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Neurogenesis in vitro of adult human olfactory epithelium
178
Epidemiological studies have indicated that viral infections may play a role in many cases of schizophrenia. The recent availability of well preserved post-mortem brain samples from diseased individuals and controls has allowed us to apply molecular techniques to address the relationship between viral infections and schizophrenia. Nucleic Acids were extracted from defined regions of cases and controls, and analyzed by the following methods: ( 1) RNA libraries expressed in phagemid messages; (2) DNA subtractive hybridization; (3) Polymerase Chain Reaction (PCR) using arbitrary primers directed at coding exons; (4) PCR using primers directed at conserved viral sequences. We have identified DNA and RNA species present in the brains of individuals with schizophrenia but absent or present in lower concentrations in analogous samples from control individuals. These studies resulted in the identification of viral and viralassociated sequences which may be associated with disease pathogenesis. Viral sequences which are identified include those homologous to enzymatic proteins of retroviruses similar to HIV and HTLV-1 as well as other mammalian viruses. We also have identified viral-associated RNAs including ones homologous to viral-induced transcriptionally active proteins and to proteins involved in the immune response. These studies point to a central role for viral infections in the pathogenesis of schizophrenia.
X.E. Developmental X.E.1 EVIDENCE
OF BRAIN
DISGENESY
IN
SCHIZOPHRENIA. A NEUROANATOMICAL CONTROLLED STUDY FROM AN ASYLUM I N S.AO P A U L O , B R A Z I L S.H. C o u r a a n d H. Elkis
Department of Psychiatry, Institute of Psychiatry, Universityof S~o Paulo, Brazil," Juquery Mental Hospital, Sao Paulo, Brazil
gender distribution between the two groups. Patients with schizophrenia had significant degrees of brain disgenesis in the following areas (results of Fisher exact test): frontal (0.02), parietal (0.0007), temporal (0.03) and occipital (0.0009). Conclusions. Since disgenesis is defined as a neurodevelopmental abnormality, this study provides some support for the neurodevelopmental hypothesis of the etiology of schizophrenia.
X.E.2 DEVELOPMENT
OF NEURONS
FETAL CEREBRAL
IN THE
CORTEX
L.J. G a r e y a n d X.X. Yah
Department of Anatomy, Charing Cross & WestminsterMedical School, London W6 8RF, U.K. Schizophrenia may involve defective development or migration of neurons in the cerebral cortex. To better understand this phenomenon, we have followed development of human fetal cortex. First, development of gamma-aminobutyric acid (GABA) neurons from 14 weeks of gestation (14W) to term was studied. At 14W few GABA cells occur in the developing cortex itself; they are mainly in the proliferative ventricular zone and the developing white matter, but by 20W the density of GABA neurons is highest in the deep layers of the definitive cortex. The GABA peak moves superficially to layer IV by 30W, after which the distribution stabilises with bands in layers IV/V and II/III. Next we studied neurons containing nitric oxide. Large neurons are first seen at 15W and achieve an adult pattern by 32W, especially in the white matter. Smaller cells become recognisible in the cortex from 32W and increase in number to term. The calcium-binding protein parvalbumin (PV) is expressed by cells in layer I by 20W but this activity is transient and mostly lost by term. PV cells appear in layer V by 20W and by 30W occupy layers IV-VI, where they form clusters from 30W.
X.E.3
Background. Post-mortem findings in brains of schizophrenics show a great variability and no specific abnormalities. Additionally, histological findings are also non specific. However, macroscopic examination of the brain may represent an important tool in the identification disgenetic abnormalities, which are supposed to be of neurodevelopmental origin. Methods. The study was carried in Juquery Mental Hospital. Twenty brains of patients with schizophrenia (mean age at death 55.00_+ 11.62; 8 males, 12 female) were examined and compared to 10 normal controls (mean age at death 45.20+20.47; 5 male, 5 female). Controls were non mentally ill patients suffering from medical diseases not related to the brain. Gyri and sulci abnormalities were assessed in both groups in the following brain areas: frontal, temporal, parietal, occipital and cingulate gyrns. All brains were also weighted. Results. No difference was found in brain age, weight and
NEUROGENESIS IN VITRO OF ADULT HUMAN OLFACTORY EPITHELIUM W. Murrell, G. Bushell, J. M c G r a t h * , P. Bates a n d A. M a c k a y - S i m
Faculty of Science and Technology, Griffith University, Nathan, QLD 4111, Australia, *ClinicalStudies Unit, WolstonPark Hospital, Wacol, QLD 4076, Australia The study of neurodevelopment disorders such as schizophrenia would benefit from examination of neural proliferation and differentiation in cultures derived from adults. Olfactory epithelium (OE) retains the ability to proliferate during adulthood and offers an attractive model to examine neurogenesis.
179
Controls Fem Frontal to temporal pole distance:
Left Right
Occipital to temporal pole distance:
Left Right
4.40 4.46 12.9 13.0
Schiz Male 4.30 4.30 13.1 13.1
OE was collected post-mortem from 12 subjects. Fragments of OE were cultured in serum-free medium for 5 days during which a flat epithelial-like outgrowth of cells appeared. At Day 5 basic fibroblast growth factor (FGF2; 50ng/ml) was added to the medium. 3H-thymidine was added to the medium at this time (0.03 microCi/ml). After 10-12 days in vitro the cultures were subject to immunocytochemistry using antibodies for several neural markers, including olfactory marker protein (OMP) which is expressed by mature olfactory sensory cells. Neurogenesis was observed in OE from adult humans. FGF2 induced the outgrowth of differentiated bipolar neurons which stained positive with antibodies to OMP, neurofilament, betatubulin, neuron-specific enolase, and microtubule associated protein-2. OMP-positive neurons were born in vitro, confirmed by 3H-thymidine autoradiography. Differences between controls and patient groups are currently being investigated. The ability to grow neurons and to observe neurogenesis in vitro from tissues from adults with serious psychiatric illness will expand the horizons of biological psychiatry. This project is supported by the Stanley Medical Research Foundation, the Garnett Passe and Rodney Williams Memorial Foundation, and the National Health and Medical Research Council of Australia.
X.F. Structural
Fem 4.74 4.72 12.5 12.4
Dep Male 4.84 4.50 12.9 13.0
Fem 4.63 4.66 12.8 13.0
Org Male 4.69 4.61 13.0 13.2
Fem 4.51 4.67 12.7 12.7
Male 4.50 4.41 12.7 13.3
Model MMIII) and interactive software (Autosketch) on a PC. Brain weight (reduced in organics) and brain length did not differ between patients with schizophrenia and controls, but measures related to position of the temporal pole did: Covaried for brain length frontal to temporal pole distance was longer in schizophrenic patients than controls (p=0.02 left; p = 0.03 right) and occipital to temporal pole distance was shorter (p = 0.13 left; p = 0.02 right). The findings in the depressive group did not differ from controls; in organics there was a trend to reduction in both measures. The findings are consistent with other studies (eg Dauphinais et al 1990); suggesting absence or loss of substance in the temporal lobe in schizophrenia.
References Dauphinais ID et al. Psychiatr Res Neuroimaging 1990; 35: 137 147.
X.F.2 A STUDY OF SYLVIAN FISSURE ASYMMETRIES IN THE RUNWELL 3 SERIES OF PSYCHOTIC BRAINS R. Highley a n d T.J. Crow
X.F.I TEMPORAL LOBE L E N G T H REDUCTIONS IN SCHIZOPHRENIA--A POST-MORTEM PHOTOGRAPHIC STUDY OF UNFIXED BRAIN FROM THE R U N W E L L 3 SERIES T.J. Crow a n d L.R. C r o w
University Department of Neurology, University Department of Neurology, The Radcliffe Hospital, Oxford, U.K Brains (the Runwell 3 series) collected at post-mortem from patients with schizophrenia (n=41) by DSMIIIR or RDC criteria, depression or suicide (n = 60), controls (n = 56) and an "organic" group (n=56, including 44 cases with a clinical diagnosis of senile or presenile dementia) were photographed from the lateral and inferior aspects before fixation. Linear measures were taken using a digitising tablet (Summa graphics
MRC Schizophrenia Research Group, Radcliffe Infirmary, Oxford, OX2 6HE, U.K. Photographs of the lateral surface of unfixed PM brains from normal controls (n = 56), patients with organic pathology (n=56), non-psychotic and psychotic depression (n=60), and schizophrenia (n = 41, the Runwell 3 Series), were used to study sylvian fissure (SF) morphology. SF length was assessed as described by Falkai et al. (1992) (method 1 ), and by addition of the two segments defined by Heschl's sulcus (method 2). Photographs were included only when available for both sides of the brain, and well orientated. An asymmetry coefficient was calculated for the SF length for each brain according to the formula: Asymmetry=(left-right)/(left+right). The mean coefficients are given below. Although there was a reduction in asymmetry in each disease group (greatest in the psychotics), the comparison with controls did not reach the 5% significance level. With method 2, there
Epidemiological studies have indicated that viral infections may play a role in many cases of schizophrenia. The recent availability of well preserved post-mortem brain samples from diseased individuals and controls has allowed us to apply molecular techniques to address the relationship between viral infections and schizophrenia. Nucleic Acids were extracted from defined regions of cases and controls, and analyzed by the following methods: ( 1) RNA libraries expressed in phagemid messages; (2) DNA subtractive hybridization; (3) Polymerase Chain Reaction (PCR) using arbitrary primers directed at coding exons; (4) PCR using primers directed at conserved viral sequences. We have identified DNA and RNA species present in the brains of individuals with schizophrenia but absent or present in lower concentrations in analogous samples from control individuals. These studies resulted in the identification of viral and viralassociated sequences which may be associated with disease pathogenesis. Viral sequences which are identified include those homologous to enzymatic proteins of retroviruses similar to HIV and HTLV-1 as well as other mammalian viruses. We also have identified viral-associated RNAs including ones homologous to viral-induced transcriptionally active proteins and to proteins involved in the immune response. These studies point to a central role for viral infections in the pathogenesis of schizophrenia.
X.E. Developmental X.E.1 EVIDENCE
OF BRAIN
DISGENESY
IN
SCHIZOPHRENIA. A NEUROANATOMICAL CONTROLLED STUDY FROM AN ASYLUM I N S.AO P A U L O , B R A Z I L S.H. C o u r a a n d H. Elkis
Department of Psychiatry, Institute of Psychiatry, Universityof S~o Paulo, Brazil," Juquery Mental Hospital, Sao Paulo, Brazil
gender distribution between the two groups. Patients with schizophrenia had significant degrees of brain disgenesis in the following areas (results of Fisher exact test): frontal (0.02), parietal (0.0007), temporal (0.03) and occipital (0.0009). Conclusions. Since disgenesis is defined as a neurodevelopmental abnormality, this study provides some support for the neurodevelopmental hypothesis of the etiology of schizophrenia.
X.E.2 DEVELOPMENT
OF NEURONS
FETAL CEREBRAL
IN THE
CORTEX
L.J. G a r e y a n d X.X. Yah
Department of Anatomy, Charing Cross & WestminsterMedical School, London W6 8RF, U.K. Schizophrenia may involve defective development or migration of neurons in the cerebral cortex. To better understand this phenomenon, we have followed development of human fetal cortex. First, development of gamma-aminobutyric acid (GABA) neurons from 14 weeks of gestation (14W) to term was studied. At 14W few GABA cells occur in the developing cortex itself; they are mainly in the proliferative ventricular zone and the developing white matter, but by 20W the density of GABA neurons is highest in the deep layers of the definitive cortex. The GABA peak moves superficially to layer IV by 30W, after which the distribution stabilises with bands in layers IV/V and II/III. Next we studied neurons containing nitric oxide. Large neurons are first seen at 15W and achieve an adult pattern by 32W, especially in the white matter. Smaller cells become recognisible in the cortex from 32W and increase in number to term. The calcium-binding protein parvalbumin (PV) is expressed by cells in layer I by 20W but this activity is transient and mostly lost by term. PV cells appear in layer V by 20W and by 30W occupy layers IV-VI, where they form clusters from 30W.
X.E.3
Background. Post-mortem findings in brains of schizophrenics show a great variability and no specific abnormalities. Additionally, histological findings are also non specific. However, macroscopic examination of the brain may represent an important tool in the identification disgenetic abnormalities, which are supposed to be of neurodevelopmental origin. Methods. The study was carried in Juquery Mental Hospital. Twenty brains of patients with schizophrenia (mean age at death 55.00_+ 11.62; 8 males, 12 female) were examined and compared to 10 normal controls (mean age at death 45.20+20.47; 5 male, 5 female). Controls were non mentally ill patients suffering from medical diseases not related to the brain. Gyri and sulci abnormalities were assessed in both groups in the following brain areas: frontal, temporal, parietal, occipital and cingulate gyrns. All brains were also weighted. Results. No difference was found in brain age, weight and
NEUROGENESIS IN VITRO OF ADULT HUMAN OLFACTORY EPITHELIUM W. Murrell, G. Bushell, J. M c G r a t h * , P. Bates a n d A. M a c k a y - S i m
Faculty of Science and Technology, Griffith University, Nathan, QLD 4111, Australia, *ClinicalStudies Unit, WolstonPark Hospital, Wacol, QLD 4076, Australia The study of neurodevelopment disorders such as schizophrenia would benefit from examination of neural proliferation and differentiation in cultures derived from adults. Olfactory epithelium (OE) retains the ability to proliferate during adulthood and offers an attractive model to examine neurogenesis.
179
Controls Fem Frontal to temporal pole distance:
Left Right
Occipital to temporal pole distance:
Left Right
4.40 4.46 12.9 13.0
Schiz Male 4.30 4.30 13.1 13.1
OE was collected post-mortem from 12 subjects. Fragments of OE were cultured in serum-free medium for 5 days during which a flat epithelial-like outgrowth of cells appeared. At Day 5 basic fibroblast growth factor (FGF2; 50ng/ml) was added to the medium. 3H-thymidine was added to the medium at this time (0.03 microCi/ml). After 10-12 days in vitro the cultures were subject to immunocytochemistry using antibodies for several neural markers, including olfactory marker protein (OMP) which is expressed by mature olfactory sensory cells. Neurogenesis was observed in OE from adult humans. FGF2 induced the outgrowth of differentiated bipolar neurons which stained positive with antibodies to OMP, neurofilament, betatubulin, neuron-specific enolase, and microtubule associated protein-2. OMP-positive neurons were born in vitro, confirmed by 3H-thymidine autoradiography. Differences between controls and patient groups are currently being investigated. The ability to grow neurons and to observe neurogenesis in vitro from tissues from adults with serious psychiatric illness will expand the horizons of biological psychiatry. This project is supported by the Stanley Medical Research Foundation, the Garnett Passe and Rodney Williams Memorial Foundation, and the National Health and Medical Research Council of Australia.
X.F. Structural
Fem 4.74 4.72 12.5 12.4
Dep Male 4.84 4.50 12.9 13.0
Fem 4.63 4.66 12.8 13.0
Org Male 4.69 4.61 13.0 13.2
Fem 4.51 4.67 12.7 12.7
Male 4.50 4.41 12.7 13.3
Model MMIII) and interactive software (Autosketch) on a PC. Brain weight (reduced in organics) and brain length did not differ between patients with schizophrenia and controls, but measures related to position of the temporal pole did: Covaried for brain length frontal to temporal pole distance was longer in schizophrenic patients than controls (p=0.02 left; p = 0.03 right) and occipital to temporal pole distance was shorter (p = 0.13 left; p = 0.02 right). The findings in the depressive group did not differ from controls; in organics there was a trend to reduction in both measures. The findings are consistent with other studies (eg Dauphinais et al 1990); suggesting absence or loss of substance in the temporal lobe in schizophrenia.
References Dauphinais ID et al. Psychiatr Res Neuroimaging 1990; 35: 137 147.
X.F.2 A STUDY OF SYLVIAN FISSURE ASYMMETRIES IN THE RUNWELL 3 SERIES OF PSYCHOTIC BRAINS R. Highley a n d T.J. Crow
X.F.I TEMPORAL LOBE L E N G T H REDUCTIONS IN SCHIZOPHRENIA--A POST-MORTEM PHOTOGRAPHIC STUDY OF UNFIXED BRAIN FROM THE R U N W E L L 3 SERIES T.J. Crow a n d L.R. C r o w
University Department of Neurology, University Department of Neurology, The Radcliffe Hospital, Oxford, U.K Brains (the Runwell 3 series) collected at post-mortem from patients with schizophrenia (n=41) by DSMIIIR or RDC criteria, depression or suicide (n = 60), controls (n = 56) and an "organic" group (n=56, including 44 cases with a clinical diagnosis of senile or presenile dementia) were photographed from the lateral and inferior aspects before fixation. Linear measures were taken using a digitising tablet (Summa graphics
MRC Schizophrenia Research Group, Radcliffe Infirmary, Oxford, OX2 6HE, U.K. Photographs of the lateral surface of unfixed PM brains from normal controls (n = 56), patients with organic pathology (n=56), non-psychotic and psychotic depression (n=60), and schizophrenia (n = 41, the Runwell 3 Series), were used to study sylvian fissure (SF) morphology. SF length was assessed as described by Falkai et al. (1992) (method 1 ), and by addition of the two segments defined by Heschl's sulcus (method 2). Photographs were included only when available for both sides of the brain, and well orientated. An asymmetry coefficient was calculated for the SF length for each brain according to the formula: Asymmetry=(left-right)/(left+right). The mean coefficients are given below. Although there was a reduction in asymmetry in each disease group (greatest in the psychotics), the comparison with controls did not reach the 5% significance level. With method 2, there