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482. Regulation of NR1 NMDA receptor splice variant expression in rat hippocampus
Saturday Abstracts
include adverse early life events and stressful events that occur in adulthood. To date, little research has focused on the effect of adverse early life events in animal models of depressive behaviour. This study was designed to assess the interaction of early and adult stresses on behavioral analogues of depression. C57BI/6J mice were maternally separated (MS) for 180 minutes per day from postnatal day 7–21 and were compared on behavioral tests to non-MS animals as adults. Sucrose preference, a baseline measure of anhedonia, demonstrated no differences between the MS mice (n ⫽ 18) and controls (n ⫽ 17), suggesting that these animals could respond normally in non-stressful situations (t ⫽ 0.67, p ⫽ 0.51). MS mice (n ⫽ 19), however, swam significantly less during the forced swim test compared to control animals (n ⫽ 22) (F ⫽ 10.79, p ⫽ 0.002). Other MS mice (n ⫽ 9), which were subjected to inescapable footshock, took longer to escape in this typical learned helplessness shock paradigm (t ⫽ 3.75, p ⫽ 0.003) when tested in the side in which they were trained. These results suggest that animals subjected to chronic stress early in life may be more susceptible to the effects of an acute stress in adulthood. Further investigation of such interactions may provide insight into the role of early trauma and neglect in humans who develop depression as adults.
482. REGULATION OF NR1 NMDA RECEPTOR SPLICE VARIANT EXPRESSION IN RAT HIPPOCAMPUS D.J. Healy, L.M. Ritter, J.H. Meador-Woodruff Mental Health Research Institute and Department of Psychiatry, University of Michigan, Ann Arbor, MI 48109 Pharmacological and neurochemical evidence implicate the NMDA subtype of glutamate receptor in the pathophysiology of schizophrenia, and alterations in NMDA receptor expression may lead to altered NMDA receptor activity. NMDA receptors are pentameric associations of subunits comprised of NR1 and NR2 subunits. NR1 subunit mRNA can be processed into eight unique splice variants containing combinations of three exons, 5, 21, and 22. These exons seem to confer unique pharmacological properties when present in the final assembled receptor. For example, exons 21 and 22 are associated with synaptic anchoring proteins, which may affect receptor insertion into the cell membrane. Therefore, the presence or absence of different exons may affect the function of NMDA receptors. Oligonucleotides specific for exons 5, 21, and 22, as well as an oligonucleotide that hybridizes to all eight splice variants, were terminally labelled with [35S]dATP and were used for both in situ and Northern hybridizations. Northern hybridization revealed that the oligonucleotide probes recognized the appropriate splice variants, and that optical density was linear with mRNA concentration. Further, we found that animals treated with either the NMDA receptor antagonist MK-801 (2–10 days) or the nonselective AMPA/kainate receptor antagonist CNQX (7 days) had increased expression of exon 21 in hippocampus, with no changes in any of the other three probes. These results suggest that there is increased insertion of exon 21 in an otherwise unchanged total pool of NR1 transcripts. Further, NR1 mRNA processing is regulatable, and is therefore a potential site of dysregulation in schizophrenia.
BIOL PSYCHIATRY 2000;47:1S–173S
147S
483. NEUROPHARMACOLOGY IN ADULT DROSOPHILA: STUDIES OF DRUG TOLERANCE H. Manev, N. Dimitrijevic, E. Simoncelli The Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, Chicago, IL, 60612 Drosophila melanogaster (fruit fly) is being widely used for genetic, molecular biologic, and behavioral studies, but the use of adult flies for neuropharmacological studies is hampered by the lack of appropriate methodology for reliable administration of accurate doses of drugs to adult flies. The commonly used method of administering drugs in a food or as a vapor does not guarantee timely and/or accurate dose delivery. We combined the methodology of single-cell injecting (using custombeveled glass pipettes coupled to a cell injector) and a fly-holder we designed, to reproducibly administer volumes of 0.2 L/fly intraabdominally to carbon-dioxide-anesthetized flies. This technique allows repeated injections without obvious injury to flies (survival time of more than 5 days), and enabled us to study tolerance to repeated administration of neuroactive substances such as ethanol and nicotine. Ethanol was injected in doses of 0.2–1.2 mol/fly. In controls, Canton-S flies, ethanol induced dose-dependent prolongation of anesthesia-triggered immobility (“sleep duration”). Pretreatment with ethanol resulted in tolerance to subsequent (4 –24 hrs later) ethanol administration, evident as shortening of the “sleep duration.” We are currently investigating the effect of ethanol on “sleep duration” in ethanol-sensitive mutant flies (amnesiac and cheapdate). Nicotine was injected in doses of 0.2– 8.0 nmol/fly; starting at 2– 4 nmol, nicotine resulted in dosedependent toxicity (about 80% lethality with 6.5 nmol). Previous treatment with 4 nmol nicotine resulted in significant tolerance to a subsequent toxic dose (administered 3– 48 hrs later). In our ongoing research we are using fruit flies to elucidate molecular mechanisms of alcohol and nicotine tolerance and dependence.
484. PARASYMPATHETIC REBOUND FOLLOWING STRESS IN FOUR RAT STRAINS V.J. Djuric (1), V.M. Kamath (2), D.H. Overstreet (3), M. Steiner (1) (1) Father Sean O’Sullivan Research Centre, St. Joseph’s Hospital and Department of Psychiatry and Behavioural Neurosciences and (2) Department of Medicine, McMaster University, Hamilton, Ontario; and (3) Department of Psychiatry and Center for Alcohol Studies, University of North Carolina, Chapel Hill Exposure to inescapable stress is accompanied by large and complex changes in autonomic nervous system functioning. Our knowledge however about genetic determinants of autonomic nervous system reactivity is still incomplete. For this purpose, we compared autonomic response to stress in four genetically defined rat strains: cholinergic hyperresponsive Flinders sensitive line rats and their control counterparts, Flinders resistant line rats; hypothalamo-pituitary-adrenal axis (HPA) hyperresponsive Fischer rats and HPA hyporesponsive Lewis rats. Autonomic function was estimated through vagal modulation of the heart by means of power spectrum analysis of a telemetrically obtained ECG signal. The ECG signal was recorded in freely moving rats before and following a 10 min forced swim procedure. Within 30 min of stress cessation the initial increase in power of low frequency spectrum component (LF, predominately sympathetic) was replaced by an increase in power of high frequency spectrum component (HF, parasympathetic).
include adverse early life events and stressful events that occur in adulthood. To date, little research has focused on the effect of adverse early life events in animal models of depressive behaviour. This study was designed to assess the interaction of early and adult stresses on behavioral analogues of depression. C57BI/6J mice were maternally separated (MS) for 180 minutes per day from postnatal day 7–21 and were compared on behavioral tests to non-MS animals as adults. Sucrose preference, a baseline measure of anhedonia, demonstrated no differences between the MS mice (n ⫽ 18) and controls (n ⫽ 17), suggesting that these animals could respond normally in non-stressful situations (t ⫽ 0.67, p ⫽ 0.51). MS mice (n ⫽ 19), however, swam significantly less during the forced swim test compared to control animals (n ⫽ 22) (F ⫽ 10.79, p ⫽ 0.002). Other MS mice (n ⫽ 9), which were subjected to inescapable footshock, took longer to escape in this typical learned helplessness shock paradigm (t ⫽ 3.75, p ⫽ 0.003) when tested in the side in which they were trained. These results suggest that animals subjected to chronic stress early in life may be more susceptible to the effects of an acute stress in adulthood. Further investigation of such interactions may provide insight into the role of early trauma and neglect in humans who develop depression as adults.
482. REGULATION OF NR1 NMDA RECEPTOR SPLICE VARIANT EXPRESSION IN RAT HIPPOCAMPUS D.J. Healy, L.M. Ritter, J.H. Meador-Woodruff Mental Health Research Institute and Department of Psychiatry, University of Michigan, Ann Arbor, MI 48109 Pharmacological and neurochemical evidence implicate the NMDA subtype of glutamate receptor in the pathophysiology of schizophrenia, and alterations in NMDA receptor expression may lead to altered NMDA receptor activity. NMDA receptors are pentameric associations of subunits comprised of NR1 and NR2 subunits. NR1 subunit mRNA can be processed into eight unique splice variants containing combinations of three exons, 5, 21, and 22. These exons seem to confer unique pharmacological properties when present in the final assembled receptor. For example, exons 21 and 22 are associated with synaptic anchoring proteins, which may affect receptor insertion into the cell membrane. Therefore, the presence or absence of different exons may affect the function of NMDA receptors. Oligonucleotides specific for exons 5, 21, and 22, as well as an oligonucleotide that hybridizes to all eight splice variants, were terminally labelled with [35S]dATP and were used for both in situ and Northern hybridizations. Northern hybridization revealed that the oligonucleotide probes recognized the appropriate splice variants, and that optical density was linear with mRNA concentration. Further, we found that animals treated with either the NMDA receptor antagonist MK-801 (2–10 days) or the nonselective AMPA/kainate receptor antagonist CNQX (7 days) had increased expression of exon 21 in hippocampus, with no changes in any of the other three probes. These results suggest that there is increased insertion of exon 21 in an otherwise unchanged total pool of NR1 transcripts. Further, NR1 mRNA processing is regulatable, and is therefore a potential site of dysregulation in schizophrenia.
BIOL PSYCHIATRY 2000;47:1S–173S
147S
483. NEUROPHARMACOLOGY IN ADULT DROSOPHILA: STUDIES OF DRUG TOLERANCE H. Manev, N. Dimitrijevic, E. Simoncelli The Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, Chicago, IL, 60612 Drosophila melanogaster (fruit fly) is being widely used for genetic, molecular biologic, and behavioral studies, but the use of adult flies for neuropharmacological studies is hampered by the lack of appropriate methodology for reliable administration of accurate doses of drugs to adult flies. The commonly used method of administering drugs in a food or as a vapor does not guarantee timely and/or accurate dose delivery. We combined the methodology of single-cell injecting (using custombeveled glass pipettes coupled to a cell injector) and a fly-holder we designed, to reproducibly administer volumes of 0.2 L/fly intraabdominally to carbon-dioxide-anesthetized flies. This technique allows repeated injections without obvious injury to flies (survival time of more than 5 days), and enabled us to study tolerance to repeated administration of neuroactive substances such as ethanol and nicotine. Ethanol was injected in doses of 0.2–1.2 mol/fly. In controls, Canton-S flies, ethanol induced dose-dependent prolongation of anesthesia-triggered immobility (“sleep duration”). Pretreatment with ethanol resulted in tolerance to subsequent (4 –24 hrs later) ethanol administration, evident as shortening of the “sleep duration.” We are currently investigating the effect of ethanol on “sleep duration” in ethanol-sensitive mutant flies (amnesiac and cheapdate). Nicotine was injected in doses of 0.2– 8.0 nmol/fly; starting at 2– 4 nmol, nicotine resulted in dosedependent toxicity (about 80% lethality with 6.5 nmol). Previous treatment with 4 nmol nicotine resulted in significant tolerance to a subsequent toxic dose (administered 3– 48 hrs later). In our ongoing research we are using fruit flies to elucidate molecular mechanisms of alcohol and nicotine tolerance and dependence.
484. PARASYMPATHETIC REBOUND FOLLOWING STRESS IN FOUR RAT STRAINS V.J. Djuric (1), V.M. Kamath (2), D.H. Overstreet (3), M. Steiner (1) (1) Father Sean O’Sullivan Research Centre, St. Joseph’s Hospital and Department of Psychiatry and Behavioural Neurosciences and (2) Department of Medicine, McMaster University, Hamilton, Ontario; and (3) Department of Psychiatry and Center for Alcohol Studies, University of North Carolina, Chapel Hill Exposure to inescapable stress is accompanied by large and complex changes in autonomic nervous system functioning. Our knowledge however about genetic determinants of autonomic nervous system reactivity is still incomplete. For this purpose, we compared autonomic response to stress in four genetically defined rat strains: cholinergic hyperresponsive Flinders sensitive line rats and their control counterparts, Flinders resistant line rats; hypothalamo-pituitary-adrenal axis (HPA) hyperresponsive Fischer rats and HPA hyporesponsive Lewis rats. Autonomic function was estimated through vagal modulation of the heart by means of power spectrum analysis of a telemetrically obtained ECG signal. The ECG signal was recorded in freely moving rats before and following a 10 min forced swim procedure. Within 30 min of stress cessation the initial increase in power of low frequency spectrum component (LF, predominately sympathetic) was replaced by an increase in power of high frequency spectrum component (HF, parasympathetic).