Biomedical subjects
Publications and source records attributed to R Freedman.
Nine multiplex schizophrenia families were genotyped with polymorphisms for the GLUR5 and NMDAR1 glutamate receptor subunit genes. Using the lod score technique, evidence of linkage was not found assuming either dominant or recessive transmission. Similarly, the non-parametric sib pair test did not yield significant evidence of linkage.
Peripheral and central aspects of motor dysfunction were assessed in 12 schizophrenic and 12 normal subjects, using a test of control of finger movement based on the widely used smooth pursuit eye movement task. This was performed in order to investigate the basis of neuromotor dysfunction in schizophrenia. In this task, subjects used finger movement to track a visual target. Simultaneously, an electromyogram of the extensor digitorum communis, the primary muscle utilized in the task, was recorded. Smooth pursuit eye movements were also assessed. Accuracy of finger-based and smooth-pursuit eye movement tracking was analyzed by fast Fourier transform and expressed as a log signal-to-noise ratio. The electromyograms were analyzed by motor unit potential discrimination and by interspike interval histography. Schizophrenics demonstrated significantly poorer finger tracking than did controls, with a mean score of 2.01 +/- 0.63 (SD) versus 2.81 +/- 0.42 (t = 3.52, d.f. = 21, p < 0.005). However, there was no evidence for motor-unit dysfunction. Schizophrenics also performed more poorly on smooth-pursuit eye movement, with a mean score of 2.06 +/- 0.62 versus 3.33 +/- 1.21 (t = 3.21, d.f. = 22, p < 0.005). Severity of extrapyramidal symptoms was correlated with poorer performance on the finger tracking task, but not smooth-pursuit eye movement. These findings support the hypothesis that schizophrenics' tracking abnormalities are due to central nervous system deficits rather than peripheral pathology.
1. To identify and isolate cDNAs encoding rat and human bradykinin-B2 receptor subtypes we isolated a human bradykinin receptor cDNA homologous to a rat B2 receptor cDNA. 2. The cDNA was expressed in the bradykinin receptor negative cell line, CHO; membranes prepared from these cells bound bradykinin and had specificity similar to that of the known rat B2 receptor. In addition, the expressed receptor has a low affinity for des-Arg9-bradykinin. Thus, the cDNA encodes a human B2-bradykinin receptor. 3. Comparison of the human and rat cDNAs suggested that the human and rat genes are composed of three exons. Cloning, sequencing and characterization of parts of the human and rat B2-bradykinin receptor genes demonstrated the postulated three-exon structure. This structure includes two 5' exons upstream of the most favorable translation initiation methionine in exon-3. 4. The two 5' exons each contain methionines, which if independently spliced to the third exon, would yield an open reading frame that includes all of exon-3. This arrangement could thus vary the amino-terminal region of the protein. Do these potential arrangements occur in human RNAs, and will they lead to proteins with differing amino-termini? 5. Reverse transcriptase-polymerase chain reactions (RT-PCR) using human mRNA, nested primers from exon-1 and exon-3, and detection of the products by hybridization using an independent exon-1 oligonucleotide showed that the arrangement of exon-1 with exon-2 and exon-3 could not be detected in eight human RNAs. Furthermore, exon-1 spliced with exon-3 was a common arrangement. 6. Low stringency examination of human and rat Southern blots revealed only bands attributable to the known human or rat B2-bradykinin receptor. 7. Reduced stringency hybridization searches of seven different genomic and cDNA libraries--including two different human genomic libraries, a rat genomic library, two different rat uterus cDNA libraries, a rat brain library and a human lung library--yielded only rat or human B2-bradykinin receptors. The results of our low stringency hybridization experiments suggest that other bradykinin receptors are less than 60% identical, on the nucleotide level, to the known B2 receptor. 8. Degenerate polymerase chain reactions using rat genomic DNA as a template and degenerate primers, designed based on the homology of a B2-bradykinin receptor with angiotensin-II type-1 receptor, identified B2-bradykinin receptors, angiotensin-II-type-1 receptors and three novel orphan receptors.
The levels of the synaptic vesicle-associated proteins, synapsin and synaptophysin, were examined in human postmortem hippocampus from the brains of schizophrenics and age-matched controls using a quantitative western blot analysis. The schizophrenic samples had significantly lower levels of synapsin I than controls. In individual data, five of the seven schizophrenic samples had extremely low levels of synapsin, whereas two of the schizophrenic samples had normal levels of synapsin. This deficit in synapsin does not appear to be due to some non-specific neuronal loss as the levels of the other synaptic vesicle marker, synaptophysin, were near normal in all seven schizophrenics. Given that synapsin is thought to regulate neurotransmitter release, it is possible that this deficit in synapsin could result in abnormal processing of neuronal information as is seen in various sensory processing abnormalities associated with schizophrenia.
Traditional diagnostic techniques may not provide all the information necessary to reveal the genetic causes of schizophrenia through linkage analysis. Use of neurophysiological indicator variables that are associated with the disease may increase the probability of detecting linkage. Such variables not only produce simpler phenotypes for analysis, but they also may be more proximal to the gene products involved in neurological dysfunctions underlying schizophrenia. We have used a previously characterized neurophysiological variable, the P50 evoked-auditory response, to search for chromosomal regions that may be of interest in the study of schizophrenia. Although our scan of over 300 markers did not show strong evidence for linkage to P50 in nine families, this exploratory analysis has revealed several chromosomal regions that may deserve further study.
Jones et al. Nature Genet 1:306-309, [1992] recently detected a C to T nucleotide transition (codon 713) in a highly conserved region of the beta-amyloid precursor gene in a single case of schizophrenia. Although the sequence variant may be a natural polymorphism, it is crucial to determine whether the mutation might be present in a small subset of schizophrenics. We isolated DNA from 86 unrelated chronic schizophrenics who had a first degree relative with chronic schizophrenia or chronic schizoaffective disorder. After PCR amplification of exon 17, we were unable to detect the presence of the codon 713 variant in these schizophrenic cases, as well as in 156 controls. Unless additional cases are found with the codon 713 mutation, it is unlikely that the sequence variant is pathogenic for schizophrenia.
Auditory stimuli repeated at short intervals result in diminished evoked responses recorded from the skull surface and from the hippocampus in the rat. The rat has been used to model diminished responses to repeated auditory stimuli--a phenomenon seen in normal human subjects, but often absent in schizophrenics. In this study, we examined the neural circuitry involved in the processing and gating of auditory responses recorded from the hippocampus of the rat. Evoked potentials and single neuron activity with diminished responses to the second of paired tones were recorded in the brainstem reticular formation in the paragigantocellular region at the caudal level of the pons, but diminished responses were not observed in the primary auditory relay nuclei. Electrical stimulation of this region of the brainstem reticular formation was able to substitute for the first, or conditioning, auditory tone to produce sensory gating of the response to the second, or test, tone when recording from the hippocampus. Stimulation of the auditory nuclei up to the level of the lateral lemniscus, but not the superior colliculus, was also able to substitute for an auditory stimulus to produce sensory gating in the hippocampus. The gating of hippocampal responses to auditory stimuli may thus involve pathways which branch from the lemniscal auditory pathway at the level of the lateral lemniscus and ascend to the hippocampus via the brainstem reticular formation.
RNA isolated from frozen human postmortem brain tissue was evaluated for its utility in molecular biological studies. Samples varying in postmortem interval, delay period before freezing, and long-term freezer storage were analyzed. It was found that storage of human postmortem brain at -70 degrees C for more than 5 years may compromise its use for oligo-dT primed library construction and in vitro expression studies. Although biological competency of the messenger RNA may be affected by long-term freezer storage of human brain, enough full-length or partial transcripts remained for amplification by the polymerase chain reaction. We conclude that human postmortem brain collections will continue to be valuable resources for the study of gene expression and isolation of nucleotide sequences.
The binding characteristics of [3H]cytisine, a putative CNS nicotinic receptor ligand, were examined in 4 regions of the human brain. [3H]Cytisine was found to bind non-cooperatively with high affinity to a single site in tissue homogenates and to exhibit low non-specific association. The binding characteristics of this ligand were evaluated in thalamus at 4 degrees C and 24 degrees C. The association constants were found to be 0.234 and 0.308 min-1 nM-1, while the dissociation constants were 0.007 and 0.098 min-1, respectively. Saturation analysis of thalamus revealed the equilibrium Kd to be 147 pM (4 degrees C) and 245 pM (24 degrees C), values in good agreement with those determined kinetically. The Hill coefficient varied slightly between brain regions; however, the mean values in all regions examined were close to 1.0 at 0.95 +/- 0.03 (4 degrees C) and 0.91 +/- 0.04 (24 degrees C). [3H]Cytisine binding could be displaced using both nicotinic agonists and antagonists. Cytisine was the most potent displacer of [3H]cytisine binding with an Ki of 250 pM. Nicotine and acetylcholine were also potent displacers with Ki values of 1.8 and 8.1 nM, respectively. The nicotinic antagonists alpha-bungarotoxin and mecamylamine were ineffective competitors for the [3H]cytisine binding site while dihydro-beta-erythroidine had an Ki value of 109 nM. Thalamus showed the highest density of cytisine binding sites of all the regions examined (48 fmol/mg protein) while the hippocampus, cingulate gyrus and the cortex showed Bmax values of 18.9, 19.3 and 8.8 fmol/mg protein, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Diminished suppression of the P50 component of the evoked potential following repeated auditory stimuli is one example of a deficit in elementary sensory processing in schizophrenia. Normal subjects suppress the P50 evoked potential to the second of two paired auditory stimuli. Although normal P50 suppression is occasionally observed in schizophrenic patients, it generally disappears with subsequent testing. The object of this experiment was to determine conditions for the reproducible normalization of P50 suppression in schizophrenic patients. After baseline recordings, 12 schizophrenic subjects were allowed to sleep for 10 minutes. The depth of sleep obtained was assessed by electroencephalography (EEG). Normalization of P50 suppression was observed for approximately 3 minutes in all subjects who entered slow wave sleep, but not in those whose EEG records remained desynchronized. Some change was even observed in subjects who had only persistent alpha waves. The amount of normalization was correlated with the deepest stage of sleep reached. Normal control subjects did not show this phenomenon but instead had a transient decrease in sensory gating after waking from sleep. The results suggest that schizophrenic patients may have a defect that causes a neuronal mechanism critical to sensory gating to fail after brief use, although its activity can be transiently restored by a short period of inactivity. A rapidly desensitized neurotransmitter receptor is one possible mechanism of such an effect.
Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF), the two best characterized members of a growing family of heparin-binding growth factors, have been shown to affect both survival of cultured neurons and regeneration of nerve terminals when applied exogenously. The endogenous expression of these growth factors in response to brain injury is not well understood. We have utilized the Swiss-Webster mouse, treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and a quantitative polymerase chain reaction assay to examine changes in endogenous synthesis of mRNA for both aFGF and bFGF in the striatum and substantia nigra. We have found that MPTP treatment results in a loss of 95% of dopaminergic function and is accompanied by an increase in expression of both aFGF and bFGF in the striatum at 1 week post-lesion. After 5 weeks, the terminals appear to be regenerating and FGF mRNA expression has returned to control levels. These results suggest that cellular reaction to chemical lesion in the brain may involve changes in growth factor expression, including both aFGF and bFGF.
Neurons in the medial septal nucleus were recorded extracellularly in response to auditory stimuli in chloral-hydrate-anesthetized rats. Two populations of neurons were identified, both of which were localized to the nucleus by horseradish peroxidase labeling. The auditory-responsive population was characterized by slow axonal conduction velocity, as measured after antidromic activation from the fornix. This population probably represents cholinergic neurons with unmyelinated or thinly myelinated axonal projections to the hippocampus. The other population was not auditory-responsive and had relatively fast conduction times. This population was most likely GABAergic neurons, which have heavily myelinated axons. The timing of discharge within the medial septal nucleus suggests that its cholinergic neurons may regulate the response of the hippocampus to auditory stimuli by influencing the activity of both pyramidal cells and interneurons. The medial septal nucleus may thus play a critical role in the gating of the response to repeated auditory response in the hippocampus.
The behavior of the P50 wave of the auditory evoked potential in a paired stimulus or conditioning-testing paradigm has been used as a measure of sensory gating disturbance in schizophrenia. Schizophrenics fail to decrement the P50 response to the second stimulus of the pair, so that the ratio of the test to the conditioning amplitude is elevated over normal values. The aim of this study was to compare this neurophysiological measure to neuropsychological measures of attention and memory. As expected, schizophrenics performed worse than controls on most measures. The time to complete a digit cancellation test, a measure of sustained attention, was found to be particularly longer in schizophrenics than in control subjects. Furthermore, the increased time to complete this task correlated with the increased ratio of the amplitude of the test P50 response to the conditioning response in the schizophrenics. Thus, a neurophysiological defect in sensory gating may relate to a disorder in sustained attention in schizophrenia. Although the P50 wave may come from the hippocampus, neuropsychological measures of verbal learning and memory were not correlated with alterations in the P50 ratio.
OBJECTIVE: Because many schizophrenic patients are heavy smokers, it has been suggested that nicotine normalizes some neuronal deficit involved in their illness. Schizophrenic subjects have various difficulties with maintenance of attention and selective processing of sensory information. One defect in sensory gating in schizophrenia has been characterized by recording auditory evoked potentials. Most normal subjects have a decrease in the evoked response to the second of two closely paired stimuli, whereas most schizophrenic subjects do not. The aim of this study was to determine whether smoking normalizes this deficit in auditory sensory gating in schizophrenia. METHOD: Changes in auditory sensory gating in response to smoking cigarettes were studied in 10 smokers without psychiatric illness and 10 schizophrenic smokers. Both groups were asked to abstain from smoking from 11:00 p.m. until 8:00 a.m. the next morning, when their auditory evoked responses to pairs of clicks were recorded. The ability to gate sensory information is reflected in a decrease in the P50 wave amplitude in response to the second of the two stimuli. After baseline recordings, the subjects smoked as much as they wished, and then two postsmoking recordings were performed. RESULTS: The schizophrenic patients had a marked but brief improvement in P50 auditory gating immediately after smoking, whereas P50 gating for the normal smokers was slightly impaired. CONCLUSIONS: This study suggests that cigarette smoking can transiently normalize the impairment of auditory sensory gating in schizophrenic patients.
Alterations in dopamine neurotransmission have been strongly implicated in the pathogenesis of schizophrenia for nearly 2 decades. Recently, the genes for five dopamine receptors have been cloned and characterized, and genetic and physical map information has become available. Using these five loci as candidate genes, we have tested for genetic linkage to schizophrenia in nine multigenerational families which include multiple affected individuals. In addition to testing conservative disease models, we have used a neurophysiological indicator variable, the P50 auditory evoked response. Deficits in gating of the P50 response have been shown to segregate with schizophrenia in this sample and may identify carriers of gene(s) predisposing for schizophrenia. Linkage results were consistently negative, indicating that a defect at any of the actual receptor sites is unlikely to be a major contributor to schizophrenia in the nine families studied.
The nicotinic cholinergic antagonist alpha-bungarotoxin (alpha-BT) binds throughout the rat hippocampal formation. The binding is displaceable by d-tubocurarine. The most heavily labeled cells are GABA-containing interneurons in the dentate and in Ammon's horn. These neurons have several different morphologies and contain several neuropeptides. alpha-BT-labeled interneurons in the dentate are small cells between the granular and molecular layers that often contain neuropeptide Y. alpha-BT-labeled interneurons in CA1 are medium-sized interneurons, occasionally found in stratum pyramidale, but more often found in stratum radiatum and stratum lacunosum moleculare. These neurons often contain cholecystokinin. The largest alpha-BT-labeled interneurons are found in CA3, in both stratum radiatum and stratum lucidum. These neurons are multipolar and frequently are autofluorescent. They often contain somatostatin or cholecystokinin. These large interneurons have been found to receive medial septal innervation and may also have projections that provide inhibitory feedback directly to the medial septal nucleus. The cholinergic innervation of the hippocampus from the medial septal nucleus is under the trophic regulation of NGF and brain-derived neurotrophic factor, even in adult life. Expression of mRNA for both these factors is increased in CA3 and the dentate after intraventricular administration of alpha-BT, but not after administration of the muscarinic antagonist atropine. alpha-BT-sensitive cholinergic receptors on inhibitory interneurons may be critical to medial septal regulation of the hippocampal activity, including the habituation of response to sensory input.
Human fetal brain tissue was obtained from first-trimester elective abortions of two women who also had schizophrenia. Portions of the embryonic hippocampus or cerebral cortex were transplanted into the anterior eye chamber of immunologically compromised athymic nude rats. In this environment, embryonic brain tissue derived from normal women generally continues organotypic growth and development for many months. Although initial survival after transplantation was normal, the tissue derived from schizophrenic women manifested less robust growth. However, cells in the transplants showed typical neuronal differentiation, with development of different neuronal types, such as pyramidal cells, granule cells, and gamma-aminobutyric acid (GABA)-containing interneurons. Rhythmic electrical activity was also observed, indicative of some local synaptic organization. The presence of messenger RNA (mRNA) for brain-derived neuronotrophic factor (BDNF) was observed using in situ hybridization. The reason for the decreased rate of growth of these transplants remains unknown and the significance of the finding cannot be assessed from only two fetuses. However, these preliminary findings suggest that fetal transplants may be a useful model system for the detection of developmental pathogenic processes in the expression and transmission of schizophrenia.
Diminished gating of the P50 auditory evoked response to repeated stimuli is a psychophysiological feature of schizophrenia, that is also present in many relatives of patients. Animal models of auditory sensory gating indicate that nicotinic cholinergic neurotransmission is a critical neuronal substrate. The aim of this experiment was to determine if the deficit in sensory gating could be reversed by nicotine administration. Nonsmoking relatives of schizophrenics with abnormal sensory gating were selected as subjects for this initial double-blind trial, to avoid effects of psychotropic medications that might complicate trials in schizophrenic patients themselves. Nicotine-containing gum increased P50 sensory gating to near normal levels within 30 min of administration. The effect was transient; the gating of P50 returned to baseline levels within 1 hr. There was no change observed after placebo administration. In one of the subjects, the anticholinesterase inhibitor physostigmine similarly normalized P50 gating. The results are consistent with the hypothesis that nicotinic cholinergic neurotransmission may mediate a familial psychophysiological deficit in schizophrenia.