Developmental Changes of Human Neural Progenitor Cells Grafted into the Ventricular System and Prefrontal Cortex of Mouse Brain in Utero.

The transplantation of neural progenitors into a host brain represents a useful tool to evaluate the involvement of cell-autonomous processes and host local cues in the regulation of neuronal differentiation during the development of the mammalian brain. Human brain development starts at the embryonic stages, in utero, with unique properties at its neotenic stages. We analyzed the engraftment and differentiation of human neuronal progenitor cells (hNPCs) transplanted in utero into the mouse brain. The influence of the environment was studied by transplanting human NPCs within the lateral ventricles (LV), compared with the prefrontal cortex (PFC) of immunocompetent mice. We developed a semi-automated method to accurately quantify the number of cell bodies and the distribution of neuronal projections among the different mouse brain structures, at 1 and 3 months post-transplantation (MPT). Our data show that human NPCs can differentiate between immature "juvenile" neurons and more mature pyramidal cells in a reproducible manner. Depending on the injection site, LV vs. PFC, specific fetal local environments could modify the synaptogenesis processes while maintaining human neoteny. The use of immunocompetent mice as host species allows us to investigate further neuropathological conditions making use of all of the engineered mouse models already available.

Genetic susceptibility to nicotine addiction: Advances and shortcomings in our understanding of the CHRNA5/A3/B4 gene cluster contribution.

Over the last decade, robust human genetic findings have been instrumental in elucidating the heritable basis of nicotine addiction (NA). They highlight coding and synonymous polymorphisms in a cluster on chromosome 15, encompassing the CHRNA5, CHRNA3 and CHRNB4 genes, coding for three subunits of the nicotinic acetylcholine receptor (nAChR). They have inspired an important number of preclinical studies, and will hopefully lead to the definition of novel drug targets for treating NA. Here, we review these candidate gene and genome-wide association studies (GWAS) and their direct implication in human brain function and NA-related phenotypes. We continue with a description of preclinical work in transgenic rodents that has led to a mechanistic understanding of several of the genetic hits. We also highlight important issues with regards to CHRNA3 and CHRNB4 where we are still lacking a dissection of their role in NA, including even in preclinical models. We further emphasize the use of human induced pluripotent stem cell-derived models for the analysis of synonymous and intronic variants on a human genomic background. Finally, we indicate potential avenues to further our understanding of the role of this human genetic variation. This article is part of the special issue on 'Contemporary Advances in Nicotine Neuropharmacology'.

A chimeric mouse model to study human iPSC-derived neurons: the case of a truncating SHANK3 mutation.

Using human induced pluripotent stem cells (iPSC), recent studies have shown that the events underlying autism spectrum disorders (ASD) can occur during neonatal development. We previously analyzed the iPSC-derived pyramidal cortical neurons of a subset of patients with ASD carrying de novo heterozygous mutations in postsynaptic SHANK3 protein, in culture. We reported altered spinogenesis of those neurons. The transplantation of human iPSC-derived neuronal precursors into mouse brain represents a novel option for in vivo analysis of mutations affecting the human brain. In this study, we transplanted the neuronal precursor cells (NPC) into the cortex of newborn mice to analyze their integration and maturation at early stages of development and studied axonal projections of transplanted human neurons into adult mouse brain. We then co-transplanted NPC from a control individual and from a patient carrying a de novo heterozygous SHANK3 mutation. We observed a reduction in cell soma size of selective neuronal categories and in axonal projections at 30 days post-transplantation. In contrast to previous in vitro studies, we did not observe any alteration in spinogenesis at this early age. The humanized chimeric mouse models offer the means to analyze ASD-associated mutations further and provide the opportunity to visualize phenotypes in vivo.

Altered spinogenesis in iPSC-derived cortical neurons from patients with autism carrying de novo SHANK3 mutations.

The synaptic protein SHANK3 encodes a multidomain scaffold protein expressed at the postsynaptic density of neuronal excitatory synapses. We previously identified de novo SHANK3 mutations in patients with autism spectrum disorders (ASD) and showed that SHANK3 represents one of the major genes for ASD. Here, we analyzed the pyramidal cortical neurons derived from induced pluripotent stem cells from four patients with ASD carrying SHANK3 de novo truncating mutations. At 40-45 days after the differentiation of neural stem cells, dendritic spines from pyramidal neurons presented variable morphologies: filopodia, thin, stubby and muschroom, as measured in 3D using GFP labeling and immunofluorescence. As compared to three controls, we observed a significant decrease in SHANK3 mRNA levels (less than 50% of controls) in correlation with a significant reduction in dendritic spine densities and whole spine and spine head volumes. These results, obtained through the analysis of de novo SHANK3 mutations in the patients' genomic background, provide further support for the presence of synaptic abnormalities in a subset of patients with ASD.

Induced pluripotent stem cells as a tool to study brain circuits in autism-related disorders.

The mammalian brain is a very complex organ containing an estimated 200 billion cells in humans. Therefore, studying human brain development has become very challenging given all the data that are available from different approaches, notably genetic studies.Recent pluripotent stem cell methods have given rise to the possibility of modeling neurodevelopmental diseases associated with genetic defects. Fibroblasts from patients have been reprogrammed into pluripotent stem cells to derive appropriate neuronal lineages. They specifically include different subtypes of cortical neurons that are at the core of human-specific cognitive abilities. The use of neurons derived from induced pluripotent stem cells (iPSC) has led to deciphering convergent and pleiotropic neuronal synaptic phenotypes found in neurodevelopmental disorders such as autism spectrum disorders (ASD) and their associated syndromes. In addition to these initial studies, remarkable progress has been made in the field of stem cells, with the major objective of reproducing the in vivo maturation steps of human neurons. Recently, several studies have demonstrated the ability of human progenitors to respond to guidance cues and signals in vivo that can direct neurons to their appropriate sites of differentiation where they become fully mature neurons.We provide a brief overview on research using human iPSC in ASD and associated syndromes and on the current understanding of new theories using the re-implantation of neural precursors in mouse brain.

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis (1). The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Contactin 4, -5 and -6 differentially regulate neuritogenesis while they display identical PTPRG binding sites.

The neural cell-adhesion molecules contactin 4, contactin 5 and contactin 6 are involved in brain development, and disruptions in contactin genes may confer increased risk for autism spectrum disorders (ASD). We describe a co-culture of rat cortical neurons and HEK293 cells overexpressing and delivering the secreted forms of rat contactin 4-6. We quantified their effects on the length and branching of neurites. Contactin 4-6 effects were different depending on the contactin member and duration of co-culture. At 4 days in culture, contactin 4 and -6 increased the length of neurites, while contactin 5 increased the number of roots. Up to 8 days in culture, contactin 6 progressively increased the length of neurites while contactin 5 was more efficient on neurite branching. We studied the molecular sites of interaction between human contactin 4, -5 or -6 and the human Protein Tyrosine Phosphatase Receptor Gamma (PTPRG), a contactin partner, by modeling their 3D structures. As compared to contactin 4, we observed differences in the Ig2 and Ig3 domains of contactin 5 and -6 with the appearance of an omega loop that could adopt three distinct conformations. However, interactive residues between human contactin 4-6 and PTPRG were strictly conserved. We did not observe any differences in PTPRG binding on contactin 5 and -6 either. Our data suggest that the differential contactin effects on neurite outgrowth do not result from distinct interactions with PTPRG. A better understanding of the contactin cellular properties should help elucidate their roles in ASD.

Contribution of α7 nicotinic receptor to airway epithelium dysfunction under nicotine exposure.

Loss or dysfunction of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to impairment of airway mucus transport and to chronic lung diseases resulting in progressive respiratory failure. Nicotinic acetylcholine receptors (nAChRs) bind nicotine and nicotine-derived nitrosamines and thus mediate many of the tobacco-related deleterious effects in the lung. Here we identify α7 nAChR as a key regulator of CFTR in the airways. The airway epithelium in α7 knockout mice is characterized by a higher transepithelial potential difference, an increase of amiloride-sensitive apical Na(+) absorption, a defective cAMP-dependent Cl(-) conductance, higher concentrations of Na(+), Cl(-), K(+), and Ca(2+) in secretions, and a decreased mucus transport, all relevant to a deficient CFTR activity. Moreover, prolonged nicotine exposure mimics the absence of α7 nAChR in mice or its inactivation in vitro in human airway epithelial cell cultures. The functional coupling of α7 nAChR to CFTR occurs through Ca(2+) entry and activation of adenylyl cyclases, protein kinase A, and PKC. α7 nAChR, CFTR, and adenylyl cyclase-1 are physically and functionally associated in a macromolecular complex within lipid rafts at the apical membrane of surface and glandular airway epithelium. This study establishes the potential role of α7 nAChR in the regulation of CFTR function and in the pathogenesis of smoking-related chronic lung diseases.

Alpha7-nicotinic receptors modulate nicotine-induced reinforcement and extracellular dopamine outflow in the mesolimbic system in mice.

RATIONALE: Nicotine is the main addictive component of tobacco and modifies brain function via its action on neuronal acetylcholine nicotinic receptors (nAChRs). The mesolimbic dopamine (DA) system, where neurons of the ventral tegmental area (VTA) project to the nucleus accumbens (ACb), is considered a core site for the processing of nicotine's reinforcing properties. However, the precise subtypes of nAChRs that mediate the rewarding properties of nicotine and that contribute to the development of addiction remain to be identified. OBJECTIVES: We investigated the role of the nAChRs containing the α7 nicotinic subunit (α7 nAChRs) in the reinforcing properties of nicotine within the VTA and in the nicotine-induced changes in ACb DA outflow in vivo. METHODS: We performed intra-VTA self-administration and microdialysis experiments in genetically modified mice lacking the α7 nicotinic subunit or after pharmacological blockade of α7 nAChRs in wild-type mice. RESULTS: We show that the reinforcing properties of nicotine within the VTA are lower in the absence or after pharmacological blockade of α7 nAChRs. We also report that nicotine-induced increases in ACb DA extracellular levels last longer in the absence of these receptors, suggesting that α7 nAChRs regulate the action of nicotine on DA levels over time. CONCLUSIONS: The present results reveal new insights for the role of α7 nAChRs in modulating the action of nicotine within the mesolimbic circuit. These receptors appear to potentiate the reinforcing action of nicotine administered into the VTA while regulating its action over time on DA outflow in the ACb.

Antibodies against extracellular domains of α4 and α7 subunits alter the levels of nicotinic receptors in the mouse brain and affect memory: possible relevance to Alzheimer's pathology.

Nicotinic acetylcholine receptors (nAChRs) of α4ß2 and α7 subtypes expressed in the brain neurons are involved in regulating memory and cognition. Their level is decreased upon several neurodegenerative disorders including Alzheimer's disease (AD), although the reasons for such a decrease are not completely understood. To test whether the nAChR-specific antibodies can affect the brain nAChRs and influence the behavior, we either immunized mice with recombinant extracellular domains of α4 and α7, subunits α4(1-209) and α7(1-208), or injected them with α7(1-208)-specific antibodies. A decrease of α4ß2- and α7-nAChRs accompanied with an increase of α4ß4-nAChRs in brain membranes of immunized mice was observed. Both α4(1-209)- and α7(1-208)-specific antibodies were detected in the brain membrane lysates of immunized mice. Antibody injection resulted in brain nAChR decrease only if mice were co-injected intraperitoneally with bacterial lipopolysaccharide. Brain sections of immunized mice were analyzed for the binding of [125I]-α-bungarotoxin and [125I]-epibatidine. A decrease in α-bungarotoxin binding in striatum (nucleus accumbens and caudate putamen) accompanied with an increase of epibatidine binding in the forebrain and caudate putamen was observed in mice immunized with either α4 or α7 nAChR domains compared to those immunized with BSA. Mice immunized with α7(1-208) demonstrated significantly worse episodic memory measured in a novel object recognition task compared to non-immunized animals but did not differ from the controls in locomotor or anxiety-related tests. These results suggest that nAChR-specific antibodies are able to penetrate the brain upon inflammation with resulting decreases of brain nAChRs and worsening episodic memory.

Functional effects of antibodies against non-neuronal nicotinic acetylcholine receptors.

Non-neuronal nicotinic acetylcholine receptors (nAChRs) are expressed in the spleen and regulate B lymphocyte propagation and activation. The aim of the present study was to investigate the cellular and physiological effects of antibodies against alpha4(1-209) and alpha7(1-208) nAChR extracellular domains. The antibodies, added in vitro, produced in vivo or injected, specifically bound mouse spleen B lymphocytes. Immunization with nAChR extracellular domains resulted in connective tissue overgrowth and infiltration of segmented neutrophils in the spleen, as well as in decreased body weight compared to mice immunized with BSA. In spite of certain cross-reactivity of alpha4(1-209)- and alpha7(1-208)-specific antibodies, all observed effects were more pronounced upon immunization with alpha7 extracellular domain. Spleens of mice injected with alpha7(1-208)-specific antibody contained decreased numbers of Annexin V-positive B lymphocytes compared to mice injected with non-specific IgG. It is concluded that alpha7 nAChRs are involved in regulating the lymphocyte survival, neutrophil migration, connective tissue overgrowth and body weight accumulation. The antibody binding triggers alpha7 nAChR signaling supporting the idea of non-channel mode of nAChR functioning in B lymphocytes.

{alpha}7 nicotinic acetylcholine receptor regulates airway epithelium differentiation by controlling basal cell proliferation.

Airway epithelial basal cells are known to be critical for regenerating injured epithelium and maintaining tissue homeostasis. Recent evidence suggests that the alpha7 nicotinic acetylcholine receptor (nAChR), which is highly permeable to Ca(2+), is involved in lung morphogenesis. Here, we have investigated the potential role of the alpha7 nAChR in the regulation of airway epithelial basal cell proliferation and the differentiation of the human airway epithelium. In vivo during fetal development and in vitro during the regeneration of the human airway epithelium, alpha7 nAChR expression coincides with epithelium differentiation. Inactivating alpha7 nAChR function in vitro increases cell proliferation during the initial steps of the epithelium regeneration, leading to epithelial alterations such as basal cell hyperplasia and squamous metaplasia, remodeling observed in many bronchopulmonary diseases. The regeneration of the airway epithelium after injury in alpha7(-/-) mice is delayed and characterized by a transient hyperplasia of basal cells. Moreover, 1-year-old alpha7(-/-) mice more frequently present basal cells hyperplasia. Modulating nAChR function or expression shows that only alpha7 nAChR, as opposed to heteropentameric alpha(x)beta(y) nAChRs, controls the proliferation of human airway epithelial basal cells. These findings suggest that alpha7 nAChR is a key regulator of the plasticity of the human airway epithelium by controlling basal cell proliferation and differentiation pathway and is involved in airway remodeling during bronchopulmonary diseases.

Regional changes in the cholinergic system in mice lacking monoamine oxidase A.

Elevated brain monoamine concentrations resulting from monoamine oxidase A genetic ablation (MAOA knock-out mice) lead to changes in other neurotransmitter systems. To investigate the consequences of MAOA deficiency on the cholinergic system, we measured ligand binding to the high-affinity choline transporter (CHT1) and to muscarinic and nicotinic receptors in brain sections of MAOA knock-out (KO) and wild-type mice. A twofold increase in [(3)H]-hemicholinium-3 ([(3)H]-HC-3) binding to CHT1 was observed in the caudate putamen, nucleus accumbens, and motor cortex in MAOA KO mice as compared with wild-type (WT) mice. There was no difference in [(3)H]-HC-3 labeling in the hippocampus (dentate gyrus) between the two genotypes. Binding of [(125)I]-epibatidine ([(125)I]-Epi), [(125)I]-alpha-bungarotoxin ([(125)I]-BGT), [(3)H]-pirenzepine ([(3)H]-PZR), and [(3)H]-AFDX-384 ([(3)H]-AFX), which respectively label high- and low-affinity nicotinic receptors, M1 and M2 muscarinic cholinergic receptors, was not modified in the caudate putamen, nucleus accumbens, and motor cortex. A small but significant decrease of 19% in M1 binding densities was observed in the hippocampus (CA1 field) of KO mice. Next, we tested acetylcholinesterase activity and found that it was decreased by 25% in the striatum of KO mice as compared with WT mice. Our data suggest that genetic deficiency in MAOA enzyme is associated with changes in cholinergic activity, which may account for some of the behavioral alterations observed in mice and humans lacking MAOA.

Sevoflurane anesthesia alters exploratory and anxiety-like behavior in mice lacking the beta2 nicotinic acetylcholine receptor subunit.

BACKGROUND: Preexisting cognitive impairment and advanced age are factors that increase the risk of developing postoperative cognitive dysfunction. Because anesthetic agents interfere with cholinergic transmission and as impairment of cholinergic function is associated with cognitive decline, the authors studied how the volatile anesthetic sevoflurane affects exploratory and anxiety-like behavior in young and aged animals with a genetically modified cholinergic system. METHODS: Young and aged wild-type and mutant mice lacking the beta2 subunit of the nicotinic cholinergic receptor (beta2KO) were anesthetized for 2 h with 2.6% sevoflurane in oxygen and compared with nonanesthetized controls. Locomotor activity and organization of movement in the open field model were assessed before and 24 h after anesthesia. Locomotor activity and anxiety-like behavior in the elevated plus maze were assessed 24 h after anesthesia. High- and low-affinity nicotinic receptor and cholinergic uptake site densities were evaluated in the hippocampus, amygdala, and forebrain regions using receptor autoradiography. RESULTS: Sevoflurane anesthesia significantly reduced locomotor activity, altered temporospatial organization of trajectories, and increased anxiety-like behavior in young beta2KO mice, whereas no such changes were observed in young wild-type mice. Aged wild-type and beta2KO mice displayed reactions that were similar, but not identical, to the reactions of young mice to sevoflurane anesthesia. However, behavioral changes were not associated with differences in nicotinic receptor or cholinergic uptake site densities. CONCLUSION: In conclusion, sevoflurane anesthesia altered exploratory and anxiety-like behavior in mice lacking the beta2 nicotinic acetylcholine receptor subunit.

Regional differential effects of chronic nicotine on brain alpha 4-containing and alpha 6-containing receptors.

Chronic nicotine upregulates central nicotinic acetylcholine receptors (nAChRs), a plasticity process thought to contribute to its addictive properties. To analyze this process in vivo, we chronically exposed mice to nicotine using minipump delivering nicotine at concentrations close to those found in tobacco smokers. Binding studies show upregulation of high-affinity nAChRs after 21 days of treatment in cortical areas, caudate putamen, nucleus accumbens, hippocampus, ventral tegmental area, and superior colliculi. No upregulation was observed in thalamus and discrete cortical areas. Using wild type and alpha 6-/- mice, we observed a downregulation of alpha 6*-nAChRs in superior colliculi and no effects in other structures. The complex pattern of upregulation/downregulation observed in this study depends on both nAChR composition and regional distribution.

Long-term effects of chronic nicotine exposure on brain nicotinic receptors.

Chronic nicotine exposure results in long-term homeostatic regulation of nicotinic acetylcholine receptors (nAChRs) that play a key role in the adaptative cellular processes leading to addiction. However, the relative contribution of the different nAChR subunits in this process is unclear. Using genetically modified mice and pharmacological manipulations, we provide behavioral, electrophysiological, and pharmacological evidence for a long-term mechanism by which chronic nicotine triggers opposing processes differentially mediated by beta2*- vs. alpha7*nAChRs. These data offer previously undescribed insights into the understanding of nicotine addiction and the treatment of several human pathologies by nicotine-like agents chronically acting on beta2*- or alpha7*nAChRs.

Nicotine and serotonin in immune regulation and inflammatory processes: a perspective.

Nicotine and serotonin modulate the innate and adaptive immune responses and the inflammatory states. Several nicotinic cholinergic and serotonergic receptor subtypes have been characterized in B and T lymphocytes, monocytes, macrophages, and dendritic cells. The use of knockout mice has allowed a better characterization of nicotinic receptors and their role in anti-inflammatory processes in these cells. Cytokines play a crucial role in controlling inflammatory reactions. Nicotine and serotonin have been reported to regulate cytokine release. Cholinergic mechanisms also play an important role in inflammation through endogenous acetylcholine. Nicotine mimics this effect by activating the cholinergic anti-inflammatory pathways. New concepts of reciprocal interactions between nicotine and serotonin are emerging. The role of nicotine as an anti-inflammatory agent has been established, whereas that of serotonin remains more controversial.

alpha3alpha5beta2-Nicotinic acetylcholine receptor contributes to the wound repair of the respiratory epithelium by modulating intracellular calcium in migrating cells.

Nicotinic acetylcholine receptors (nAChRs), present in human bronchial epithelial cells (HBECs), have been shown in vitro to modulate cell shape. Because cell spreading and migration are important mechanisms involved in the repair of the bronchial epithelium, we investigated the potential role of nAChRs in the wound repair of the bronchial epithelium. In vivo and in vitro, alpha3alpha5beta2-nAChRs accumulated in migrating HBECs involved in repairing a wound, whereas alpha7-nAChRs were predominantly observed in stationary confluent cells. Wound repair was improved in the presence of nAChR agonists, nicotine, and acetylcholine, and delayed in the presence of alpha3beta2 neuronal nAChR antagonists, mecamylamine, alpha-conotoxin MII, and kappa-bungarotoxin; alpha-bungarotoxin, an antagonist of alpha7-nAChR, had no effect. Addition of nicotine to a repairing wound resulted in a dose-dependent transient increase of intracellular calcium in migrating cells that line the wound edge. Mecamylamine and kappa-bungarotoxin inhibited both the cell-migration speed and the nicotine-induced intracellular calcium increase in wound-repairing migrating cells in vitro. On the contrary alpha-bungarotoxin had no significant effect on migrating cells. These results suggest that alpha3alpha5beta2-nAChRs actively contribute to the wound repair process of the respiratory epithelium by modulating intracellular calcium in wound-repairing migrating cells.

Altered map of visual space in the superior colliculus of mice lacking early retinal waves.

During the development of the mammalian retinocollicular projection, a coarse retinotopic map is set up by the graded distribution of axon guidance molecules. Subsequent refinement of the initially diffuse projection has been shown to depend on the spatially correlated firing of retinal ganglion cells. In this scheme, the abolition of patterned retinal activity is not expected to influence overall retinotopic organization, but this has not been investigated. We used optical imaging of intrinsic signals to visualize the complete retinotopic map in the superior colliculus (SC) of mice lacking early retinal waves, caused by the deletion of the beta2 subunit of the nicotinic acetylcholine receptor. As expected from previous anatomical studies in the SC of beta2(-/-) mice, regions activated by individual visual stimuli were much larger and had less sharp borders than those in wild-type mice. Importantly, however, we also found systematic distortions of the entire retinotopic map: the map of visual space was expanded anteriorly and compressed posteriorly. Thus, patterned neuronal activity in the early retina has a substantial influence on the coarse retinotopic organization of the SC.

5-HT activates ERK MAP kinase in cultured-human peripheral blood mononuclear cells via 5-HT1A receptors.

In the present work, we tested the hypothesis that serotonin (5-hydroxytryptamine = 5-HT) might activate the extracellular signal-regulated kinase (ERK) pathway in human peripheral blood mononuclear cells (PBMC). PBMC were maintained in culture for 72 hrs at 37 degrees C prior to the addition of 5-HT. Our results showed an increase in ERK activation by 5-HT with a peak effect at 30 min and maximal stimulation with 5-HT at 1microM. This activation of ERK did not occur in adherent monocytes suggesting that the effect was on lymphocytes. In addition, p38 MAP kinase was not activated under these conditions. The effect of 5-HT on ERK activation appeared to be mediated through the activation of 5-HT1A receptors since similar results were obtained with R-+-8-hydroxy-DPAT, a selective 5-HT1A receptor agonist and WAY100635, a selective 5-HT1A receptor antagonist, reversed the 5-HT and the R-+-8-hydroxy-DPAT effects. Results from Western blot analysis confirmed the presence of 5-HT1A receptors on the PBMC. A 5-HT2A antagonist, ketanserin, and a 5-HT transport inhibitor, fluoxetine, both failed to block the activation of ERK by 5-HT. Our results indicate that 5-HT activates ERK, but not p38, MAP kinase of human PBMC via a 5-HT1A receptor.