- Email: [email protected]
Clinical research in neuropsychiatry
Clinical Research in Neuropsychiatry Michael H. Ebert, M.D. David Goldman, M.D. Linda E. Nee, M.S.W. Section on Experimental Bethesda, Ma yland
Therapeutics,
Laboratory
of Clinical Science, National Institute of Mental Health,
Abstract: This paper presents the thesis that rapidly developing areas of knowledge in neuroscience will rekindle interest in neuropsychiaty, and increase scientific and clinical interaction betweenpsychiatrists and neurologists in teaching hospitals. A neuropsychiatric clinical research unit at the National Institute of Mental Health is described. Examples of research conducted on the unit illustrate areas of biological science that are likely to increase the interface between psychiaf y and neurology. Hopefully, the explosion of knowledge in the basic neurosciences in the last decade will be followed in this decade by clinical research of increasing specificity and sophistication on central nervous system disorders.
Introduction There is increasing commentary on the scientific and clinical interface between psychiatry and neurology. This is so in spite of the fact that, as Norman Geschwind points out, “this common ground unfortunately bears more resemblance to a no-man’s land than to an open border. While neurologists tend to mutter darkly about the failure of psychiatrists to be aware of the brain as the organ of the mind, psychiatrists, perhaps somewhat defensively, have stressed their awarenessof the whole man, biologic as well as psychologic. Unfortunately, few members of either group have, in fact, really interested themselves in the borderland area, and too frequently interactions between them are educationally disappointing, whether at the level of mutual consultation or in the interchange of residents for training“ (1). Rapidly expanding areas of knowledge in basic General Hospital Psychiatry 5, 105-109, 1983 Q Elsevier Science Publishing Co., Inc. 1983 52 Vanderbilt Avenue, New York, NY 10017
neuroscience have led to a renewal of interest in neuropsychiatry. New knowledge concerning neurotransmitter biochemistry and physiology has stimulated neurochemical hypotheses about the etiology of a variety of neurological and psychiatric illnesses. New technologies are available to study the function of the central nervous system in clinical research including neurochemical assays, neurophysiological procedures such as averaged evoked potentials, and various radiological scanning procedures. These developments have stimulated clinical research on disorders such as Huntington’s chorea, Gilles de la Tourette’s syndrome, Parkinson’s disease, tardive dyskinesia, and Alzheimer’s dementia. During the same time, but for different reasons, there has been heightened public interest in some of these diseases. Lay interest groups such as the Committee to Combat Huntington’s Disease, the Tourettes Syndrome Association, and the Alzheimer’s Disease and Related Disorders Association have stimulated clinical research through public education efforts, fund raising, and lobbying. Recognition of the growing medical problems that the aging population presents to our society has stimulated research in the presenile dementias. Currently both neurologists and psychiatrists in academic settings are seeking additional training in areas of basic and clinical neuroscience. Furthermore, some diseases and syndromes, whose pathophysiology appears to be related to changes in neurotransmitter function, require both psychiatric and neurological clinical skills for optimal management of the patient. These facts may encourage the 105
ISSN 0163-8343/&X3%3.00
M. Ebert et. al. formation of treatment teams in academic hospitals and research institutes where neurologists, psychiatrists, and individuals trained in both specialties are working together and sharing common laboratory facilities. They may also stimulate a modest increase in the number of individuals seeking board certification in both psychiatry and neurology.
Interface Between Psychiatry Neurology
and
This paper focuses on the experience of operating a neuropsychiatric ward at the Clinical Center of the NIH over the past five years. The ward was established to carry out clinical research on some of the syndromes that occupy the interface between psychiatry and neurology. Clinical pharmacology has been the major scientific discipline on the ward, encompassing pharmacokinetics, clinical neurochemistry, and clinical neuropharmacology. Other scientific disciplines, particularly neuropsychology and clinical genetics have also played an important role. The ward has served as a base for offering fellowship training to psychiatrists and neurologists in clinical pharmacology. This clinical unit has been a successful example of operating in the interface between psychiatry and neurology with regard to clinical teaching and research training of post doctoral fellows and medical students. Clinical research from the unit has been published in approximately equal proportions in journals specialized in psychiatry, neurology, internal medicine, and pharmacology. Hopefully this neuropsychiatric unit offers a model that is pertinent to exploring the scientific and clinical relationship between departments of psychiatry and neurology in medical schools. The essential ingredients for such an operation are a critical mass of individuals engaged in clinical research and sufficient clinical research technology in place to conduct such studies. The ward was established with an interdisciplinary staff. There have been l-3 neurologists, l-3 psychiatrists, and 1 internist during each year of its operation. Most of the medical staff are in rotating fellowship positions for training in clinical research after having completed residency training. Several individuals have had training in both psychiatry and neurology. During the majority of the year there are three senior medical students on the ward. The interdisciplinary nature of the medical staff allows a comfortable margin of clinical knowledge to deal with neurological and medical patients 106
as well as psychiatric problems. After difficulty with a traditional psychiatric nursing staff, a satisfactory nursing group was established by incorporating a mixture of psychiatric and medical-surgical nurses. During the last several years, the scope of disorders studied on the unit has been as follows: Several presenile dementias have been studied from a neuropsychological and neuropharmacological point of view. They include Huntington’s chorea, Alzheimer’s dementia, and Korsakoff’s psychosis. Patients with Gilles de la Tourette’s syndrome have been studied with clinical genetic, neuropharmacological, and neurochemical techniques. Patients with anorexia nervosa and bulemia have been studied in the context of an inpatient behavioral treatment program from a neurochemical, neuroendocrine, and neuropharmacological standpoint. Patients with several forms of orthostatic hypotension (Shy-Drager’s syndrome and idiopathic orthostatic hypotension) have been studied from the point of view of the function and biochemistry of the autonomic nervous system. Collaborative projects on the psychopharmacology of the treatment of children with attention deficit disorder (or hyperkinetic syndrome) have been carried out using the ward as a day program for diagnostic workups and clinical trials. Several clinical studies that we have carried out in the illnesses discussed above illustrate areas of biological science that are particularly suitable to bridge the gap between clinical psychiatry and neurology. Although not an exclusive list, clinical and biochemical genetics, clinical neuropharmacology, and clinical neurochemistry are three important areas of investigation in this regard. In the area of biochemical genetics we have made an effort to apply the new technology of two dimensional electrophoresis to the study of central nervous system diseases. The search for trait-specific molecular markers is an important area of research in neuropsychiatry because such markers can refine diagnostic categories and give clues to etiology. Two-dimensional electrophoresis with computerized microdensitometry permits the quantitative characterization of up to 1000 cellular proteins for quantitative or positional variation. Using this technique, four categories of molecular variation can be sought in an individual with a disease. First, the mutant polypeptide responsible for a genetic disease or altered phenotype may be detected. In this way, a mutation in the @actin gene was detected in human fibroblasts and some of the altered properties of the mutant S-actin were char-
Clinical Research in Neuropsychiatry
acterized (2). Second, a characteristic pattern of secondary protein modulation in a metabolic disease may be noted. Modulations have now been quantitatively characterized using two dimensional electrophoresis in cells exposed to hormones (3), heat shock (4), neoplastic transformation (5), inborn errors of metabolism, as in the Lesch-Nyhan syndrome (6), and alterations in chromosome number, as in trisomy 21(7,8). Third, a protein produced by an infectious agent may be detected. Fourth, visualization of new groups of related polypeptides allows the detection of additional protein polymorphisms that can be used in linkage analysis. On a fundamental level, detection of these molecular variants by two dimensional electrophoresis will assist in the construction of the human genetic map, and in refining estimates of human genetic variation . The technique of two dimensional electrophoresis, developed by O’Farrell (9), involves two-stage electrophoresis, with isoelectric focusing in the first dimension to separate the denatured proteins on the basis of charge, and polyacrylamide gel electrophoresis in the second dimension for a separation on the basis of molecular weight. Proteins are visualized by staining or autoradiography. Staining is usually accomplished using a dye such as Coomassie blue or an ultrasensitive silver stain for proteins and other polymers (10). Autoradiography depends on radiolabelling of the proteins. This is usually done prior to electrophoresis by having cells incorporate radiolabelled precursors during protein synthesis or posttranslational modification. Autoradiograms are prepared by exposing a piece of x-ray film next to the gel. Computerized analysis enables proteins to be quantitatively characterized for positional or densitometric variation. It is necessitated by the quantitative nature of some problems and the need to correct for overall variations in pattern intensity, the large number of measurements that need to be made, and the need to objectify analysis. It is accomplished by scanning the image with a high resolution densitometer or video camera and then measuring individual protein spots in automated or semiautomated fashion. Our group has used two dimensional electrophoresis for the analysis of both tissue fluid and cellular proteins. We have identified 26 proteins in the protein pattern of cerebrospinal fluid and 4 proteins that appear in cerebrospinal fluid but not plasma (11). We have identified a number of protein variations in primate cerebrospinal fluid which
depend on the level from which it is obtained (12). These studies can help form a basis for understanding cerebrospinal fluid protein variations in pathological states. We have completed two large, computer-assisted surveys of cellular proteins in disease. In clinical studies completed in our laboratory, 11 quantitative protein modulations were demonstrated among 400 lymphocyte proteins from patients with the Lesch-Nyhan syndrome (13). This is an X-linked recessive disease marked by spastic@, hyperuricemia, and self-mutilation, and is one of the few metabolic diseases with neurological symptoms for which the molecular origin is known. Patients with the Lesch-Nyhan syndrome have a deficiency or absence of the enzyme, hypoxanthinequanine phosphoribosyl transferase, an enzyme of purine metabolism. In a study of genetic markers for Huntington’s disease we surveyed 300 lymphocyte proteins in 28 individuals (14). No quantitative or qualitative protein abnormalities were observed among the 13 patients and 2 individuals at risk we studied. However, a number of new protein polymorphisms that can potentially be applied to genetic marker studies (15) were discovered during the course of the survey. The application of one dimensional electrophoresis had a dramatic impact on our ability to understand diseases at the molecular level. With its power to definitively resolve most proteins from others with similar mobility and the availability of high sensitivity image production and analytic methods, two dimensional electrophoresis offers the opportunity to expand this window to a more than tenfold greater number of gene products. In the area of clinical genetics, we have conducted family studies in Gilles de la Tourette’s syndrome and Alzheimer’s dementia to determine if pedigree evidence exists for genetic transmission of the disease. When we began collecting pedigree data on Tourette’s patients, very few genetic studies on this disorder had been conducted. Patients and their families were recruited through the Tourette Syndrome Association and through various forms of media advertising. The initial evaluation by a physician and social worker involved an examination of the patient and information gathered from the patient and family regarding family history. Telephone interviews were used to gather data from distant family members. Data collected included history of illness and of pharmacological treatment, family history, neurological and psychiatric workup, response to neuroleptic treatment, 107
M. Ebert et. al. and side effects (usually haloperidol). After 69 individuals were evaluated for Tourette’s syndrome and the diagnosis confirmed on 50, the data were analyzed. Of the 50 patients with a confirmed diagnosis of Tourette’s syndrome, 16 patients had a family history of Tourette’s syndrome and an additional 16 had a family history of tics. Twenty-four families had more than 2 members with Tourette’s syndrome or tics. In contrast to the findings of other investigators, a, preponderance of families of eastern European origin was not revealed. Thirty four patients had obsessive compulsive behavior, using DSM III criteria. Among the 50 patients there was a high frequency of sleep disturbance, learning disability, self-destructive behavior, inappropriate sexual activity, and antisocial behavior. Family history of Tourette’s syndrome was significantly related to the occurrence of sleep disturbance, obsessive-compulsive behavior, positive haloperidol response, and a low incidence of side effects of haloperidol (16). The study indicated that a subgroup of Tourette’s patients have family histories that suggest genetic transmission of the disease. The data support the concept that Tourette’s syndrome is a heterogenous disorder with multiple etiologies. The subgroup with a strong family history, obsessive-compulsive behavior, and a good therapeutic response to neuroleptics may be the most appropriate patients in which to conduct neurochemical investigations. This concept received further support from a second study in which we investigated 30 adult patients with Tourette’s syndrome of 10 years or more duration. Since the symptoms of Tourette’s syndrome sometimes wane and completely abate, it was felt that these patients might represent a subgroup with a different etiology. This group of 30 adults manifest a 90% incidence of obsessive-compulsive behavior compared to an overall incidence of 68% in the first group of 50 patients, an incidence of 80% for a positive family history of Tourette’s syndrome or tics, and a relatively high incidence of self-inflicted pain behavior (females more frequently than males (17)). A second neuropsychiatric disorder in which we have conducted clinical genetic studies is familial Alzheimer’s dementia. About 50 such families have been reported in the medical literature. A large Canadian family with several members known to have Alzheimer’s disease was referred to our research group. Three index cases all had neurologi108
cal and neuropsychological findings that were indistinguishable from patients with idiopathic Alzheimer’s dementia. Neuropathologicai findings on several family members who had died of the disease were reviewed and were found to be characteristic of Alzheimer’s disease. Interviews of family members and review of medical and historical records revealed that 52 members of the family met the diagnostic criteria for Alzheimer’s disease. The pedigree was constructed back more than 200 years and became the largest pedigree of Alzheimer’s disease of which we are aware. The pattern of transmission in the pedigree fulfilled clinical criteria for autosomal dominant inheritance (18). The family study laid the ground work for genetic marker studies using technologies such as two dimensional electrophoresis. Clinical studies of neurotransmitter and neuropeptide function and metabolism are one of the most promising areas of interface between psychiatry and neurology. A large number of new assays have been reported for neuroactive compounds and their metabolites in cerebrospinal fluid (19). Clinical studies utilizing these measurements have not been firmly established in most academic medical centers, however, because of the highly specialized laboratories that need to be established. Most assays of this type use complex equipment such as high performance liquid chromatography or gas chromatography-mass spectrometry, and operate at high levels of sensitivity, i.e., low nanogram to picogram range of detection. As a result of small numbers of investigators working in the field, there is little consensus regarding which tests should be used to assess the functional state of most brain neurotransmitter systems, even the “classical” neurotransmitters such as catecholamines or indoleamines. Our experience in these areas of clinical neurochemistry has focused on developing new clinical tests of brain neurotransmitter metabolism and conducting neurochemical studies of psychosomatic and neurological syndromes of unknown etiology. An example of our research in the former area is a series of studies designed to test the assumption that the excretion rate of a urinary norepinephrine metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG), can be used to estimate the brain production rate of norepinephrine. It had been shown in a number of preclinical studies that MHPG was the principal metabolic product of radiolabelled norepinephrine administered into the central nervous system.
Clinical Research in Neuropsychiatry
We have conducted studies of the clearance and metabolic fate of MHPG, using deuterium-labeled MHPG administered intravenously and gas chrospectrometry assays. These matography-mass studies demonstrated that over half of the administered MHPG is converted to VMA. About half of the urinary VMA was derived from MHPG, and the other half from sources not in equilibrium with circulating MHPG. The results indicated that only about one fifth of urinary MHPG is derived from the brain, and that urinary MHPG cannot be used as a valid index of brain norepinephrine metabolism (20). An example of our research in the latter area is a series of studies on the neurobiology of anorexia nervosa. Evidence has accumulated, particularly from endocrinology, that the more severe and chronic forms of anorexia nervosa become a biological syndrome with clear evidence of hypothalamic dysfunction. We have employed various neurochemical tests to define the changes that occur in various stages of anorexia nervosa in the metabolism of various neurotransmitter systems known to be involved in the regulation of mood and appetite. A clinical research design was employed in which acutely ill patients with anorexia nervosa were studied at low weight, and again after weight gain in a behavior modification program. A second group under study were anorexia patients who had psychological manifestations of anorexia nervosa for years, but were not severely underweight (chronic anorexics). Normal age matched women served as controls. Brain metabolism of serotonin and dopamine were assessed by measuring the level of their acid metabolites in cerebrospinal fluid. There was clear evidence of decreased brain metabolism of these transmitters in the acute underweight stage of anorexia nervosa. Brain norepinephrine metabolism was assessed by measuring the level of norepinephrine in cerebrospinal fluid. A wide range of levels of this transmitter were found in acutely ill patients, but the group of chronic anorexics had consistently low levels, indicating decreased production of norepinephrine in this stage of the disorder (21). The endogenous opioid system is another brain neurotransmitter system known to be involved in the regulation of eating behavior in animals. To assess this system we used the measurement of total opioid activity in cerebrospinal fluid, measured by radioreceptor assay. High levels of opioid activity were found in the
acutely underweight stage of anorexia nervosa. This may be a compensatory response to weight loss or may be etiologically related to the syndrome (22). These findings add evidence to the hypothesis that a complex neurobiological syndrome exists in anorexia nervosa that may contribute to the chronic, tenacious course of the illness.
References 1.
2. 3. 4. 6. 7. 8. 9.
10. 11.
12. 13. 14. 15. 16. 17.
18. 19. 20. 21. 22.
Geschwind N.: In Benson DF, Blumer D (eds). Psychiatric Aspects of Neurologic Disease. New York, Grune and Stratton, 1975, pp. l-9 Leavitt J, Goldman D, Merril C, Kakunaga T: Carcinogenesis 3:61-70, 1982 Garrels JI: J Biol Chem 254:7978-7985, 1979 Miller MJ, Xuong NH, Geiduschek EP: Proc Nat1 Acad Sci USA 76:5222-5225, 1979 Merril CR, Goldman D, Ebert M: Proc Nat1 Acad Sci USA 786471-6475, 1981 Van Keuren ML, Goldman D, Merril CR NY Acad Sci. In press Weil J, Epstein CJ: Am J Hum Genet 7~478-488, 1979 O’FarreII PH: J Biol Chem 2504007-4021, 1975 Merril CR, Goldman D, Van Keuren ML: Electrophoresis 1982 in press Goldman D, MerriI CR, Ebert MH: Clin Chem 26:1317-1322, 1980 Merril CR, Goldman D, Sedman SA, Ebert MH: Science 211:1437-1438, 1981 Merril CR, Goldman D, Ebert M: Proc Nat1 Acad Sci USA 786471-6475, 1981 Goldman D, Merril CR, Polinsky RJ, Ebert MH: Clin Chem. 28:1021-1025, 1982 Goldman D, Merril CR: Proc Nat1 Acad Sci USA. In press Nee LE, Caine ED, Polinsky RJ, Eldridge R, Ebert MH: Ann Neurol 7:41-49, 1980 Nee LE, Polinsky RJ Ebert MH: In Chase TN, Friedhoff AJ, (eds) GiIIes de la Tourette Syndrome. New York, Raven Press, pp 291-295 Nee LE, Polinsky RJ, Eldridge R, Weingartner H, Smallberg S, Ebert M: Ann Neurol. In press Wood JH (ed) Neurobiology of Cerebrospinal Fluid. New York, Plenum Press, 1980 Blombery PA, Kopin IJ, Gordon EK, Markey SP, Ebert MH: Arch Gen Psychiatry 37:1095-1098, 1980 Kaye WH, Ebert MH, Lake CR, Raleigh M: Arch Gen Psychiatry. In press Kaye WH, Pickar D, Naber D, Ebert MH: Am J Psychiatry 139643-645, 1982
Direct reprint reqests to: Michael H. Ebert, M.D. Room 3N 234, Bldg. 10 NIMH Bethesda, Maryland, 20205
109
Therapeutics,
Laboratory
of Clinical Science, National Institute of Mental Health,
Abstract: This paper presents the thesis that rapidly developing areas of knowledge in neuroscience will rekindle interest in neuropsychiaty, and increase scientific and clinical interaction betweenpsychiatrists and neurologists in teaching hospitals. A neuropsychiatric clinical research unit at the National Institute of Mental Health is described. Examples of research conducted on the unit illustrate areas of biological science that are likely to increase the interface between psychiaf y and neurology. Hopefully, the explosion of knowledge in the basic neurosciences in the last decade will be followed in this decade by clinical research of increasing specificity and sophistication on central nervous system disorders.
Introduction There is increasing commentary on the scientific and clinical interface between psychiatry and neurology. This is so in spite of the fact that, as Norman Geschwind points out, “this common ground unfortunately bears more resemblance to a no-man’s land than to an open border. While neurologists tend to mutter darkly about the failure of psychiatrists to be aware of the brain as the organ of the mind, psychiatrists, perhaps somewhat defensively, have stressed their awarenessof the whole man, biologic as well as psychologic. Unfortunately, few members of either group have, in fact, really interested themselves in the borderland area, and too frequently interactions between them are educationally disappointing, whether at the level of mutual consultation or in the interchange of residents for training“ (1). Rapidly expanding areas of knowledge in basic General Hospital Psychiatry 5, 105-109, 1983 Q Elsevier Science Publishing Co., Inc. 1983 52 Vanderbilt Avenue, New York, NY 10017
neuroscience have led to a renewal of interest in neuropsychiatry. New knowledge concerning neurotransmitter biochemistry and physiology has stimulated neurochemical hypotheses about the etiology of a variety of neurological and psychiatric illnesses. New technologies are available to study the function of the central nervous system in clinical research including neurochemical assays, neurophysiological procedures such as averaged evoked potentials, and various radiological scanning procedures. These developments have stimulated clinical research on disorders such as Huntington’s chorea, Gilles de la Tourette’s syndrome, Parkinson’s disease, tardive dyskinesia, and Alzheimer’s dementia. During the same time, but for different reasons, there has been heightened public interest in some of these diseases. Lay interest groups such as the Committee to Combat Huntington’s Disease, the Tourettes Syndrome Association, and the Alzheimer’s Disease and Related Disorders Association have stimulated clinical research through public education efforts, fund raising, and lobbying. Recognition of the growing medical problems that the aging population presents to our society has stimulated research in the presenile dementias. Currently both neurologists and psychiatrists in academic settings are seeking additional training in areas of basic and clinical neuroscience. Furthermore, some diseases and syndromes, whose pathophysiology appears to be related to changes in neurotransmitter function, require both psychiatric and neurological clinical skills for optimal management of the patient. These facts may encourage the 105
ISSN 0163-8343/&X3%3.00
M. Ebert et. al. formation of treatment teams in academic hospitals and research institutes where neurologists, psychiatrists, and individuals trained in both specialties are working together and sharing common laboratory facilities. They may also stimulate a modest increase in the number of individuals seeking board certification in both psychiatry and neurology.
Interface Between Psychiatry Neurology
and
This paper focuses on the experience of operating a neuropsychiatric ward at the Clinical Center of the NIH over the past five years. The ward was established to carry out clinical research on some of the syndromes that occupy the interface between psychiatry and neurology. Clinical pharmacology has been the major scientific discipline on the ward, encompassing pharmacokinetics, clinical neurochemistry, and clinical neuropharmacology. Other scientific disciplines, particularly neuropsychology and clinical genetics have also played an important role. The ward has served as a base for offering fellowship training to psychiatrists and neurologists in clinical pharmacology. This clinical unit has been a successful example of operating in the interface between psychiatry and neurology with regard to clinical teaching and research training of post doctoral fellows and medical students. Clinical research from the unit has been published in approximately equal proportions in journals specialized in psychiatry, neurology, internal medicine, and pharmacology. Hopefully this neuropsychiatric unit offers a model that is pertinent to exploring the scientific and clinical relationship between departments of psychiatry and neurology in medical schools. The essential ingredients for such an operation are a critical mass of individuals engaged in clinical research and sufficient clinical research technology in place to conduct such studies. The ward was established with an interdisciplinary staff. There have been l-3 neurologists, l-3 psychiatrists, and 1 internist during each year of its operation. Most of the medical staff are in rotating fellowship positions for training in clinical research after having completed residency training. Several individuals have had training in both psychiatry and neurology. During the majority of the year there are three senior medical students on the ward. The interdisciplinary nature of the medical staff allows a comfortable margin of clinical knowledge to deal with neurological and medical patients 106
as well as psychiatric problems. After difficulty with a traditional psychiatric nursing staff, a satisfactory nursing group was established by incorporating a mixture of psychiatric and medical-surgical nurses. During the last several years, the scope of disorders studied on the unit has been as follows: Several presenile dementias have been studied from a neuropsychological and neuropharmacological point of view. They include Huntington’s chorea, Alzheimer’s dementia, and Korsakoff’s psychosis. Patients with Gilles de la Tourette’s syndrome have been studied with clinical genetic, neuropharmacological, and neurochemical techniques. Patients with anorexia nervosa and bulemia have been studied in the context of an inpatient behavioral treatment program from a neurochemical, neuroendocrine, and neuropharmacological standpoint. Patients with several forms of orthostatic hypotension (Shy-Drager’s syndrome and idiopathic orthostatic hypotension) have been studied from the point of view of the function and biochemistry of the autonomic nervous system. Collaborative projects on the psychopharmacology of the treatment of children with attention deficit disorder (or hyperkinetic syndrome) have been carried out using the ward as a day program for diagnostic workups and clinical trials. Several clinical studies that we have carried out in the illnesses discussed above illustrate areas of biological science that are particularly suitable to bridge the gap between clinical psychiatry and neurology. Although not an exclusive list, clinical and biochemical genetics, clinical neuropharmacology, and clinical neurochemistry are three important areas of investigation in this regard. In the area of biochemical genetics we have made an effort to apply the new technology of two dimensional electrophoresis to the study of central nervous system diseases. The search for trait-specific molecular markers is an important area of research in neuropsychiatry because such markers can refine diagnostic categories and give clues to etiology. Two-dimensional electrophoresis with computerized microdensitometry permits the quantitative characterization of up to 1000 cellular proteins for quantitative or positional variation. Using this technique, four categories of molecular variation can be sought in an individual with a disease. First, the mutant polypeptide responsible for a genetic disease or altered phenotype may be detected. In this way, a mutation in the @actin gene was detected in human fibroblasts and some of the altered properties of the mutant S-actin were char-
Clinical Research in Neuropsychiatry
acterized (2). Second, a characteristic pattern of secondary protein modulation in a metabolic disease may be noted. Modulations have now been quantitatively characterized using two dimensional electrophoresis in cells exposed to hormones (3), heat shock (4), neoplastic transformation (5), inborn errors of metabolism, as in the Lesch-Nyhan syndrome (6), and alterations in chromosome number, as in trisomy 21(7,8). Third, a protein produced by an infectious agent may be detected. Fourth, visualization of new groups of related polypeptides allows the detection of additional protein polymorphisms that can be used in linkage analysis. On a fundamental level, detection of these molecular variants by two dimensional electrophoresis will assist in the construction of the human genetic map, and in refining estimates of human genetic variation . The technique of two dimensional electrophoresis, developed by O’Farrell (9), involves two-stage electrophoresis, with isoelectric focusing in the first dimension to separate the denatured proteins on the basis of charge, and polyacrylamide gel electrophoresis in the second dimension for a separation on the basis of molecular weight. Proteins are visualized by staining or autoradiography. Staining is usually accomplished using a dye such as Coomassie blue or an ultrasensitive silver stain for proteins and other polymers (10). Autoradiography depends on radiolabelling of the proteins. This is usually done prior to electrophoresis by having cells incorporate radiolabelled precursors during protein synthesis or posttranslational modification. Autoradiograms are prepared by exposing a piece of x-ray film next to the gel. Computerized analysis enables proteins to be quantitatively characterized for positional or densitometric variation. It is necessitated by the quantitative nature of some problems and the need to correct for overall variations in pattern intensity, the large number of measurements that need to be made, and the need to objectify analysis. It is accomplished by scanning the image with a high resolution densitometer or video camera and then measuring individual protein spots in automated or semiautomated fashion. Our group has used two dimensional electrophoresis for the analysis of both tissue fluid and cellular proteins. We have identified 26 proteins in the protein pattern of cerebrospinal fluid and 4 proteins that appear in cerebrospinal fluid but not plasma (11). We have identified a number of protein variations in primate cerebrospinal fluid which
depend on the level from which it is obtained (12). These studies can help form a basis for understanding cerebrospinal fluid protein variations in pathological states. We have completed two large, computer-assisted surveys of cellular proteins in disease. In clinical studies completed in our laboratory, 11 quantitative protein modulations were demonstrated among 400 lymphocyte proteins from patients with the Lesch-Nyhan syndrome (13). This is an X-linked recessive disease marked by spastic@, hyperuricemia, and self-mutilation, and is one of the few metabolic diseases with neurological symptoms for which the molecular origin is known. Patients with the Lesch-Nyhan syndrome have a deficiency or absence of the enzyme, hypoxanthinequanine phosphoribosyl transferase, an enzyme of purine metabolism. In a study of genetic markers for Huntington’s disease we surveyed 300 lymphocyte proteins in 28 individuals (14). No quantitative or qualitative protein abnormalities were observed among the 13 patients and 2 individuals at risk we studied. However, a number of new protein polymorphisms that can potentially be applied to genetic marker studies (15) were discovered during the course of the survey. The application of one dimensional electrophoresis had a dramatic impact on our ability to understand diseases at the molecular level. With its power to definitively resolve most proteins from others with similar mobility and the availability of high sensitivity image production and analytic methods, two dimensional electrophoresis offers the opportunity to expand this window to a more than tenfold greater number of gene products. In the area of clinical genetics, we have conducted family studies in Gilles de la Tourette’s syndrome and Alzheimer’s dementia to determine if pedigree evidence exists for genetic transmission of the disease. When we began collecting pedigree data on Tourette’s patients, very few genetic studies on this disorder had been conducted. Patients and their families were recruited through the Tourette Syndrome Association and through various forms of media advertising. The initial evaluation by a physician and social worker involved an examination of the patient and information gathered from the patient and family regarding family history. Telephone interviews were used to gather data from distant family members. Data collected included history of illness and of pharmacological treatment, family history, neurological and psychiatric workup, response to neuroleptic treatment, 107
M. Ebert et. al. and side effects (usually haloperidol). After 69 individuals were evaluated for Tourette’s syndrome and the diagnosis confirmed on 50, the data were analyzed. Of the 50 patients with a confirmed diagnosis of Tourette’s syndrome, 16 patients had a family history of Tourette’s syndrome and an additional 16 had a family history of tics. Twenty-four families had more than 2 members with Tourette’s syndrome or tics. In contrast to the findings of other investigators, a, preponderance of families of eastern European origin was not revealed. Thirty four patients had obsessive compulsive behavior, using DSM III criteria. Among the 50 patients there was a high frequency of sleep disturbance, learning disability, self-destructive behavior, inappropriate sexual activity, and antisocial behavior. Family history of Tourette’s syndrome was significantly related to the occurrence of sleep disturbance, obsessive-compulsive behavior, positive haloperidol response, and a low incidence of side effects of haloperidol (16). The study indicated that a subgroup of Tourette’s patients have family histories that suggest genetic transmission of the disease. The data support the concept that Tourette’s syndrome is a heterogenous disorder with multiple etiologies. The subgroup with a strong family history, obsessive-compulsive behavior, and a good therapeutic response to neuroleptics may be the most appropriate patients in which to conduct neurochemical investigations. This concept received further support from a second study in which we investigated 30 adult patients with Tourette’s syndrome of 10 years or more duration. Since the symptoms of Tourette’s syndrome sometimes wane and completely abate, it was felt that these patients might represent a subgroup with a different etiology. This group of 30 adults manifest a 90% incidence of obsessive-compulsive behavior compared to an overall incidence of 68% in the first group of 50 patients, an incidence of 80% for a positive family history of Tourette’s syndrome or tics, and a relatively high incidence of self-inflicted pain behavior (females more frequently than males (17)). A second neuropsychiatric disorder in which we have conducted clinical genetic studies is familial Alzheimer’s dementia. About 50 such families have been reported in the medical literature. A large Canadian family with several members known to have Alzheimer’s disease was referred to our research group. Three index cases all had neurologi108
cal and neuropsychological findings that were indistinguishable from patients with idiopathic Alzheimer’s dementia. Neuropathologicai findings on several family members who had died of the disease were reviewed and were found to be characteristic of Alzheimer’s disease. Interviews of family members and review of medical and historical records revealed that 52 members of the family met the diagnostic criteria for Alzheimer’s disease. The pedigree was constructed back more than 200 years and became the largest pedigree of Alzheimer’s disease of which we are aware. The pattern of transmission in the pedigree fulfilled clinical criteria for autosomal dominant inheritance (18). The family study laid the ground work for genetic marker studies using technologies such as two dimensional electrophoresis. Clinical studies of neurotransmitter and neuropeptide function and metabolism are one of the most promising areas of interface between psychiatry and neurology. A large number of new assays have been reported for neuroactive compounds and their metabolites in cerebrospinal fluid (19). Clinical studies utilizing these measurements have not been firmly established in most academic medical centers, however, because of the highly specialized laboratories that need to be established. Most assays of this type use complex equipment such as high performance liquid chromatography or gas chromatography-mass spectrometry, and operate at high levels of sensitivity, i.e., low nanogram to picogram range of detection. As a result of small numbers of investigators working in the field, there is little consensus regarding which tests should be used to assess the functional state of most brain neurotransmitter systems, even the “classical” neurotransmitters such as catecholamines or indoleamines. Our experience in these areas of clinical neurochemistry has focused on developing new clinical tests of brain neurotransmitter metabolism and conducting neurochemical studies of psychosomatic and neurological syndromes of unknown etiology. An example of our research in the former area is a series of studies designed to test the assumption that the excretion rate of a urinary norepinephrine metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG), can be used to estimate the brain production rate of norepinephrine. It had been shown in a number of preclinical studies that MHPG was the principal metabolic product of radiolabelled norepinephrine administered into the central nervous system.
Clinical Research in Neuropsychiatry
We have conducted studies of the clearance and metabolic fate of MHPG, using deuterium-labeled MHPG administered intravenously and gas chrospectrometry assays. These matography-mass studies demonstrated that over half of the administered MHPG is converted to VMA. About half of the urinary VMA was derived from MHPG, and the other half from sources not in equilibrium with circulating MHPG. The results indicated that only about one fifth of urinary MHPG is derived from the brain, and that urinary MHPG cannot be used as a valid index of brain norepinephrine metabolism (20). An example of our research in the latter area is a series of studies on the neurobiology of anorexia nervosa. Evidence has accumulated, particularly from endocrinology, that the more severe and chronic forms of anorexia nervosa become a biological syndrome with clear evidence of hypothalamic dysfunction. We have employed various neurochemical tests to define the changes that occur in various stages of anorexia nervosa in the metabolism of various neurotransmitter systems known to be involved in the regulation of mood and appetite. A clinical research design was employed in which acutely ill patients with anorexia nervosa were studied at low weight, and again after weight gain in a behavior modification program. A second group under study were anorexia patients who had psychological manifestations of anorexia nervosa for years, but were not severely underweight (chronic anorexics). Normal age matched women served as controls. Brain metabolism of serotonin and dopamine were assessed by measuring the level of their acid metabolites in cerebrospinal fluid. There was clear evidence of decreased brain metabolism of these transmitters in the acute underweight stage of anorexia nervosa. Brain norepinephrine metabolism was assessed by measuring the level of norepinephrine in cerebrospinal fluid. A wide range of levels of this transmitter were found in acutely ill patients, but the group of chronic anorexics had consistently low levels, indicating decreased production of norepinephrine in this stage of the disorder (21). The endogenous opioid system is another brain neurotransmitter system known to be involved in the regulation of eating behavior in animals. To assess this system we used the measurement of total opioid activity in cerebrospinal fluid, measured by radioreceptor assay. High levels of opioid activity were found in the
acutely underweight stage of anorexia nervosa. This may be a compensatory response to weight loss or may be etiologically related to the syndrome (22). These findings add evidence to the hypothesis that a complex neurobiological syndrome exists in anorexia nervosa that may contribute to the chronic, tenacious course of the illness.
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10. 11.
12. 13. 14. 15. 16. 17.
18. 19. 20. 21. 22.
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