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P.1.a.015 Strain differences in susceptibility to unpredictable chronic mild stress as assessed by hippocampal gene expression profiling
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P.1.a Basic neuroscience – Genetics and genomics
about 10 years ago, many studies have focused on the mechanism of delayed action of antidepressants to the intracellular signal transductions related to the structural plasticity and neuronal adaptation in the central nervous system. Though fluoxetine increases serotonin and/or norpinephrine, many studies have suggested that therapeutic effects might be achieved by gene expression regulations through complicated processes of the neurogenesis. The aim of this study is to identify expressed gene differences in rat hippocampus between the experimental group and control group using cDNA microarray chip techniques during the 5 and 14 day after fluoxetine or distilled water administration especially with the relation of neurogenesis. By these results, we are trying to make some implications about what kind of genes acts on the delayed actions of antidepressants and how it does. Methods: By using 7 week old (200~250 g) SPF (specific pathogen free) Sprague-Dawley line male rats, we identified differently regulated genes in rat hippocmapus between fluoxetine administration group and control group using cDNA microarray chip techniques. For the experimental group rats, 10 mg per Kg fluoxetine dissolved in distilled water (40 mg/10 ml) contained in the syringe was administrated per oral. And for the control group rats, same amount of distilled water only was administrated. We analyzed expression pattern of genes after fluoxetine in rat during 5 and 14 days. And, a part of the regulated genes in cDNA microarray results were confirmed by real-time RT-PCR. Results: When we analyzed expression pattern of genes during 5 days and 14 days after fluoxetine treatment in rat hippocampus, some genes of fluoxetine treated rat hippocampus increased dramatically especially during 14 days compared to that of control. Camk2d, one of isotypes of Camk2 that is known to play important role in the survival of neural cell, synaptic plasticity and memory in hippocampus, in fluoxetine-treated rat hippocampus during 14 days were 2.6 times higher than in control rat by cDNA microarray chip assessment. mRNA of Map2k2, one intermediate of BNDF pathway, also increased 1.8 times higher than in control during 14 days after fluoxetine treatment. It is known that patients with depression have lower level of transthyretin that plays an important role in the transport of thyroid hormone in the central nervous system. In this study, mRNA transthyretin expression increased during 5 and 14 days after fluoxetine treatment. Conclusion: This results imply that chronic treatment of fluoxetine promotes expression of genes related to the survival of neural cell and neurogenensis in addition to the transport of thyroid hormone. It suggests that expression of some of important genes which play important roles in the neurogenesis and intracellular signal transduction cascade might be related to the delayed actions of antidepressants. References [1] Duman RS, Malberg J, Nakagawa S, Di’Sa C, 2000, Neuronal plasticity and survival in mood disorders, Biol Psychiatry, 48, 732–739. [2] Landgrebe J, Welzl G, Metz T, Van Gaalen MM, Ropers H, Wurst W, Holsboer F, 2002, Molecular characterization of antidepressant effects in the mouse brain using gene expression profiling, J Psychiatr Res, 36, 119–129. [3] Mason GA, Bondy SC, Nemeroff CB, Walker CH, Prange AJ, 1987, The effects of thyroid state on beta-adrenergic and serotonergic receptors in rat brain, Psychoneuroendocrinology, 12, 261–270.
P.1.a.015 Strain differences in susceptibility to unpredictable chronic mild stress as assessed by hippocampal gene expression profiling J.L. Paya-Cano1 ° , F. Sluyter1 , Y.S. Mineur2 , C. Fernandes1 , W.E. Crusio3 , L.C. Schalkwyk1 . 1 Institute of Psychiatry/King’s College, SGDP, London, United Kingdom; 2 Yale School of Medicine, Pyschiatry, Pharmacology and Neurobiology, New Haven, USA; 3 Centre National de la Recherche Scientifique, Laboratoire de Neurosciences Cognitives, Talence, France Introduction: Exposure to unpredictable chronic mild stress (UCMS) induces depression-like behaviours in rodents. Furthermore, it has been shown that inbred strains of mice present different thresholds of susceptibility to UCMS (Pothion et al. 2004). On the other hand, gene expression profiling across strains constitute a useful tool for dissecting the molecular mechanisms underlying behavioural variation found between inbred mice (Fernandes et al. 2004). In the present experiment we assessed gene expression profiles across two inbred strains of mice subjected to either UCMS or no treatment in order to examine the potential effects of UCMS on gene expression in the hippocampus. Materials and methods: At the age of two months 16 mice from C57BL/6J (B6) and BALB/cJ (Balb) strains were assigned to one of two treatment groups, controls (ctrl, n = 8) and UCMS (n = 8). The stress procedure lasted for 4 weeks prior to the behavioural testing. Mice were subjected to different kinds of stressors such as: cage tilting, damp sawdust, predator sounds, placement in an empty cage, placement in an empty cage with water on the bottom, inversion of the light/dark cycle, lights on for a short period of time during the dark phase, and switching cages. Stressors continued to be applied during the testing phase, except on testing days to avoid effects of acute stress. In the 5th and 6th weeks of UCMS, animals were tested in an anxiety test (dark-light box) and a learning task (cued and contextual fear conditioning), respectively. In the 7th week of UCMS, animals were subjected to the tail suspension test and 24 hours later to the forced swim test (results not shown). After testing, left and right hippocampi were dissected out, pooled and stored at −800ºC. A total of 16 MOE430A GeneChip arrays corresponding to individual animals were used. Analysis: Based on neuropharmacological and other evidence on the aetiology of affective disorders (Hattori et al. 2005), we selected 252 candidate genes. Most of these genes have a mouse homologue and most of these homologues are represented on the 430A array. These probesets were analyzed using a 2-way ANOVA with strain (B6, Balb) and treatment (UCMS, ctrl) as main factors. Results: After Bonferroni correction for multiple comparisons, our results showed effects of strain (26 probesets), treatment (1 probeset) and strain × treatment interaction (7 probesets) in gene expression in the hippocampus. Conclusions. As expected, the analysis of gene expression profiling of the selected set of candidate genes for affective disorders revealed a greater number of differences related to strain than for the environmental UCMS manipulation. References [1] Fernandes C., Paya-Cano J.L., Sluyter F., D’Souza U., Plomin R., Schalkwyk L.C., 2004, Hippocampal gene expression profiling across eight mouse inbred strains: Towards understanding the molecular basis for behaviour, Eur. J. Neurosci., 19, 9, 2576–2582. [2] Hattori E., Liu C., Zhu H., Gershon E.S., 2005, Genetic tests of biologic systems in affective disorders, Mol. Psychiatry, 10, 8, 719–740.
P.1.b Basic neuroscience – Neuroanatomy and neurophysiology [3] Pothion S., Bizot J.C., Trovero F., Belzung C., 2004, Strain differences in sucrose preference and in the consequences of unpredictable chronic mild stress, Behav. Brain Res., 155, 1, 135–146.
P.1.a.016 Genetic and biological markers of families of patients with schizophrenia L.A. Ryadovaya, E.V. Gutkevich ° , L.P. Pitina, L.A. Gorbacevich. Mental Health Research Institute, Tomsk Science Centre SB RAMSci, Tomsk, Russian Federation Objective: Study genetic and biological markers of families of patients with schizophrenia’s disorders for the increase efficiency of medical-genetic aid. Methods: 90 families of patients with schizophrenia were investigated. The distribution of the diagnoses among the patients are the following: paranoid schizophrenia compose 36.7%, simple schizophrenia – 18.9%, schizotypal disorders – 12.2%, schizoaffective disorders – 7.8%. The clinical and genealogical analysis, the analysis of groups of blood by system ABO and a Rhesus factor, estimate of processes of apoptosis at receptor and celllike levels for the patients and relatives of the first degree of relationship have been carried out. Results: Data about the state of health of 1506 relatives of five degrees of relationship of patients have been received. 1.2 siblings; 0.3 children; 0.1 grandsons; 0.3 spouses corresponds to 1 patient. Middle age of a sibling equals 33.0 years, of a child – 18.4 years, of a grandson – 6.8 years. The risk of occurrence schizophrenia for siblings of patients makes from 1.3% up to 9.9%, for children of patients – from 1.4% up to 7.7%. Thus, siblings and children of patients concern to the group of average and high risk of occurrence schizophrenia. Spreading of a pathology among relatives was about 6.24% (schizophrenia – in 0.68% of cases). There was the accumulation of repeated cases of disorders in these families and basic share of pathology was marked among the first degrees of relationships. The distribution of groups of blood among patients was following: the group O (I) compose 38.9%, A (II) – 27.4%, B (III) – 24.2%, AB (IV) – 9.5%. 86.3% of patients had Rh+ and 13.7% ones had rh−, i.e. in comparison with the general population among patients there was the increase of number of persons with groups of blood O (I) (p = 0.2435) and AB (IV) and persons with Rh+. These groups of a blood correlate with such somatopathies as diseases of a gastrointestinal tract, cardiovascular and kidneys. We have observed statistically significant increase of expression of a receptor CD95 in patients with schizophrenia in comparison with control (p < 0.05), for the relatives of patients this metric statistically did not differ from control (17.71±1.06%, 15.56±1.04% and 12.00±0.77%, accordingly). The level of spontaneous apoptosis of neutrophils for the persons with schizophrenia differed from values of control, the tendency to increase of this metric is characteristic for their relatives in comparison with control (1.50±0.37%, 0.70±0.34% and 0.25±0.12%, accordingly). Such neutrophils had the smaller size, sometimes in cytoplasm a little large vacuoles were allocated on one pole. Its nucleus had the smaller size with condensation and granulation chromatine on perimeter of its border. In blood of patients and their relatives the lymphocytes with fragmented nucleus were observed reliably more often in comparison with control (2.67±0.62%, 4.04±1.12% and 0.97±0.35%, accordingly, p < 0.05). Conclusions: Schizophrenia – multifactorial disorder, its development depend on genetic, biological and external factors.
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P.1.b Neuroanatomy and neurophysiology P.1.b.001 Registration of multiunit activity and analysis of neuro-neuronal, cardio-neuronal and respiratory-neuronal temporal relationships H. Prudnikau ° . Belarusian State Medical University, Normal Physiology, Minsk, Belarus The goal was to elaborate computerized system for registration of multiunit activity and statistical analysis of temporal neuroneuronal, cardio-neuronal and respiratory-neuronal relationships. The capabilities of created system are: registration of multiunit activity, ECG and external respiration; identification of separate neuron spikes in multiunit records and the spike sorting by single, double, triple or quadruple template matching algorithms (which allow to find out spikes of 2−4 neurons with using single microelectrode); the quality control of spike identification and sorting by estimation its sensitivity and specificity; computing all parameters of ECG and time of respiratory cycles outset [3]. The following complex of statistical processing is possible: 1. computing of neuronal discharges frequency and variability of neuronal firing rate; 2. detection of neuronal discharges patterns diversity, maximal quantity of inter-spike intervals in patterns, degree of contrast of generated patterns; 3. computing of diversity of the neuronal group functional conditions (“functional mosaic” of neuronal groups), degree of contrast of these conditions; 4. detection of temporal relationships between the separate neurons activity by using cross-correlograms [2]; 5. detection of temporal relationships between neuronal activity and heart by cardiac-cycle triggered correlograms as well as between neuronal activity and external respiration by respiratoryneuronal correlograms [1]; 6. detection of relationships between neuronal activity or external respiration and changes of ECG’s parameters by parametric histogram; 7. detection of accordingly reorganized temporal neuronal relationships by spectral analysis of cross-intervals instant frequencies; 8. analysis of accordance of the separate neuron discharges patterns or “functional mosaic” of neuronal network activity with the certain conditions of neuro-neuronal (cardio-neuronal and respiratory-neuronal) temporal relationships; 9. analysis of accordance of the separate neuron activity or heart (external respiration) rate with changes of neuro-neuronal (cardio-neuronal, respiratory-neuronal) temporal relationships by computing correlation between spectral components of the cross-intervals instant frequencies and spectral components of the instant firing rates of neuronal discharges or instant frequencies of external respiration and heart contractions; 10. examination of state of the separate neuro-neuronal (cardioneuronal or respiratory-neuronal) temporal relationships after discharges of certain neurons by special parametric histograms; 11. estimation of possible influences of certain neurons on activity of adjacent neurons and its neuro-neuronal (cardio-neuronal or respiratory-neuronal) temporal relationships efficacy by realistic post-experimental modeling. The multiunit activity (twenty neuronal group, 2−4 neurons in each group) was recorded in dorsal medulla (NTS) of anesthetized rats. Sensitivity of spike identification was 96−99%. Specificity
P.1.a Basic neuroscience – Genetics and genomics
about 10 years ago, many studies have focused on the mechanism of delayed action of antidepressants to the intracellular signal transductions related to the structural plasticity and neuronal adaptation in the central nervous system. Though fluoxetine increases serotonin and/or norpinephrine, many studies have suggested that therapeutic effects might be achieved by gene expression regulations through complicated processes of the neurogenesis. The aim of this study is to identify expressed gene differences in rat hippocampus between the experimental group and control group using cDNA microarray chip techniques during the 5 and 14 day after fluoxetine or distilled water administration especially with the relation of neurogenesis. By these results, we are trying to make some implications about what kind of genes acts on the delayed actions of antidepressants and how it does. Methods: By using 7 week old (200~250 g) SPF (specific pathogen free) Sprague-Dawley line male rats, we identified differently regulated genes in rat hippocmapus between fluoxetine administration group and control group using cDNA microarray chip techniques. For the experimental group rats, 10 mg per Kg fluoxetine dissolved in distilled water (40 mg/10 ml) contained in the syringe was administrated per oral. And for the control group rats, same amount of distilled water only was administrated. We analyzed expression pattern of genes after fluoxetine in rat during 5 and 14 days. And, a part of the regulated genes in cDNA microarray results were confirmed by real-time RT-PCR. Results: When we analyzed expression pattern of genes during 5 days and 14 days after fluoxetine treatment in rat hippocampus, some genes of fluoxetine treated rat hippocampus increased dramatically especially during 14 days compared to that of control. Camk2d, one of isotypes of Camk2 that is known to play important role in the survival of neural cell, synaptic plasticity and memory in hippocampus, in fluoxetine-treated rat hippocampus during 14 days were 2.6 times higher than in control rat by cDNA microarray chip assessment. mRNA of Map2k2, one intermediate of BNDF pathway, also increased 1.8 times higher than in control during 14 days after fluoxetine treatment. It is known that patients with depression have lower level of transthyretin that plays an important role in the transport of thyroid hormone in the central nervous system. In this study, mRNA transthyretin expression increased during 5 and 14 days after fluoxetine treatment. Conclusion: This results imply that chronic treatment of fluoxetine promotes expression of genes related to the survival of neural cell and neurogenensis in addition to the transport of thyroid hormone. It suggests that expression of some of important genes which play important roles in the neurogenesis and intracellular signal transduction cascade might be related to the delayed actions of antidepressants. References [1] Duman RS, Malberg J, Nakagawa S, Di’Sa C, 2000, Neuronal plasticity and survival in mood disorders, Biol Psychiatry, 48, 732–739. [2] Landgrebe J, Welzl G, Metz T, Van Gaalen MM, Ropers H, Wurst W, Holsboer F, 2002, Molecular characterization of antidepressant effects in the mouse brain using gene expression profiling, J Psychiatr Res, 36, 119–129. [3] Mason GA, Bondy SC, Nemeroff CB, Walker CH, Prange AJ, 1987, The effects of thyroid state on beta-adrenergic and serotonergic receptors in rat brain, Psychoneuroendocrinology, 12, 261–270.
P.1.a.015 Strain differences in susceptibility to unpredictable chronic mild stress as assessed by hippocampal gene expression profiling J.L. Paya-Cano1 ° , F. Sluyter1 , Y.S. Mineur2 , C. Fernandes1 , W.E. Crusio3 , L.C. Schalkwyk1 . 1 Institute of Psychiatry/King’s College, SGDP, London, United Kingdom; 2 Yale School of Medicine, Pyschiatry, Pharmacology and Neurobiology, New Haven, USA; 3 Centre National de la Recherche Scientifique, Laboratoire de Neurosciences Cognitives, Talence, France Introduction: Exposure to unpredictable chronic mild stress (UCMS) induces depression-like behaviours in rodents. Furthermore, it has been shown that inbred strains of mice present different thresholds of susceptibility to UCMS (Pothion et al. 2004). On the other hand, gene expression profiling across strains constitute a useful tool for dissecting the molecular mechanisms underlying behavioural variation found between inbred mice (Fernandes et al. 2004). In the present experiment we assessed gene expression profiles across two inbred strains of mice subjected to either UCMS or no treatment in order to examine the potential effects of UCMS on gene expression in the hippocampus. Materials and methods: At the age of two months 16 mice from C57BL/6J (B6) and BALB/cJ (Balb) strains were assigned to one of two treatment groups, controls (ctrl, n = 8) and UCMS (n = 8). The stress procedure lasted for 4 weeks prior to the behavioural testing. Mice were subjected to different kinds of stressors such as: cage tilting, damp sawdust, predator sounds, placement in an empty cage, placement in an empty cage with water on the bottom, inversion of the light/dark cycle, lights on for a short period of time during the dark phase, and switching cages. Stressors continued to be applied during the testing phase, except on testing days to avoid effects of acute stress. In the 5th and 6th weeks of UCMS, animals were tested in an anxiety test (dark-light box) and a learning task (cued and contextual fear conditioning), respectively. In the 7th week of UCMS, animals were subjected to the tail suspension test and 24 hours later to the forced swim test (results not shown). After testing, left and right hippocampi were dissected out, pooled and stored at −800ºC. A total of 16 MOE430A GeneChip arrays corresponding to individual animals were used. Analysis: Based on neuropharmacological and other evidence on the aetiology of affective disorders (Hattori et al. 2005), we selected 252 candidate genes. Most of these genes have a mouse homologue and most of these homologues are represented on the 430A array. These probesets were analyzed using a 2-way ANOVA with strain (B6, Balb) and treatment (UCMS, ctrl) as main factors. Results: After Bonferroni correction for multiple comparisons, our results showed effects of strain (26 probesets), treatment (1 probeset) and strain × treatment interaction (7 probesets) in gene expression in the hippocampus. Conclusions. As expected, the analysis of gene expression profiling of the selected set of candidate genes for affective disorders revealed a greater number of differences related to strain than for the environmental UCMS manipulation. References [1] Fernandes C., Paya-Cano J.L., Sluyter F., D’Souza U., Plomin R., Schalkwyk L.C., 2004, Hippocampal gene expression profiling across eight mouse inbred strains: Towards understanding the molecular basis for behaviour, Eur. J. Neurosci., 19, 9, 2576–2582. [2] Hattori E., Liu C., Zhu H., Gershon E.S., 2005, Genetic tests of biologic systems in affective disorders, Mol. Psychiatry, 10, 8, 719–740.
P.1.b Basic neuroscience – Neuroanatomy and neurophysiology [3] Pothion S., Bizot J.C., Trovero F., Belzung C., 2004, Strain differences in sucrose preference and in the consequences of unpredictable chronic mild stress, Behav. Brain Res., 155, 1, 135–146.
P.1.a.016 Genetic and biological markers of families of patients with schizophrenia L.A. Ryadovaya, E.V. Gutkevich ° , L.P. Pitina, L.A. Gorbacevich. Mental Health Research Institute, Tomsk Science Centre SB RAMSci, Tomsk, Russian Federation Objective: Study genetic and biological markers of families of patients with schizophrenia’s disorders for the increase efficiency of medical-genetic aid. Methods: 90 families of patients with schizophrenia were investigated. The distribution of the diagnoses among the patients are the following: paranoid schizophrenia compose 36.7%, simple schizophrenia – 18.9%, schizotypal disorders – 12.2%, schizoaffective disorders – 7.8%. The clinical and genealogical analysis, the analysis of groups of blood by system ABO and a Rhesus factor, estimate of processes of apoptosis at receptor and celllike levels for the patients and relatives of the first degree of relationship have been carried out. Results: Data about the state of health of 1506 relatives of five degrees of relationship of patients have been received. 1.2 siblings; 0.3 children; 0.1 grandsons; 0.3 spouses corresponds to 1 patient. Middle age of a sibling equals 33.0 years, of a child – 18.4 years, of a grandson – 6.8 years. The risk of occurrence schizophrenia for siblings of patients makes from 1.3% up to 9.9%, for children of patients – from 1.4% up to 7.7%. Thus, siblings and children of patients concern to the group of average and high risk of occurrence schizophrenia. Spreading of a pathology among relatives was about 6.24% (schizophrenia – in 0.68% of cases). There was the accumulation of repeated cases of disorders in these families and basic share of pathology was marked among the first degrees of relationships. The distribution of groups of blood among patients was following: the group O (I) compose 38.9%, A (II) – 27.4%, B (III) – 24.2%, AB (IV) – 9.5%. 86.3% of patients had Rh+ and 13.7% ones had rh−, i.e. in comparison with the general population among patients there was the increase of number of persons with groups of blood O (I) (p = 0.2435) and AB (IV) and persons with Rh+. These groups of a blood correlate with such somatopathies as diseases of a gastrointestinal tract, cardiovascular and kidneys. We have observed statistically significant increase of expression of a receptor CD95 in patients with schizophrenia in comparison with control (p < 0.05), for the relatives of patients this metric statistically did not differ from control (17.71±1.06%, 15.56±1.04% and 12.00±0.77%, accordingly). The level of spontaneous apoptosis of neutrophils for the persons with schizophrenia differed from values of control, the tendency to increase of this metric is characteristic for their relatives in comparison with control (1.50±0.37%, 0.70±0.34% and 0.25±0.12%, accordingly). Such neutrophils had the smaller size, sometimes in cytoplasm a little large vacuoles were allocated on one pole. Its nucleus had the smaller size with condensation and granulation chromatine on perimeter of its border. In blood of patients and their relatives the lymphocytes with fragmented nucleus were observed reliably more often in comparison with control (2.67±0.62%, 4.04±1.12% and 0.97±0.35%, accordingly, p < 0.05). Conclusions: Schizophrenia – multifactorial disorder, its development depend on genetic, biological and external factors.
S217
P.1.b Neuroanatomy and neurophysiology P.1.b.001 Registration of multiunit activity and analysis of neuro-neuronal, cardio-neuronal and respiratory-neuronal temporal relationships H. Prudnikau ° . Belarusian State Medical University, Normal Physiology, Minsk, Belarus The goal was to elaborate computerized system for registration of multiunit activity and statistical analysis of temporal neuroneuronal, cardio-neuronal and respiratory-neuronal relationships. The capabilities of created system are: registration of multiunit activity, ECG and external respiration; identification of separate neuron spikes in multiunit records and the spike sorting by single, double, triple or quadruple template matching algorithms (which allow to find out spikes of 2−4 neurons with using single microelectrode); the quality control of spike identification and sorting by estimation its sensitivity and specificity; computing all parameters of ECG and time of respiratory cycles outset [3]. The following complex of statistical processing is possible: 1. computing of neuronal discharges frequency and variability of neuronal firing rate; 2. detection of neuronal discharges patterns diversity, maximal quantity of inter-spike intervals in patterns, degree of contrast of generated patterns; 3. computing of diversity of the neuronal group functional conditions (“functional mosaic” of neuronal groups), degree of contrast of these conditions; 4. detection of temporal relationships between the separate neurons activity by using cross-correlograms [2]; 5. detection of temporal relationships between neuronal activity and heart by cardiac-cycle triggered correlograms as well as between neuronal activity and external respiration by respiratoryneuronal correlograms [1]; 6. detection of relationships between neuronal activity or external respiration and changes of ECG’s parameters by parametric histogram; 7. detection of accordingly reorganized temporal neuronal relationships by spectral analysis of cross-intervals instant frequencies; 8. analysis of accordance of the separate neuron discharges patterns or “functional mosaic” of neuronal network activity with the certain conditions of neuro-neuronal (cardio-neuronal and respiratory-neuronal) temporal relationships; 9. analysis of accordance of the separate neuron activity or heart (external respiration) rate with changes of neuro-neuronal (cardio-neuronal, respiratory-neuronal) temporal relationships by computing correlation between spectral components of the cross-intervals instant frequencies and spectral components of the instant firing rates of neuronal discharges or instant frequencies of external respiration and heart contractions; 10. examination of state of the separate neuro-neuronal (cardioneuronal or respiratory-neuronal) temporal relationships after discharges of certain neurons by special parametric histograms; 11. estimation of possible influences of certain neurons on activity of adjacent neurons and its neuro-neuronal (cardio-neuronal or respiratory-neuronal) temporal relationships efficacy by realistic post-experimental modeling. The multiunit activity (twenty neuronal group, 2−4 neurons in each group) was recorded in dorsal medulla (NTS) of anesthetized rats. Sensitivity of spike identification was 96−99%. Specificity