Molecular cloning and characterization of hNP22: a gene up-regulated in human alcoholic brain

An improved differential display technique was used to search for changes in gene expression in the superior frontal cortex of alcoholics, A cDNA fragment was retrieved and cloned. Further sequence of the cDNA was determined from 5' RACE and screening of a human brain cDNA library. The gene was named hNP22 (human neuronal protein 22). The deduced protein sequence of hNP22 has an estimated molecular mass of 22.4 kDa with a putative calcium-binding site, and phosphorylation sites for casein kinase II and protein kinase C. The deduced amino acid sequence of hNP22 shares homology (from 67% to 42%) with four other proteins, SM22 alpha, calponin, myophilin and mp20. Sequence homology suggests a potential interaction of hNP22 with cytoskeletal elements. hNP22 mRNA was expressed in various brain regions but in alcoholics, greater mRNA expression occurred in the superior frontal cortex, but not in the primary motor cortex or cerebellum. The results suggest that hNP22 may have a role in alcohol-related adaptations and may mediate regulatory signal transduction pathways in neurones.

GABA(A) receptor alpha-subunit proteins in human chronic alcoholics

Antibodies were raised against specific peptides from N-terminal regions of the alpha (1) and alpha (3) isoforms of the GABA(A) receptor, and used to assess the relative expression of these proteins in the superior frontal and primary motor cortices of 10 control, nine uncomplicated alcoholic and six cirrhotic alcoholic cases were matched for age and post-mortem delay. The regression of expression on post-mortem delay was not statistically significant for either isoform in either region. In both cortical areas, the regression of a, expression on age differed significantly between alcoholic cases, which showed a decrease, and normal controls, which did not. Age had no effect on alpha (3) expression. The alpha (1) and alpha (3) isoforms were found to be expressed differentially across cortical regions and showed a tendency to be expressed differentially across case groups. In cirrhotic alcoholics, alpha (1) expression was greater in superior frontal than in motor cortex, whereas this regional difference was not significant in controls or uncomplicated alcoholics. In uncomplicated alcoholics, alpha (3) expression was significantly lower in superior frontal than in motor cortex. Expression of alpha (1) was significantly different from that Of alpha (3) in the superior frontal cortex of alcoholics, but not in controls. In motor cortex, there were no significant differences in expression between the isoforms in any case group.

Reliability of the input-output properties of the cortico-spinal pathway obtained from transcranial magnetic and electrical stimulation

The purpose of this experiment was to assess the test-retest reliability of input-output parameters of the cortico-spinal pathway derived from transcranial magnetic (TMS) and electrical (TES) stimulation at rest and during muscle contraction. Motor evoked potentials (MEPs) were recorded from the first dorsal interosseous muscle of eight individuals on three separate days. The intensity of TMS at rest was varied from 5% below threshold to the maximal output of the stimulator. During trials in which the muscle was active, TMS and TES intensities were selected that elicited MEPs of between 150 and 300 X at rest. MEPs were evoked while the participants exerted torques up to 50% of their maximum capacity. The relationship between MEP size and stimulus intensity at rest was sigmoidal (R-2 = 0.97). Intra-class correlation coefficients (ICC) ranged between 0.47 and 0.81 for the parameters of the sigmoid function. For the active trials, the slope and intercept of regression equations of MEP size on level of background contraction were obtained more reliably for TES (ICC = 0.63 and 0.78, respectively) than for TMS (ICC = 0.50 and 0.53, respectively), These results suggest that input-output parameters of the cortico-spinal pathway may be reliably obtained via transcranial stimulation during longitudinal investigations of cortico-spinal plasticity. (C) 2001 Elsevier Science B.V. All rights reserved.

Phasic modulation of corticomotor excitability during passive movement of the upper limb: effects of movement frequency and muscle specificity

Modulations in the excitability of spinal reflex pathways during passive rhythmic movements of the lower limb have been demonstrated by a number of previous studies [4]. Less emphasis has been placed on the role of supraspinal pathways during passive movement, and on tasks involving the upper limb. In the present study, transcranial magnetic stimulation (TMS) was delivered to subjects while undergoing passive flexion-extension movements of the contralateral wrist. Motor evoked potentials (MEPs) of flexor carpi radialis (FCR) and abductor pollicus brevis (APB) muscles were recorded. Stimuli were delivered in eight phases of the movement cycle during three different frequencies of movement. Evidence of marked modulations in pathway excitability was found in the MEP amplitudes of the FCR muscle, with responses inhibited and facilitated from static values in the extension and flexion phases, respectively. The results indicated that at higher frequencies of movement there was greater modulation in pathway excitability. Paired-pulse TMS (sub-threshold conditioning) at short interstimulus intervals revealed modulations in the extent of inhibition in MEP amplitude at high movement frequencies. In the APE muscle, there was some evidence of phasic modulations of response amplitude, although the effects were less marked than those observed in FCR. It is speculated that these modulatory effects are mediated via Ia afferent pathways and arise as a consequence of the induced forearm muscle shortening and lengthening. Although the level at which this input influences the corticomotoneuronal pathway is difficult to discern, a contribution from cortical regions is suggested. (C) 2001 Published by Elsevier Science B.V.

Synaptic vesicle transport and synaptic membrane transporter sites in excitatory amino acid nerve terminals in Alzheimer disease

The apparent L-[H-3]glutamate uptake rate (v') was measured in synaptic vesicles isolated from cerebral cortex synaptosomes prepared from autopsied Alzheimer and non-Alzheimer dementia cases, and age-matched controls. The initial synaptosome preparations exhibited similar densities of D-[H-3]aspartate membrane binding sites (B-MAX values) in the three groups. In control brain the temporal cortex D-[H-3]aspartate B-MAX was 132% of that in motor cortex, parallel with the L- [H-3]glutamate v' values (temporal = 139% of motor; NS). Unlike D- [H-3]aspartate B-MAX values, L- [H-3]glutamate v' values were markedly and selectively lower in Alzheimer brain preparations than in controls, particularly in temporal cortex. The difference could not be attributed to differential effects of autopsy interval or age at death. Non-Alzheimer dementia cases resembled controls. The selective loss of vesicular glutamate transport is consistent with a dysfunction in the recycling of transmitter glutamate.

Genes and gene expression in the brain of the alcoholic

Chronic alcoholism leads to localized brain damage, which is prominent in superior frontal cortex but mild in motor cortex. The likelihood of developing alcohol dependence is associated with genetic markers. GABA(A) receptor expression differs between alcoholics and controls, whereas glutamate receptor differences are muted. We determined whether genotype differentiated the localized expression of glutamate and gamma-aminobutyric acid (GABA) receptors to influence the severity of alcohol-induced brain damage. Cerebrocortical tissue was obtained at autopsy from alcoholics without alcohol-related disease, alcoholics with cirrhosis, and matched controls. DRD2A, DRD2B, GABB2, EAAT2, and 5HTT genotypes did not divide alcoholic cases and controls on N-methyl-D-aspartate (NMDA) receptor parameters. In contrast, alcohol dehydrogenase (ADH)3 genotype interacted significantly with NMDA receptor efficacy and affinity in a region-specific manner. EAAT2 genotype interacted significantly with local GABAA receptor subunit mRNA expression, and GABB2 and DRD2B genotypes with p subunit isoform protein expression. Genotype may modulate amino acid transmission locally so as to mediate neuronal vulnerability. This has implications for the effectiveness of pharmacological interventions aimed at ameliorating brain damage and, possibly, dependence. (C) 2004 Elsevier Ltd. All rights reserved

Gene expression in profiling in individual cases reveals consistent transcriptional changes in alcoholic human brain

Chronic alcohol exposure induces lasting behavioral changes, tolerance, and dependence. This results, at least partially, from neural adaptations at a cellular level. Previous genome-wide gene expression studies using pooled human brain samples showed that alcohol abuse causes widespread changes in the pattern of gene expression in the frontal and motor cortices of human brain. Because these studies used pooled samples, they could not determine variability between different individuals. In the present study, we profiled gene expression levels of 14 postmortem human brains (seven controls and seven alcoholic cases) using cDNA microarrays (46 448 clones per array). Both frontal cortex and motor cortex brain regions were studied. The list of genes differentially expressed confirms and extends previous studies of alcohol responsive genes. Genes identified as differentially expressed in two brain regions fell generally into similar functional groups, including metabolism, immune response, cell survival, cell communication, signal transduction and energy production. Importantly, hierarchical clustering of differentially expressed genes accurately distinguished between control and alcoholic cases, particularly in the frontal cortex.

GABAA receptor beta subunit mRNA expression in the human alcoholic brain

A competitive RT-PCR assay was used to quantify the expression of the GABA(A) receptor beta(1), beta(2) and beta(3) isoform mRNA transcripts in the superior frontal cortex and motor cortex of 21 control and 22 alcoholic cases. A single set of primers was designed that permitted amplification of all three transcripts and the internal standard simultaneously; differentiation of the individual transcripts was achieved by restriction enzyme digestion. Construction of a standard curve, using the internal standard and a concentration range of beta(2) cRNA-enabled quantitation of mRNA expression levels. No significant difference in mRNA expression was found between the control and alcoholic case groups in either the superior frontal or motor cortex for the beta(2) or beta(3) isoforms. A significant interaction was found between isoform and area, although, the two case groups did not partition on this measure. The interaction was due to a significant difference between superior frontal and motor cortex for the beta(3) isoform; this regional comparison was not significant for beta(2) mRNA. Age at death and post-mortem delay (PMD) had no significant effect on beta mRNA expression in either case group in either region. A beta(1) signal could not be detected in the RT-PCR assay. (C) 2004 Elsevier Ltd. All rights reserved.

Genetic study of alcoholism and novel gene expression in the alcoholic brain

Alcohol dependence may result from neuroadaptation involving alteration of gene expression after long-term alcohol exposure. The systematic study of gene expression profiles of the human alcoholic brain was initiated using the method of polymerase chain reaction (PCR)-differential display and was followed by DNA microarray. To date, more than 100 alcohol-responsive genes have been identified from the frontal cortex, motor cortex and nucleus accumbens of the human brain. These genes have a wide range of functions in the brain and indicate diverse actions of alcohol on neuronal function. This review discusses the current information on the genetic basis of alcoholism and the induction and characterization of these alcohol-responsive genes.

Visual feedback alters the variations in corticospinal excitability that arise from rhythmic movements of the opposite limb

Augmented visual feedback can have a profound bearing on the stability of bimanual coordination. Indeed, this has been used to render tractable the study of patterns of coordination that cannot otherwise be produced in a stable fashion. In previous investigations (Carson et al. 1999), we have shown that rhythmic movements, brought about by the contraction of muscles on one side of the body, lead to phase-locked changes in the excitability of homologous motor pathways of the opposite limb. The present study was conducted to assess whether these changes are influenced by the presence of visual feedback of the moving limb. Eight participants performed rhythmic flexion-extension movements of the left wrist to the beat of a metronome (1.5 Hz). In 50% of trials, visual feedback of wrist displacement was provided in relation to a target amplitude, defined by the mean movement amplitude generated during the immediately preceding no feedback trial. Motor potentials (MEPs) were evoked in the quiescent muscles of the right limb by magnetic stimulation of the left motor cortex. Consistent with our previous observations, MEP amplitudes were modulated during the movement cycle of the opposite limb. The extent of this modulation was, however, smaller in the presence of visual feedback of the moving limb (FCR omega(2) =0.41; ECR omega(2)=0.29) than in trials in which there was no visual feedback (FCR omega(2)=0.51; ECR omega(2)=0.48). In addition, the relationship between the level of FCR activation and the excitability of the homologous corticospinal pathway of the opposite limb was sensitive to the vision condition; the degree of correlation between the two variables was larger when there was no visual feedback of the moving limb. The results of the present study support the view that increases in the stability of bimanual coordination brought about by augmented feedback may be mediated by changes in the crossed modulation of excitability in homologous motor pathways.

Specialization of pyramidal cell structure in the visual areas V1, V2 and V3 of the South American rodent, Dasyprocta primnolopha

Marked phenotypic variation has been reported in pyramidal cells in the primate cerebral cortex. These extent and systematic nature of these specializations suggest that they are important for specialized aspects of cortical processing. However, it remains unknown as to whether regional variations in the pyramidal cell phenotype are unique to primates or if they are widespread amongst mammalian species. In the present study we determined the receptive fields of neurons in striate and extrastriate visual cortex, and quantified pyramidal cell structure in these cortical regions, in the diurnal, large-brained, South American rodent Dasyprocta primnolopha. We found evidence for a first, second and third visual area (V1, V2 and V3, respectively) forming a lateral progression from the occipital pole to the temporal pole. Pyramidal cell structure became increasingly more complex through these areas, suggesting that regional specialization in pyramidal cell phenotype is not restricted to primates. However, cells in V1, V2 and V3 of the agouti were considerably more spinous than their counterparts in primates, suggesting different evolutionary and developmental influences may act on cortical microcircuitry in rodents and primates. (c) 2006 Elsevier B.V. All rights reserved.

Comorbidity and genotype modify receptor expression in human alcoholics

Chronic alcohol misuse leads to both widespread and localized damage in human cerebral cortex. The latter, as neuronal loss, is marked in superior frontal cortex (SFC) but milder in primary motor cortex (PMC) and elsewhere. Quantitative morphometry by Harper et al showed that neuronal loss is greater in alcoholics with comorbidity (Wernicke Korsakoff syndrome, liver cirrhosis). Previous work revealed a paradox: the marked differences in GABAA receptor density, pharmacology, and expression between alcoholics without cormorbidity and controls are muted or absent in cirrhotic alcoholics. This concurs with work by the Butterworth group on hepatic encephalopathy cases — most of whom had an alcoholic ætiology — who show only minor differences from controls. Glutamate receptor differences are muted in many autopsy studies, though we have evidence that NMDA site pharmacology may vary in cirrhotic alcoholics. Here we used Real-Time PCR normalized to GAPDH deltaCT to quantify NMDA NR1, NR2A and NR2B subunit expression in SFC and PMC samples obtained at autopsy from alcoholics with and without comorbid cirrhosis and matched controls. Overall subunit transcript expression was signifi cantly lower in alcoholic cirrhotics than in either of the other groups (F2,42 = 12.942, P < 0.001). The effect was most marked for the NR1 subunit; males differed from females, particularly in SFC. The data suggest that if excitotoxicity mediates neuronal loss in SFC, it may be implemented differently: passively in uncomplicated alcoholics, by altered GABAergic transmission; actively in cirrhotic alcoholics, by altered glutamatergic transmission. We also subdivided cases on a panel of genetic markers. Different genotypes interacted with NMDA and GABAA pharmacology and expression. Cirrhotic and uncomplicated alcoholics may differ pathogenically because of inherent characteristics in addition to possible neurotoxic sequelæ to the liver damage.

Neurobiological alterations in the human alcoholic brain: effect of genotype

Long-term alcohol abuse by human subjects leads to selective brain damage that is restricted in extent and variable in severity. Within the cerebral cortex, neuronal loss is most marked in the superior frontal cortex and relatively mild in motor cortex. Cirrhotic alcoholics and subjects with alcohol-related Wernicke-Korsakoff syndrome show more severe and more extensive damage than do uncomplicated cases. Accumulating evidence suggests that the likelihood of developing alcohol dependency is associated with one or more genetic markers. In previous work we showed that GABAA receptor functionality, and the subunit isoform expression that underlies this, differed in region- and disease-specific ways between alcoholics and controls. By contrast, glutamate receptor (NMDA, KA, AMPA) differences were muted or absent. Here we asked if genotype differentiated the form, pharmacology, or expression of glutamate and GABA receptors in pathologically vulnerable and spared cortical regions, with a view to determining whether such subject factors might influence the severity of alcohol-induced brain damage. Cerebrocortical tissue was obtained at autopsy under informed, written consent from uncomplicated and alcoholic-cirrhotic Caucasian (predominantly Anglo-Celtic) cases, together with matched controls and cases with cirrhosis of non-alcoholic origin. All subjects had pathological confirmation of liver and brain diagnosis; none had been polydrug abusers. Samples were processed for synaptic membrane receptor binding, mRNA analysis by quantitative RT-PCR, and protein analysis by Western blot. Genotyping was performed by PCR methods, in the main using published primers. Several genetic markers differentiated between our alcoholic and control subjects, including the GABAA receptor 2 subunit (GABB2) gene ( 2 (3) 10.329, P 0.01), the dopamine D2 receptor B1 (DRD2B) allele ( 2 (3) 10.109, P 0.01) and a subset of the alcohol dehydrogenase-3 (ADH3) alleles ( 2 (2) 4.730, P 0.05). Although neither the type-2 glutamate transporter (EAAT2) nor the serotonin transporter (5HTT) genes were significantly associated with alcoholism, only EAAT2 heterozygotes showed a significant association between ADH3 genotype and alcoholism ( 2 (3) 7.475, P 0.05). Other interactions between genotypes were also observed. DRD2A, DRD2B, GABB2, EAAT2 and 5HTT genotypes did not divide alcoholic cases and controls on NMDA receptor parameters, although in combined subjects there was a significant DRD2B X Area Interaction with glutamateNMDA receptor efficacy (F(1,57) 4.67; P 0.05), measured as the extent of glutamate-enhanced MK801 binding. In contrast, there was a significant Case-group X ADH3 X Area Interaction with glutamateNMDA receptor efficacy (F(3,57) 2.97; P 0.05). When GABAA receptor subunit isoform expression was examined, significant Case-group X Genotype X Area X Isoform interactions were found for EAAT2 with subunit mRNA (F(1,37) 4.22; P0.05), for GABB2 with isoform protein (F(1,37) 5.69; P 0.05), and for DRD2B with isoform protein (F(2,34)5.69; P0.05). The results suggest that subjects’ genetic makeup may modulate the effectiveness of amino acid-mediated transmission in different cortical regions, and thereby influence neuronal vulnerability to excitotoxicity.

Microarray and proteomic analysis of the human alcoholic brain

The superior frontal cortex (SFC) is selectively damaged in chronic alcohol abuse, with localized neuronal loss and tissue atrophy. Regions such as motor cortex show little neuronal loss except in severe co-morbidity (liver cirrhosis or WKS). Altered gene expression was found in microarray comparisons of alcoholic and control SFC samples [1]. We used Western blots and proteomic analysis to identify the proteins that also show differential expression. Tissue was obtained at autopsy under informed, written consent from uncomplicated alcoholics and age- and sex-matched controls. Alcoholics had consumed 80 g ethanol/day chronically (often, 200 g/day for 20 y). Controls either abstained or were social drinkers ( 20 g/day). All subjects had pathological confirmation of liver and brain diagnosis; none had been polydrug abusers. Samples were homogenized in water and clarified by brief centrifugation (1000g, 3 min) before storage at –80°C. For proteomics the thawed suspensions were centrifuged (15000g, 50 min) to prepare soluble fractions. Aliquots were pooled from SFC samples from the 5 chronic alcoholics and 5 matched controls used in the previous microarray study [1]. 2-Dimensional electrophoresis was performed in triplicate using 18 cm format pH 4–7 and pH 6–11 immobilized pH gradients for firstdimension isoelectric focusing. Following second-dimension SDS-PAGE the proteins were fluorescently stained and the images collected by densitometry. 182 proteins differed by 2-fold between cases and controls. 141 showed lower expression in alcoholics, 33 higher, and 8 were new or had disappeared. To date 63 proteins have been identified using MALDI-MS and MS-MS. Western blots were performed on uncentrifuged individual samples from 76 subjects (controls, uncomplicated alcoholics and cirrhotic alcoholics). A common standard was run on every gel. After transfer, immunolabeling, and densitometry, the intensities of the unknown bands were compared to those of the standards. We focused on proteins from transcripts that showed clear differences in a series of microarray studies, classified into common sets including Regulators of G-protein Signaling and Myelin-associated proteins. The preponderantly lower level of differentially expressed proteins in alcoholics parallels the microarray mRNA analysis in the same samples. We found that mRNA and protein expression do not frequently correspond; this may help identify pathogenic processes acting at the level of transcription, translation, or post-translationally.

Genes and gene expression in human alcoholic brain

Chronic alcoholism leads to localized brain damage, which is prominent in superior frontal cortex but mild in motor cortex. The likelihood of developing alcohol dependence is associated with genetic markers. GABA-A receptor expression differs between alcoholics and controls, whereas glutamate receptor differences are muted. We determined whether genotype differentiated the localized expression of glutamate N-methyl-D-aspartate (NMDA) and GABA-A receptors to influence the severity of alcohol-induced brain damage. Cerebral cortex tissue was obtained at autopsy from alcoholics without disease comorbid with alcoholics, alcoholics with cirrhosis, and matched controls. DRD2A, DRD2B, GABRB2, SLC1A2, and 5HTT genotypes did not divide alcoholic cases and controls on NMDA receptor parameters. In contrast, a specific alcohol dehydrogenase (ADHIC) genotype interacted significantly with NMDA efficacy and affinity in a region-specific manner SLC1A2 (glutamate transporter-2) genotype interacted significantly with local GABAA receptor b subunit mRNA expression, and ADHIC, DRD2B, SLC1A2, and APOE genotypes with b subunit isoform protein expression. In the latter instance, possession of the alcoholism- associated allele altered b isoform protein expression patterns toward a less-efficacious form of the GABA-A receptor in the pathologically vulnerable region. GABRB2 and GRIN2B (NMDA receptor 2B subunit} Genotypes were associated with significant regional difference in the pattern of b subunit protein isoform expression, but this was not influenced by alcoholism status. Genotype may modulate amino acid transmission locally so as to mediate neuronal vulnerability. This has implications for the effectiveness of pharmacological interventions aimed at ameliorating brain damage and, possibly, dependence.