP) neither reduced seizure threshold doses of either convulsant studied nor did this drug (16-128 micrograms/kg IP) reliably decrease the number of lever presses under the punishment schedule. The present study provides no further evidence for a dose-related biphasic effect of diazepam which could give new insight into the functioning of benzodiazepine-coupled brain processes.

Autoradiography of serotonin receptor subtypes in the central nervous system.

The autoradiographic technique is the most relevant approach for the visualization at the light microscope level of the different classes (5-HT(1A), 5-HT(1B), 5-HT(1C), 5-HT(1D), 5-HT(2) and 5-HT(3)) of receptors for the monoamine neurotransmitter serotonin (5-HT) in the central nervous system of mammals, including man. The only exception is the 5-HT(4) subtype for which no satisfactory radioligand has been developed to date. Quantitative estimates of receptor labelling can be achieved by measurements of optical density on autoradiographic films of brain sections incubated with specific radioligands. This review summarizes the most significant contributions of quantitative autoradiography to the current knowledge of the respective regional distributions and differential regulations of the various classes of central 5-HT receptors.

Localization of 5-HT3 receptors in the rat spinal cord: immunohistochemistry and in situ hybridization.

Specific antibodies raised against a fusion protein containing the amino acid sequence of the putative second intracellular loop of the cloned 5-HT3-A receptor subunit were used for the immunohistochemical visualization of 5-HT3 receptors in the rat spinal cord. A dense 5-HT3-like immunoreactivity was found in the superficial layers of the dorsal horn, which closely matched the labelling of 5-HT3 binding sites by [125I]iodo-zacopride. This immunostaining was markedly decreased following unilateral rhizotomy, consistently with a preferential location of 5-HT3 receptors on terminals of primary afferent fibres, and with the presence of 5-HT3 mRNA in dorsal root ganglia. However, a significant proportion of 5-HT3 receptors persisted after rhizotomy, and the corresponding mRNA was found in the dorsal horn of the spinal cord. 5-HT3 receptors are therefore also located on intrinsic neurones of the spinal cord.

Electrophysiological, biochemical, neurohormonal and behavioural studies with WAY-100635, a potent, selective and silent 5-HT1A receptor antagonist.

Although considerable progress has been made in characterising the 5-HT1A receptor using agonists, partial agonists or non-selective antagonists, further studies of 5-HT1A receptor function have been hindered by the lack of highly selective antagonists. The term 'silent' antagonist has been used for such compounds in order to distinguish them unequivocally from several 5-HT1A receptor partial agonists which were initially designated 'antagonists'. In this report we provide a comprehensive review of the biochemical, pharmacological and behavioural properties of the first potent, selective and silent 5-HT1A receptor antagonist, WAY-100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-N-(2- pyridinyl)cyclohexanecarboxamide trihydrochloride). WAY-100635 had an IC50 (displacement of specific [3H]8-OH-DPAT binding to 5-HT1A receptors in the rat hippocampus) of 1.35 nM and was > 100-fold selective for the 5-HT1A site relative to a range of other CNS receptors. [3H]WAY-100635 was also characterised as the first 5-HT1A antagonist radioligand, displaying the same regional distribution of binding sites as [3H]8-OH-DPAT in rat brain. As would be expected for the binding of an antagonist to a G-protein-coupled receptor, the Bmax of [3H]WAY-100635 specific binding was consistently 50-60% greater than that of the agonist radioligand, [3H]8-OH-DPAT. Mn2+, but not guanine nucleotides, inhibited [3H]WAY-100635-specific binding. [3H]WAY-100635 was also shown to bind selectively to brain 5-HT1A receptors in vivo, following intravenous administration to mice. In vitro electrophysiological studies demonstrated that WAY-100635 had no 5-HT1A receptor agonist actions, but dose-dependently blocked the effects of agonists at both the postsynaptic 5-HT1A receptor in the CA1 region of the hippocampus, and the somatodendritic 5-HT1A receptor located on dorsal raphe 5-HT neurones. In vivo, WAY-100635 also dose-dependently blocked the ability of 8-OH-DPAT to inhibit the firing of dorsal raphe 5-HT neurones, and to induce the '5-HT syndrome', hypothermia, hyperphagia and to elevate plasma ACTH levels. In the mouse light/dark box anxiety model, WAY-100635 induced anxiolytic-like effects. WAY-100635 had no intrinsic effect on cognition in the delayed-matching-to-position model of short-term memory in the rat, but reversed the disruptive effects of 8-OH-DPAT on motor motivational performance. These data clearly demonstrate that WAY-100635 is the first potent, selective and silent 5-HT1A receptor antagonist. Furthermore, [3H]WAY-100635 is the first antagonist radioligand to become available for 5-HT1A receptor binding studies both in vitro and in vivo. The positive effects of WAY-100635 in an anxiety model also indicate that a postsynaptic 5-HT1A receptor antagonist action may contribute to the anxiolytic properties of 5-HT1A receptor partial agonists.