Serotonin (5-HT), a major neurotransmitter, has a large number of G protein-coupled receptors in mammals. On activation by exposure to their ligand, 5-HT 2 receptor subtypes increase IP3 levels and undergo desensitization and internalization. To visualize the receptor in cells during these processes, we have constructed a 5-HT 2A-enhanced GFP (SR2-GFP) fusion receptor. We show that this fusion receptor undergoes internalization on exposure to its natural ligand, 5-HT. Because 5-HT2A receptors activate the phospholipase C pathway, we studied the effect of protein kinase C (PKC) on the internalization process and found that activation of PKC by its specific activator phorbol 12-myristate 13-acetate, in the absence of 5-HT, leads to internalization of the receptor. Moreover, inhibition of PKC by its inhibitor sphingosine in the presence of 5-HT prevents the internalization process, suggesting that activation of PKC is sufficient and necessary for the internalization of 5-HT 2A receptors. We also show that SR2-GFP recycles back to the plasma membrane after 5-HT-dependent internalization, suggesting a mechanism for resensitization. In addition, receptors that have been internalized on addition of phorbol 12-myristate 13-acetate in the absence of 5-HT also recycle to the surface, with a time course similar to that seen after activation of the receptors by 5-HT. Our study suggests that 5-HT2A receptors internalize and return to the surface after both serotonin-and PKC-mediated processes. This study reveals a role for PKC in receptor internalization and also shows that 5-HT2A receptors are recycled.general feature of G protein-coupled receptors (GPCRs) is the existence of complex regulatory mechanisms that modulate receptor responsiveness. These mechanisms underlie important physiological phenomena such as signal transduction and plasticity. Many GPCRs also demonstrate rapid desensitization in the continued presence of agonists, due to uncoupling of the receptor from the G protein involved (1). In some systems, such as opiate and ␤ 2 adrenergic receptors, sequestration or internalization of receptors appears to occur as a part of the overall process of agonist-induced desensitization (2-9).The phenomena of receptor desensitization and downregulation are well described in 5-HT receptors (10-15). For example, phosphatidylinositol hydrolysis in bovine aorta mediated by 5-HT 2 receptors is markedly attenuated after brief treatments with agonists (16). 5-HT 2 receptor-induced Ca 2ϩ mobilization is rapidly diminished after agonist treatment of rat C6BU-1 glioma cells, and rapid decreases in 5-HT-induced currents are also seen in Xenopus oocytes expressing 5-HT 2A receptors (17,18).Sequestration or internalization of 5-HT 2A receptors is also thought to occur as part of the overall process of agonist-induced desensitization of 5-HT 2A receptors (19,20). The process of resensitization would require the recovery of the ability of the receptor to couple to G proteins. Although it is not clear that internalization of desensitized ...

We report an optical tweezers based approach for efficient and controlled manipulation of neuronal growth cones. The approach exploits asymmetric transverse gradient force created in a line optical tweezers to transport actin monomers in the desired growth direction. With this approach induction of artificial growth cones from the neuronal cell body and enhancement of the growth rate of the natural growth cones have been achieved. The use of this approach to bring two growth cones into close proximity for establishing a neuronal connection is also discussed.

Using electrophysiological techniques, we studied interactions of dopamine and selected dopaminergic drugs with serotonin (5- Serotoninergic systems are widespread in mammalian brain, and their dysfunction has been implicated in a variety of neurological disorders (e.g., refs. 1 and 2). Serotonin (5-hydroxytryptamine; 5-HT) has been shown to act through numerous receptor subtypes, which show differential patterns of expression in brain and are coupled to different ion channels and intracellular messenger pathways (e.g., ref. 3).hydroxytyptamEarly studies indicated that poly(A)+ RNA extracted from rat or human brain expressed functional 5-HT receptors in Xenopus oocytes, thus providing an additional approach for investigations into the molecular biology and pharmacology of these proteins (4). When assayed by electrical recording techniques, exogenous 5-HT receptors were found to mediate at least three membrane current responses in oocytes: two involving increases in membrane conductance to Cl-and one involving a reduction in conductance to K+ (4-6). The dominant electrical response was elicited through activation of an endogenous intracellular messenger pathway, which was triggered by receptor-stimulated hydrolysis of inositol phospholipids, liberation of intracellular Ca2l by inositol polyphosphates, and activation of endogenous Ca2+-gated Cl-channels in the oocyte membrane (7,8). In addition, inositol 1,4,5-trisphosphate was found to regulate Ca2' channels in the plasma membrane, stimulating entry of extracellular Ca2l and further activation of C1-currents (9). The oocyte expression system was subsequently exploited in "expression-cloning" procedures used to isolate clones encoding 5-HT1I receptors from mouse and rat choroid plexus cDNA libraries (10,11). Sequence data from the 5-HT1, receptor clone was then used to isolate cDNAs encoding the 5-HT2 receptor from rat cerebral cortex (12, 13).In the present study, effects of dopamine and selected dopaminergic drugs were assayed on rat 5-HT1c and 5-HT2 4708The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

G protein-coupled receptor (GPCR) signaling is modulated by endocytosis and endosomal sorting of receptors between degradation and recycling. Differential regulation of these processes by endogenous ligands and synthetic drugs is a poorly understood area of GPCR signaling. Here, we describe remarkable diversity in the regulation of trafficking of GPCR induced by multiple ligands. We show that the serotonin receptor 2A (5-HT 2A ), a prototypical GPCR in the study of functional selectivity at a signaling receptor, is functionally selective in endocytosis and recycling in response to five ligands tested: endogenous agonists serotonin (5-HT) and dopamine (DA), synthetic agonist 1-(2,5-dimethoxy-4-iodophenyl)-aminopropane (DOI), antagonist ketanserin, and inverse agonist and antipsychotic drug clozapine. Only four ligands (5-HT, DA, DOI, and clozapine) bring about receptor endocytosis. As we have earlier described with 5-HT and DA, there is ligand-specific requirement for protein kinase C (PKC) in endocytosis. We now show 5-HT 2A phosphorylation by PKC is necessary for 5-HT-mediated and DOI-mediated receptor endocytosis, but DA-mediated and clozapine-mediated internalization is not affected if PKC is inhibited. Internalized receptors are recycled to the cell surface, but there is variability in the time course of recycling. 5-HT-and DA-internalized receptors are recycled in 2.5 hours while agonist DOI and antagonist clozapine bring about recycling in 7.5 hours. Recycling in response to those ligands that require PKC activation to effect receptor endocytosis is dependent on receptor dephosphorylation by protein phosphatase 2A (PP2A). Thus, internalization and phosphorylation/dephosphorylation cycles may play a significant role in the regulation of 5-HT 2A by functionally and therapeutically important ligands.

BackgroundThere is emerging evidence that there are shared genetic, environmental and developmental risk factors in psychiatry, that cut across traditional diagnostic boundaries. With this background, the Discovery biology of neuropsychiatric syndromes (DBNS) proposes to recruit patients from five different syndromes (schizophrenia, bipolar disorder, obsessive compulsive disorder, Alzheimer’s dementia and substance use disorders), identify those with multiple affected relatives, and invite these families to participate in this study. The families will be assessed: 1) To compare neuro-endophenotype measures between patients, first degree relatives (FDR) and healthy controls., 2) To identify cellular phenotypes which differentiate the groups., 3) To examine the longitudinal course of neuro-endophenotype measures., 4) To identify measures which correlate with outcome, and 5) To create a unified digital database and biorepository.MethodsThe identification of the index participants will occur at well-established specialty clinics. The selected individuals will have a strong family history (with at least another affected FDR) of mental illness. We will also recruit healthy controls without family history of such illness. All recruited individuals (N = 4500) will undergo brief clinical assessments and a blood sample will be drawn for isolation of DNA and peripheral blood mononuclear cells (PBMCs). From among this set, a subset of 1500 individuals (300 families and 300 controls) will be assessed on several additional assessments [detailed clinical assessments, endophenotype measures (neuroimaging- structural and functional, neuropsychology, psychophysics-electroencephalography, functional near infrared spectroscopy, eye movement tracking)], with the intention of conducting repeated measurements every alternate year. PBMCs from this set will be used to generate lymphoblastoid cell lines, and a subset of these would be converted to induced pluripotent stem cell lines and also undergo whole exome sequencing.DiscussionWe hope to identify unique and overlapping brain endophenotypes for major psychiatric syndromes. In a proportion of subjects, we expect these neuro-endophenotypes to progress over time and to predict treatment outcome. Similarly, cellular assays could differentiate cell lines derived from such groups. The repository of biomaterials as well as digital datasets of clinical parameters, will serve as a valuable resource for the broader scientific community who wish to address research questions in the area.

Serotonin is reported to be present in early embryos of many species and plays an important role in early patterning. Since it is a fluorophore, it can be directly visualized using fluorescence microscopy. Here, we use three-photon microscopy to image serotonin in live pre-implantation mouse embryos. We find that it is present as puncta averaging 1.3 square microns and in concentrations as high as 442 mM. The observed serotonin puncta were found to co-localize with mitochondria. Live embryos pre-incubated with serotonin showed a higher mitochondrial potential, indicating that it can modulate mitochondrial potential. Pre-implantation mouse embryos were also examined at various developmental stages for the presence of transcripts of the peripheral and neuronal forms of tryptophan hydroxylase (Tph1 and Tph2 respectively) and the classical serotonin transporter (Slc6a4). Transcripts of Tph2 were seen in oocytes and in two-cell stages, whereas transcripts of Tph1 were not detected at any stage. Transcripts of the transporter, Slc6a4, were present in all pre-implantation stages investigated. These results suggest that serotonin in embryos can arise from a combination of synthesis and uptake from the surrounding milieu.

'AT(4) receptors' through which Angiotensin IV (Ang IV) improves memory acquisition, were recently identified as insulin regulated aminopeptidase (IRAP). Radioligand binding studies have hitherto been performed with iodinated Ang IV in the presence of divalent cation chelators EDTA and 1,10-phenanthrolin. Hence, they referred to the apo-form of IRAP. Presently, binding of [(3)H]Ang IV and [(3)H]AL-11, a stable Ang IV analog, was compared on Chinese hamster ovary (CHO-K1) and mouse hippocampal (P40H1) cell membranes. With chelators, their high affinity sites showed the same pharmacological profile as for [(125)I]Ang IV binding. Without chelators, only high affinity binding was perceived for [(3)H]AL-11. The same pharmacological profile was recorded in both membrane preparations; it was different from the one in the presence of chelators and corresponded to catalytically active IRAP (despite the concurrent presence of aminopeptidase N (APN) in P40H1 cell membranes). This confirms that the active and apo-forms of IRAP have a distinct pharmacological profile.

Abbreviations used: 5-HT, 5-hydroxytryptamine; 5-HT 2A R, serotonin receptor subtype 2A; CFP, cyan fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; GPCR, G-protein coupled receptor; GRK-2, G-protein receptor kinase 2; HEK, human embryonic kidney; HRP, horse radish peroxidase; PLC, phospholipase C; SDS, sodium dodecyl sulfate. AbstractInternalization and recycling of G-protein coupled receptors are important cellular processes regulating receptor function. These are receptor-subtype and cell type-specific. Although important, trafficking variations between receptor isoforms of different species has received limited attention. We report here, differences in internalization and recycling between rat and human serotonin 2A receptor (5-HT 2A R) isoforms expressed in human embryonic kidney 293 cells in response to serotonin. Although the human and rat 5-HT 2A Rs differ by only a few amino acids, the human receptor takes longer to recycle to the cell surface after internalization, with the additional involvement of b arrestin-2 and G-protein receptor kinase 2. The interaction of b arrestin-2 with the human receptor causes the delay in recycling and is dependent on a primate-specific ASK motif present in the C-terminus of the receptor. Conversion of this motif to NCT, the corresponding sequence present in the rat isoform, results in the human isoform trafficking like the rat receptor. Replacing the serine 457 with alanine in the ASK motif of human isoform resulted in faster recycling, although with continued arrestin-dependent internalization. This study establishes significant differences between the two isoforms with important implications in our understanding of the human 5-HT 2A R functions; and indicates that extrapolating results from non-human receptor isoforms to human subtypes is not without caveats.