- Email: [email protected]
Agonist-induced internalization of the 5HT2A receptor in vitro
Abstracts
107. A ONE YEAR RANDOMIZED TRIAL OF CLOZAPINE VS. USUAL CARE IN BIPOLAR I PATIENTS T. Suppes'v A], Rush l • A. Webb l , T. Carmody', & H. Kraemer IThc University cf Texas Southwestern Medical Center. Dalla.... TX
75:!35~ ~Stanfonl University, Stanford, CA 94305
Patients with severe, persistent bipolar or schizoaffcctive disorder (i.e. symptoms despite lithium I> 0.8 mEq/L] combined with an anticonvulsant nt therapeutic levels lind. if psychotic. a neuroleptic chlorpromazine ;::: 500 mgor equivalent) entered a randomized trial of either "treatmentas-usual" (TAU) orclozapine forone year. TAU allowedany medication except elozapine; clozapine patients received clozapine plus llny medications.Twenty-five women and 16 men were randomized. Two patients did not return for their firstvisit and arc excluded from the analysis. TIm majority or patlems were bipolar I (73%) with n current median age of 38 years. All patients had a IonS history of illness with a medianof 16 years from flrst treatrnent and 13 years from first hospitulizmion, Patients wen: evaluated monthly using standard symptom scales: BPRS (IS·item). HRS·n (24·ilcm), the Bcch-Raphaclsen Mania Scale (BRMS), the COl. and a
108. RCBF CHANGES WITH CLOZAPINE TREATMENT IN REFRACTORY BIPOLAR
DISORDER M.H. Trivedi, T. Suppes. M.D. Devous, Sr., T.S. Harris, & A.J. Rush Department of Psychiatry. U.T. Southwestern Medical Center, Dallas. TX 75235·9101
DIOL PSYCHIATRY 1996:39:500-666
531
signifieanl limilations. To address these limitations. we developed n 3·dimcnsional voxel-bascd analysis in which a modified Pelizarri algorithm was integrated into an automated coreglstraticn system.Dam were automatically normalized to whole brain counts and corcgistered, with the user choosing only slice range for inclusion and two initial transfermatlon parameters. The system yields an accuracy of -2mm between lOOdel and registered images. Patient groups images were evaluated parametrically (t-tcsts and z-tests) against themselves in n pre,{posltreatment paradigm. We found neural-circuits showing decreased rCBF (involving left caudate anti bilateral temporal lobes) 3S well :15 areas of increased rCBF. Funded in pan by NARSAD(MHT&TS) and Mental Health Connecuons.
109. INTERMITTENT LOW DOSE CLOZAPINE SENSITIZES (KINDLES?) MYOCLONUS I
I
..,
D. D eoney, J.R. Stevens, & P, Szot.. 'Oregon Health Sciences University. Portland. OR 97201 : :!University of Washington. Seattle, WA 98195
Clozapine in clinical usage may provoke slowing and paroxysmal aclivily in the EEG, epileptic seizures. and myoclonus. With the hypolhesis thai these markers of cent....l1 nervous system excitation may relate to the therapeutic efficacy of clozaplne, we have studied the proconvulsive properties of thls drug in the rot. In previous experiments we have shown a dose response relationship for the production of myoclonus by clozapine in the panlally restrained rat (Denney D. Stevens JR (1995): Bioi Psyc!liatry 37:427-433). In a second series of experiments. repeated I mgmlkg injections at alternate day or 6 day Intervals. a dose that inilially failed to elicit myoclonus. resulted in progressive increases in myoclonic jcrks (MJs> reaching rarcs of 100140per hour by the IDth lnjection (StevensJR, Denney 0, 51.01 P (1995): Epilep.vy R(·.~. In press). This result is consistent with sensitization or kindling of the myoclonic response, 11/ sitll hybridlwtion demonstrates increased expression of the early gene c-fos mRNA in ventral segmental area and anterior thalamic nuclei of clozuplne sensitized (kindled) rats, Further studies of this model demonstrate ihat while motor myoclonus is only manifest in partially restrained animals, sensitization (kindling) occurs in unrestrained rats given the same low dose. Low dose intermittent clozaplue sensitization mayhave therapeutic potenrlal by slimulllling or disinhibiting neuronal populations that arc directly related to the palhophysiology or schizophrenia.
110. AGONIST-INDUCED INTERNALIZATION OF THE 5HT2A RECEPTOR IN VITRO S.A. Berry, N. Khan, & B.L. Roth
lillie is known about regional cerebral blood now (rCBF) or glucose metabolic abnormalhles in symptomatic bipolar disorderas well as rCBF changes accompanying successful treatment In patients with severe lind persistent bipolar illness. As part of lin ongoing lrial of clozapine for treatment refractory bipolar disorder, we examined rCBF abnormalitles (Tc·99m·I~MPAO, Amershan; high resolution SPECT. PRISM30005) in IS patients (Bipolar: n=lIi Sehizoll!fcctivc: n""4) before and at 6 months on clozuplnc, Patients were 38.6 :t 7 years old. and included 10 females. Their mean BPRS (:!li·i1~m) was 39 :t 6. which decreased to 31 :!: 8 by 6 months on clozaplne, The Bech Rataclson Mania Scale SCore decreased from 6 ± 4 to 2.5 ± 2 after treatment. Studles of PET nnd SPECT have utilized regions-or·interest based analyses. which have
Case Western Reserve University. Cleveland. OU 44106 An understanding of serotonin receptor regulation and signal transduction is essential 10 elucidate the biological basis of chronic psychiatric illnesses. SHTu receptors are regulaled by many psychoactive drugs (clozapine, risperidone, LSD), but the molecular details responsible for these processes arc unknown. Here we report on ngonlsr-Induccd Internalizalion of lhe 5HT;u. receptor in GF·6 cells, a cell line which over-expresses the SHT~ receptor. Cells were exposed to 10 j.l.M quJpazinc lit 3711C for S. IS. 30. 120 and 180 minutes. A polyclonal
532
Abstracts
DIOL PSYCHIATRY 1996;39:500-666
antibody directed against the amino terminus of the 5HTu , receptor was used for immunocytochemical localization of receptors in fixed cells. Confocal lasermicroscopic imagesof the agonist-exposed cells revealed a redistribution of 5HTM receptors as measured by a statistically significant increase in intracellular fluorescence compared to vehicletreated control cells (p
However. the lmponant question is whether upregulation of the receptor is a consequence of the disease or the 5HT2A receptor is involved, at least partly, in Iho development of the disease. In order to study the link between SHT21\ receptor and behavior, we have generated receptor deficient knock-out mice. Receptor deflcienr mice were also produced by antisense oligonucleotide injection. We studied the behavior or receptor deficicm animals in stressful situations suchas escape directed behavior in the forced swim testand fear.and "anxiety" in the elevated maze. The forced swimand the elevated maze testsarc pharmacologically validated animal models of depression and anxiety, respectively. The behavioral experiments demonstrated that reduced 5HT2A receptor level alone is sufficient to induce a behavioral slate characterized by less immobility and more •anxiety,• In conclusion, the result with the genetically modified and antisense oligonucleotide treated animals suggests an important role for the 5HT2A receptor in the regulation of certain behaviors.
1I I. REGULATION OF PLATELET SEROTONIN TRANSPORTER BY PROTEIN KINASE C D. Marazzlti'. M.R. Mazzoni', L. Pulegc', A. Rotondo I, A. Rossi", & A. Lucacchinl" Psychiatry lind :"(stituto Pelicattcdra Discipline Biologiche", University ot' Pisa, Pisa, Italy II nsututc of
Some data show thut different factors may affect the serotonin (S·HT) uptake rate. Our study aimed at evnluating the possible role of protein klnnse activators, such as 4-p-1 2-tctnldecmloyl-phorbol·13-11cctate (/3TPA) on platele; S-IIT uptnke, Human platelets and 5-HT uptake were carried out according to the method of Arora and Meltzer (1979) and ~H-puroxetine ('U-Par) binding was performed following Marazziti et nl's method (1995). The resultsshowed that B·TPA reduced significantly the maximal velocity (Vmax) of S·UT uptake, wilh no change in the Michaells constum or in the lH_ Par binding parameters. These findings indicate that protein kinase C decreases the S·HT uptake rate by modifying the phosph07;lation state of the transporter. which docs not influence thenumber of' H-Par bindingsites.Sincepreliminary results in patients with anxiety disorders are at variance with what observed in healthy controls, it is tempting to speculate that protein kinase C is altered in these conditions, or. attcmutlvcly, that S-HT uptake has n different regulation in pathological states, but further data nrc needed in order to substantlute this observation,
112. GENETIC APPROACHES TO ELUCIDATE SEROTONIN 2A (5HT2A) RECEPTOR FUNCTION M. Toth. E. Sibille, Z. Gyulai, & 1. Gal Cornell University Medical College. New York, NY. 10021 Department of Pharmacology Uprcgulntion of the SHT2A receptors. besides hypercortisolcrnia, is n reproducible correlate of depression. at least in a subgroup of patlcnts,
113. CELLULAR SUBSTRATES FOR SEROTONERGIC MODULATION OF GABA IN THE NUCLEUS ACCUMBENS
EJ. Van Bockstaele & V.M. Pickel Cornell University Medical College, New York, NY, 10021 Serotonin (S-hydroxylf}'ptllminc: 5·HT) and OABA have been strongly hnpllcatcd in modulating a variety of common motivational and motorrelated functions prominently subserved bythe nucleus accumbens (Acb) ill the venunl striatum. The shell subregion of the Acb, in partlcular, has been ussociatcd with emotions and limbic-related processes and is of speclal interest in relation to depression as well u.~ in mediating certain forms of stress. We used immunoelectron microscopy to determine whether there were cellular substrates that might indicate more specific sites for functional interactions involving these transmitters in the shell subregion of the Acb. Approximately half (187/366) of the s-mimmunorcactlve axon terminals apposed or formed synaptic junctions with postsynaptic neurons. Thesejunctions were mainly of the symmetric-type (83/187) eharacteristic of inhibitory transmhters, and were equally prevalent on dendrites with and without detectable gold-silver labeling for GABA. Of the 187 S-HT axon terminals with recognized synaptic contacts, 36% also showed convergence with a GADA-Iabelcd axon terminal as seen in single sections. In addition, 5·HT and GADA axon terminals werecommonly (33/366) identified in directapposition 10 one another. Within a single plane of section. 41% of the apposed GADA-immunoreactive axon terminals formed symmetric-type junctions with unlabeled dendrites or somata; whereas, the apposed S-HTlabeled axon terminals rarely showed postsynaptic contacts. Theseresults indicate'tha; 5-HT axon terrnlnals postsynaptically inhlblt GABAergic neurons and their targets within the shell of the rat nucleus accumbens. Additionally. our results strongly suggest thnt, in this brain region. S-HT may presynaptlcally modulate or be modulated by QABA.
107. A ONE YEAR RANDOMIZED TRIAL OF CLOZAPINE VS. USUAL CARE IN BIPOLAR I PATIENTS T. Suppes'v A], Rush l • A. Webb l , T. Carmody', & H. Kraemer IThc University cf Texas Southwestern Medical Center. Dalla.... TX
75:!35~ ~Stanfonl University, Stanford, CA 94305
Patients with severe, persistent bipolar or schizoaffcctive disorder (i.e. symptoms despite lithium I> 0.8 mEq/L] combined with an anticonvulsant nt therapeutic levels lind. if psychotic. a neuroleptic chlorpromazine ;::: 500 mgor equivalent) entered a randomized trial of either "treatmentas-usual" (TAU) orclozapine forone year. TAU allowedany medication except elozapine; clozapine patients received clozapine plus llny medications.Twenty-five women and 16 men were randomized. Two patients did not return for their firstvisit and arc excluded from the analysis. TIm majority or patlems were bipolar I (73%) with n current median age of 38 years. All patients had a IonS history of illness with a medianof 16 years from flrst treatrnent and 13 years from first hospitulizmion, Patients wen: evaluated monthly using standard symptom scales: BPRS (IS·item). HRS·n (24·ilcm), the Bcch-Raphaclsen Mania Scale (BRMS), the COl. and a
108. RCBF CHANGES WITH CLOZAPINE TREATMENT IN REFRACTORY BIPOLAR
DISORDER M.H. Trivedi, T. Suppes. M.D. Devous, Sr., T.S. Harris, & A.J. Rush Department of Psychiatry. U.T. Southwestern Medical Center, Dallas. TX 75235·9101
DIOL PSYCHIATRY 1996:39:500-666
531
signifieanl limilations. To address these limitations. we developed n 3·dimcnsional voxel-bascd analysis in which a modified Pelizarri algorithm was integrated into an automated coreglstraticn system.Dam were automatically normalized to whole brain counts and corcgistered, with the user choosing only slice range for inclusion and two initial transfermatlon parameters. The system yields an accuracy of -2mm between lOOdel and registered images. Patient groups images were evaluated parametrically (t-tcsts and z-tests) against themselves in n pre,{posltreatment paradigm. We found neural-circuits showing decreased rCBF (involving left caudate anti bilateral temporal lobes) 3S well :15 areas of increased rCBF. Funded in pan by NARSAD(MHT&TS) and Mental Health Connecuons.
109. INTERMITTENT LOW DOSE CLOZAPINE SENSITIZES (KINDLES?) MYOCLONUS I
I
..,
D. D eoney, J.R. Stevens, & P, Szot.. 'Oregon Health Sciences University. Portland. OR 97201 : :!University of Washington. Seattle, WA 98195
Clozapine in clinical usage may provoke slowing and paroxysmal aclivily in the EEG, epileptic seizures. and myoclonus. With the hypolhesis thai these markers of cent....l1 nervous system excitation may relate to the therapeutic efficacy of clozaplne, we have studied the proconvulsive properties of thls drug in the rot. In previous experiments we have shown a dose response relationship for the production of myoclonus by clozapine in the panlally restrained rat (Denney D. Stevens JR (1995): Bioi Psyc!liatry 37:427-433). In a second series of experiments. repeated I mgmlkg injections at alternate day or 6 day Intervals. a dose that inilially failed to elicit myoclonus. resulted in progressive increases in myoclonic jcrks (MJs> reaching rarcs of 100140per hour by the IDth lnjection (StevensJR, Denney 0, 51.01 P (1995): Epilep.vy R(·.~. In press). This result is consistent with sensitization or kindling of the myoclonic response, 11/ sitll hybridlwtion demonstrates increased expression of the early gene c-fos mRNA in ventral segmental area and anterior thalamic nuclei of clozuplne sensitized (kindled) rats, Further studies of this model demonstrate ihat while motor myoclonus is only manifest in partially restrained animals, sensitization (kindling) occurs in unrestrained rats given the same low dose. Low dose intermittent clozaplue sensitization mayhave therapeutic potenrlal by slimulllling or disinhibiting neuronal populations that arc directly related to the palhophysiology or schizophrenia.
110. AGONIST-INDUCED INTERNALIZATION OF THE 5HT2A RECEPTOR IN VITRO S.A. Berry, N. Khan, & B.L. Roth
lillie is known about regional cerebral blood now (rCBF) or glucose metabolic abnormalhles in symptomatic bipolar disorderas well as rCBF changes accompanying successful treatment In patients with severe lind persistent bipolar illness. As part of lin ongoing lrial of clozapine for treatment refractory bipolar disorder, we examined rCBF abnormalitles (Tc·99m·I~MPAO, Amershan; high resolution SPECT. PRISM30005) in IS patients (Bipolar: n=lIi Sehizoll!fcctivc: n""4) before and at 6 months on clozuplnc, Patients were 38.6 :t 7 years old. and included 10 females. Their mean BPRS (:!li·i1~m) was 39 :t 6. which decreased to 31 :!: 8 by 6 months on clozaplne, The Bech Rataclson Mania Scale SCore decreased from 6 ± 4 to 2.5 ± 2 after treatment. Studles of PET nnd SPECT have utilized regions-or·interest based analyses. which have
Case Western Reserve University. Cleveland. OU 44106 An understanding of serotonin receptor regulation and signal transduction is essential 10 elucidate the biological basis of chronic psychiatric illnesses. SHTu receptors are regulaled by many psychoactive drugs (clozapine, risperidone, LSD), but the molecular details responsible for these processes arc unknown. Here we report on ngonlsr-Induccd Internalizalion of lhe 5HT;u. receptor in GF·6 cells, a cell line which over-expresses the SHT~ receptor. Cells were exposed to 10 j.l.M quJpazinc lit 3711C for S. IS. 30. 120 and 180 minutes. A polyclonal
532
Abstracts
DIOL PSYCHIATRY 1996;39:500-666
antibody directed against the amino terminus of the 5HTu , receptor was used for immunocytochemical localization of receptors in fixed cells. Confocal lasermicroscopic imagesof the agonist-exposed cells revealed a redistribution of 5HTM receptors as measured by a statistically significant increase in intracellular fluorescence compared to vehicletreated control cells (p
However. the lmponant question is whether upregulation of the receptor is a consequence of the disease or the 5HT2A receptor is involved, at least partly, in Iho development of the disease. In order to study the link between SHT21\ receptor and behavior, we have generated receptor deficient knock-out mice. Receptor deflcienr mice were also produced by antisense oligonucleotide injection. We studied the behavior or receptor deficicm animals in stressful situations suchas escape directed behavior in the forced swim testand fear.and "anxiety" in the elevated maze. The forced swimand the elevated maze testsarc pharmacologically validated animal models of depression and anxiety, respectively. The behavioral experiments demonstrated that reduced 5HT2A receptor level alone is sufficient to induce a behavioral slate characterized by less immobility and more •anxiety,• In conclusion, the result with the genetically modified and antisense oligonucleotide treated animals suggests an important role for the 5HT2A receptor in the regulation of certain behaviors.
1I I. REGULATION OF PLATELET SEROTONIN TRANSPORTER BY PROTEIN KINASE C D. Marazzlti'. M.R. Mazzoni', L. Pulegc', A. Rotondo I, A. Rossi", & A. Lucacchinl" Psychiatry lind :"(stituto Pelicattcdra Discipline Biologiche", University ot' Pisa, Pisa, Italy II nsututc of
Some data show thut different factors may affect the serotonin (S·HT) uptake rate. Our study aimed at evnluating the possible role of protein klnnse activators, such as 4-p-1 2-tctnldecmloyl-phorbol·13-11cctate (/3TPA) on platele; S-IIT uptnke, Human platelets and 5-HT uptake were carried out according to the method of Arora and Meltzer (1979) and ~H-puroxetine ('U-Par) binding was performed following Marazziti et nl's method (1995). The resultsshowed that B·TPA reduced significantly the maximal velocity (Vmax) of S·UT uptake, wilh no change in the Michaells constum or in the lH_ Par binding parameters. These findings indicate that protein kinase C decreases the S·HT uptake rate by modifying the phosph07;lation state of the transporter. which docs not influence thenumber of' H-Par bindingsites.Sincepreliminary results in patients with anxiety disorders are at variance with what observed in healthy controls, it is tempting to speculate that protein kinase C is altered in these conditions, or. attcmutlvcly, that S-HT uptake has n different regulation in pathological states, but further data nrc needed in order to substantlute this observation,
112. GENETIC APPROACHES TO ELUCIDATE SEROTONIN 2A (5HT2A) RECEPTOR FUNCTION M. Toth. E. Sibille, Z. Gyulai, & 1. Gal Cornell University Medical College. New York, NY. 10021 Department of Pharmacology Uprcgulntion of the SHT2A receptors. besides hypercortisolcrnia, is n reproducible correlate of depression. at least in a subgroup of patlcnts,
113. CELLULAR SUBSTRATES FOR SEROTONERGIC MODULATION OF GABA IN THE NUCLEUS ACCUMBENS
EJ. Van Bockstaele & V.M. Pickel Cornell University Medical College, New York, NY, 10021 Serotonin (S-hydroxylf}'ptllminc: 5·HT) and OABA have been strongly hnpllcatcd in modulating a variety of common motivational and motorrelated functions prominently subserved bythe nucleus accumbens (Acb) ill the venunl striatum. The shell subregion of the Acb, in partlcular, has been ussociatcd with emotions and limbic-related processes and is of speclal interest in relation to depression as well u.~ in mediating certain forms of stress. We used immunoelectron microscopy to determine whether there were cellular substrates that might indicate more specific sites for functional interactions involving these transmitters in the shell subregion of the Acb. Approximately half (187/366) of the s-mimmunorcactlve axon terminals apposed or formed synaptic junctions with postsynaptic neurons. Thesejunctions were mainly of the symmetric-type (83/187) eharacteristic of inhibitory transmhters, and were equally prevalent on dendrites with and without detectable gold-silver labeling for GABA. Of the 187 S-HT axon terminals with recognized synaptic contacts, 36% also showed convergence with a GADA-Iabelcd axon terminal as seen in single sections. In addition, 5·HT and GADA axon terminals werecommonly (33/366) identified in directapposition 10 one another. Within a single plane of section. 41% of the apposed GADA-immunoreactive axon terminals formed symmetric-type junctions with unlabeled dendrites or somata; whereas, the apposed S-HTlabeled axon terminals rarely showed postsynaptic contacts. Theseresults indicate'tha; 5-HT axon terrnlnals postsynaptically inhlblt GABAergic neurons and their targets within the shell of the rat nucleus accumbens. Additionally. our results strongly suggest thnt, in this brain region. S-HT may presynaptlcally modulate or be modulated by QABA.