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Perinatal asphyxia can cause neuronal loss and depletion of neurotransmitters within the striatum. The striatum plays an important role in motor control, sensorimotor integration and learning. In the present study we investigated whether perinatal asphyxia leads to motor deficits related to striatal damage, and in particular to the loss of GABAergic neurons. Perinatal asphyxia was induced in time-pregnant Wistar rats on the day of delivery by placing the uterus horns, containing the pups, in a 37 degrees C water bath for 20 min. Three motor performance tasks (open field, grip test and walking pattern) were performed at 3 and 6 weeks of age. Antibodies against calbindin and parvalbumin were used to stain GABAergic striatal projection neurons and interneurons, respectively. The motor tests revealed subtle effects of perinatal asphyxia, i.e. small decrease in motor activity. Analysis of the walking pattern revealed an increase in stride width at 6 weeks of age after perinatal asphyxia. Furthermore, a substantial loss of calbindin-immunoreactive (-22%) and parvalbumin-immunoreactive (-43%) cells was found in the striatum following perinatal asphyxia at two months of age. GABA(A) receptor autoradiography revealed no changes in GABA binding activity within the striatum, globus pallidus or substantia nigra. We conclude that perinatal asphyxia resulted in a loss of GABAergic projection neurons and interneurons in the striatum without alteration of GABA(A) receptor affinity. Despite a considerable loss of striatal neurons, only minor deficits in motor performance were found after perinatal asphyxia.

Huntington's disease is a dominantly inherited neurological disorder where specific neurodegeneration is caused by an extended polyglutamine stretch in the huntingtin protein. Proteins with expanded polyglutamine regions have the ability to self-aggregate and previous work in our laboratory, and by others, revealed sparse amyloid-like deposits in the Huntington's disease brain, supporting the hypothesis that the polyglutamine stretches may fold into regular beta-sheet structures. This process of folding has similarities to other neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and the prion diseases which all exhibit beta-sheet protein accumulation. We were therefore interested in testing the hypothesis that TATA-binding protein may play a role in Huntington's disease as it contains an elongated polymorphic polyglutamine stretch that ranges in size from 26 to 42 amino acids in normal individuals. A proportion of TBP alleles fall within the range of glutamine length that causes neurodegeneration when located in the huntingtin protein. In this study the distribution and cellular localisation of TATA-binding protein was compared to the distribution and cellular localisation of the huntingtin protein in the middle frontal gyrus of Huntington's disease and neurologically normal subjects. Seven different morphological forms of TATA-binding protein-positive structures were detected in Huntington's disease but not in control brain. TATA-binding protein labelling was relatively more abundant than huntingtin labelling and increased with the grade of the disease. At least a proportion of this accumulated TBP exists as insoluble protein. This suggests that TBP may play a role in the disease process.

The G-protein coupled metabotropic glutamate receptor mGlu5 plays a pivotal role as a modulator of synaptic plasticity, ion channel activity and excitotoxicity. Two splice variants, hmGlu5a and -5b have been reported previously. During screening of a human brain cDNA library for hmGlu5a, we identified a novel variant (hmGlu5d) generated by alternative splicing at the C-terminal domain. The predicted hmGlu5d protein has a C-terminal 267 amino acid shorter than that of hmGlu5a. The pattern of mRNA expression of mGluR5 variants in human brain were analyzed by RT-PCR and in situ hybridization histochemistry. RT-PCR analysis demonstrated the presence of the hmGlu5d transcript, although at low level, in human whole brain, cerebellum, cerebral cortex and hippocampus. [3H]Quisqualate displayed similar affinity at the hmGlu5 splice variants (K(D) values of 80+/-8 and 54+/-17 nM for hmGlu5a and -5d receptors, respectively). For the five mGlu agonists studied, a similar rank order of potency was observed on both hmGlu5a and -5d receptors: quisqualate>glutamate>DHPG>L-CCGI approximately ACPD. MPEP inhibited the glutamate (2 microM)-induced [Ca(2+)](i) response in hmGlu5a and -5d-HEK293 cells also with similar potency (IC(50) values 25+/-1.5 and 20+/-1.4 nM, respectively). Therefore, the large truncation of the C-terminal tail of mGlu5 does not have any apparent major effect on the potency and efficacy of agonists as measured by the [Ca(2+)](i) responses or by activation of recombinant G-protein coupled inwardly rectifying K(+) (GIRK) channel currents. The only major functional difference is the increased sensitivity of hmGlu5d to protein kinase C (PKC)-mediated desensitization, relative to hmGlu5a.

We screened a custom-made candidate gene cDNA array comprising 300 genes. Genes chosen have either been implicated in schizophrenia, make conceptual sense in the light of the current understanding of the disease, or are located on high-susceptibility chromosome locations. The array screen using prefrontal cortex tissue from 10 schizophrenia and 10 control brains revealed robust up-regulation of apolipoprotein L1 (apo L1) by 2.6-fold. The finding was cross-validated in a blinded quantitative PCR study using prefrontal cortex tissue from the Stanley Foundation brain collection, Bethesda, MD. This collection consists of 15 schizophrenia, 15 bipolar disorder, 15 major depression, and 15 control individuals, all 60 brains being well-matched on conventional parameters, with antipsychotic drug exposure in the schizophrenia and bipolar disorder groups. Significant up-regulation of apo L1 gene expression in schizophrenia was confirmed. Using quantitative PCR, expression profiles of other members of the apo L family (apo L2-L6) were investigated, showing that apo L2 and L4 were highly significantly up-regulated in schizophrenia. Results were then confirmed in an independent set of 20 schizophrenia and 20 control brains from Japan and New Zealand. Apo L proteins belong to the group of high density lipoproteins, with all six apo L genes located in close proximity to each other on chromosome 22q12, a confirmed high-susceptibility locus for schizophrenia and close to the region associated with velocardiofacial syndrome that includes symptoms of schizophrenia.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder accounting for about 50% of all dementias, yet its pathogenic mechanisms remain poorly understood. In order to provide a more complete picture of pathogenesis in AD, we analysed six human brain regions for alterations in their proteomes. Quantitative proteome analysis was used to compare signals corresponding to individual proteins between post mortem brain tissues from persons with AD, and those from age-matched nondemented control (NC) tissues. In severely injured brain regions, 76 proteins were differentially expressed in AD hippocampus compared with NC, 62 proteins were differentially expressed in temporal cortex, and 39 proteins were differentially expressed in entorhinal cortex. Significant differences were also present in relatively spared regions. Thus, 34 proteins were differentially expressed in AD cerebellum compared with NC, 125 proteins were differentially expressed in cingulate gyrus, and 75 proteins were differentially expressed in sensorimotor cortex. The identity of 37 of these proteins was determined, and the possible relevance of changes in key pathogenic pathways analysed. These studies provide a unique snapshot illustrating the complexity of interrelated disease mechanisms at work in a complex, multifactorial disease, and show that comparative proteome analysis is a method with the power to develop important new insights into pathogenic mechanisms in the dementias.

We have recently described the progressive and selective loss of the presynaptic protein complexin II in brains of mice (R6/2) transgenic for the Huntington's disease (HD) mutation. Here we have determined the expression of components of the synaptic vesicle fusion machinery in the striatum and hippocampus from post-mortem brains of HD cases and neurologically normal controls. As in the brains of R6/2 mice, complexin II was markedly depleted in the HD striatum; the depletion was compartmentally organized, with complexin II-poor regions corresponding with areas of low immunoreactivity toward the matrix marker calbindin D(28K). Decreases in the levels of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) protein synaptobrevin 2 and of rab3A were also seen, but none of the other proteins tested was significantly affected. In the hippocampus, levels of complexin II, synaptobrevin 2, rab3A, and also of alpha-SNAP, were markedly elevated in HD brains. We suggest that the observed abnormalities in the expression of proteins known to be involved in the control of neurotransmitter release, including both modulators and core components of the vesicle fusion machinery, might account for at least some of the functional abnormalities seen in HD.

Insulin-like growth factor-1 (IGF-1) is known to be important for oligodendrocyte survival and myelination. In the current study, the authors examined the hypothesis that exogenous IGF-1 could reduce postischemic white matter injury. Bilateral brain injury was induced in near-term fetal sheep by 30 minutes of reversible carotid artery occlusion. Ninety minutes after ischemia, either vehicle (n = 8) or a single dose of 3 microg IGF-1 (n = 9) was infused intracerebroventricularly over 1 hour. White matter changes were assessed after 4 days recovery in the parasagittal intragyral white matter and underlying corona radiata. Proteolipid protein (PLP) mRNA staining was used to identify bioactive oligodendrocytes. Glial fibrillary acidic protein (GFAP) and isolectin B-4 immunoreactivity were used to label astrocytes and microglia, respectively. Myelin basic protein (MBP) density and the area of the intragyral white matter tracts were determined by image analysis. Insulin-like growth factor-1 treatment was associated with significantly reduced loss of oligodendrocytes in the intragyral white matter (P < 0.05), with improved MBP density (P < 0.05), reduced tissue swelling, and increased numbers of GFAP and isolectin B-4 positive cells compared with vehicle treatment. After ischemia there was a close association of PLP mRNA labeled cells with reactive astrocytes and macrophages/microglia. In conclusion, IGF-1 can prevent delayed, postischemic oligodendrocyte cell loss and associated demyelination.

Antisera directed against unique peptide regions from each of the human brain voltage-gated sodium channel alpha subunits were generated. In immunoblots these were found to be highly specific for the corresponding recombinant polypeptides and to recognise the native holoprotein in human brain membrane preparations. These antisera were used to perform a comparative immunohistochemical distribution analysis of all four brain sodium channel subtypes in selected human CNS regions. Distinct but heterogeneous distribution patterns were observed for each of the alpha subunits. In general, these were complimentary to that previously shown for the corresponding human mRNAs. A high degree of conservation with respect to the distribution found in rat was also evident. The human alpha subunit proteins exhibited distinct subcellular localisation patterns. Types I, III and VI immunoreactivity was predominantly in neuronal cell bodies and proximal processes, whereas type II was concentrated along axons. This is similar to rat brain and suggests the different the sodium channel subtypes have distinct functions which are highly conserved between human and rodents. A notable difference was that the type III protein was detected in all human brain regions examined, unlike in rat brain where expression in adults is very restricted. Also in contrast to rat brain, the human type VI protein was not detected in axons of unmyelinated neurons. These differences may reflect true species variation and could have important implications for understanding the function of the sodium channel subtypes and their roles in human disease.

Studies with animal seizure models have indicated that changes in temporal and spatial expression of voltage-gated sodium channels may be important in the pathology of epilepsy. Here, by using in situ hybridisation with previously characterised subtype-selective oligonucleotide probes [Whitaker et al. (2000) J. Comp. Neurol. 422, 123-139], we have compared the cellular expression of all four brain alpha-subunit sodium channel mRNAs in "normal" and epileptic hippocampi from humans. Neuronal cell loss was observed in all regions of the hippocampus of diseased patients, indicating that sclerosis had occurred. Losses of up to 40% compared to post-mortem controls were observed which were statistically significant in all regions studied (dentate gyrus, hilus, and CA1-3). To assess mRNA levels of the different alpha-subtypes in specific subregions, control and diseased tissue sections were hybridised to subtype-specific probes. To quantify any changes in expression while allowing for cell loss, the sections were processed for liquid emulsion autoradiography and grain counts were performed on populations of individual neurones in different subregions. No significant differences were found in the expression of type I and VI mRNAs. In contrast, a significant down-regulation of type II mRNA was observed in the epileptic tissue in the remaining pyramidal cells of CA3 (71+/-7% of control, P<0.01), CA2 (81+/-8% of control, P<0.05) and CA1 (72+/-6% of control, P<0.05) compared with control tissue. Additionally, a significant up-regulation in type III mRNA in epileptic CA4 pyramidal cells (145+/-7% of control, P<0.05) was observed. It is not clear whether these changes play a causal role in human epilepsy or whether they are secondary to seizures or drug treatment; further studies are necessary to investigate these alternatives. However, it is likely that such changes would affect the intrinsic excitability of hippocampal neurones.

A disturbance in glutamatergic transmission has been suggested to contribute to the pathophysiology of schizophrenia and recent studies on ionotropic glutamate receptors are consistent with altered glutamatergic function in the hippocampus of schizophrenics. In order to investigate this hypothesis further, the expression of two 'glutamatergic' markers, the mRNAs of metabotropic glutamate receptor 5 (mGluR5) and human excitatory amino acid transporter (EAAT2) were compared in the hippocampus of control subjects and schizophrenics. We examined the regional/cellular mRNA expression of mGluR5 and EAAT2 in postmortem hippocampal sections from schizophrenics and control subjects, using in situ hybridization. Regions of interests were dentate gyrus, cornu ammonis 4, 3, 1 and parahippocampal gyrus. The regional/cellular mGluR5 mRNA content was not different between the two groups. The cellular EAAT2 mRNA content was significantly decreased in schizophrenic parahippocampal gyrus, but not in other hippocampal regions. Furthermore, only in the parahippocampal gyrus, schizophrenics had a significantly increased mGluR5/EAAT2 ratio at both the regional and cellular mRNA level. The results suggest that a disturbance of glutamatergic neurotransmission in schizophrenia was not apparent using these indices in the hippocampus, but 'hypo-glutamatergic' neurotransmission may be present in the schizophrenic parahippocampal gyrus.

We have investigated the binding properties of [(3)H]quisqualate to rat metabotropic glutamate (mGlu) 1a and 5a receptors and to rat and human brain sections. Saturation isotherms gave K:(D) values of 27 +/- 4 and 81 +/- 22 nM: for mGlu1a and mGlu5a receptors, respectively. Several compounds inhibited the binding to mGlu1a and mGlu5a receptors concentration-dependently. (S:)-4-Carboxyphenylglycine, (S:)-4-carboxy-3-hydroxyphenylglycine, and (R,S)-1-aminoindan-1,5-dicarboxylic acid, which completely inhibited [(3)H]quisqualate binding to the mGlu5a receptor, were inactive in a functional assay using this receptor. The distribution and abundance of binding sites in rat and human brain sections were studied by quantitative receptor radioautography and image analysis. Using 10 nM: [(3)H]quisqualate, a high density of binding was detected in various brain regions with the following rank order of increasing levels: medulla, thalamus, olfactory bulb, cerebral cortex, spinal cord dorsal horn, olfactory tubercle, dentate gyrus molecular layer, CA1-3 oriens layer of hippocampus, striatum, and cerebellar molecular layer. The ionotropic component of this binding could be inhibited by 30 microM: kainate, revealing the distribution of mGlu1+5 receptors. The latter were almost completely inhibited by the group I agonist (S:)-3,5-dihydroxyphenylglycine. The binding profile correlated well with the cellular sites of synthesis and regional expression of the respective group I receptor proteins revealed by in situ hybridization histochemistry and immunohistochemistry, respectively.

Somatostatin is known to have diverse neurophysiological effects in the mammalian CNS. To date, genes for five different receptors, termed sst(1-5), have been isolated. Recently several reports have been published on the localisation of the individual receptor protein in the rat CNS, but their localisation in the human CNS remains largely unknown. Until now little information about the function of the sst(4) receptor is available, and there is a lack of receptor specific agonists and antagonists. Here, we report for the first time the immunohistochemical localisation of the sst(4) receptor in selected human brain areas using an anti-peptide antibody raised against a carboxy-terminal portion of the receptor protein. Strong receptor immunoreactivity was found in several brain regions, including the hippocampal formation, the cerebellar cortex and the medulla. Further immunohistochemical labelling was observed in the cerebral cortex, the red nucleus and the globus pallidus. Somatodendritic as well as axonal staining was observed. Specific signals were entirely absent following antibody pre-adsorption with the synthetic peptide. The results are in good agreement with the previously published immunohistochemical localisation of the sst(4) receptor in the rat brain. This is the first immunohistochemical study of the localisation of the sst(4) receptor in the human brain, and implicates this receptor in the function of higher centres of the human nervous system.

A number of studies have suggested that disturbance in glutamatergic transmission in the cerebral cortex may underlie, or contribute to the pathophysiology of schizophrenia. In this study we examined expression of the postsynaptic density protein 95 (PSD95) mRNA in the prefrontal cortex and hippocampus in postmortem material from neuroleptic-treated schizophrenics and normal controls. PSD95 is known to bind to NMDA receptor subunits and is known to be involved in synaptic plasticity. In situ hybridization analysis showed that the expression of PSD95 was significantly decreased in Brodmann area 9 of the prefrontal cortex but not in the hippocampus. These results further implicate the prefrontal cortex in the pathophysiology of schizophrenia and suggest dysfunction of NMDA receptors in the schizophrenic cortex.

Unlabelled: Calcium-binding proteins show a heterogeneous distribution in the mammalian central nervous system and are useful markers for identifying neuronal populations. The distribution of the three major calcium-binding proteins - calbindin-D28k (calbindin), calretinin and parvalbumin - has been investigated in eight neurologically normal human thalami using standard immunohistochemical techniques. Most thalamic nuclei show immunoreactive cell bodies for at least two of the three calcium-binding proteins; the only nucleus showing immunoreactivity for one calcium-binding protein is the centre médian nucleus (CM) which is parvalbumin-positive. Overall, the calcium-binding proteins show a complementary staining pattern in the human thalamus. In general terms, the highest density of parvalbumin staining is in the component nuclei of the ventral nuclear group (i.e. in the ventral anterior, ventral lateral and ventral posterior nuclear complexes) and in the medial and lateral geniculate nuclear groups. Moderate densities of parvalbumin staining are also present in regions of the mediodorsal nucleus (MD). By contrast, calbindin and calretinin immunoreactivity both show a similar distribution of dense staining in the thalamus which appears to complement the pattern of intense parvalbumin staining. That is, calbindin and calretinin staining is most dense in the rostral intralaminar nuclear group and in the patchy regions of the MD which show very low levels of parvalbumin staining. However, calbindin and calretinin also show low levels of staining in the ventral nuclear complex and in the medial and lateral geniculate bodies which overlaps with the intense parvalbumin staining in these regions. These results show that the calcium-binding proteins are heterogeneously distributed in a complementary fashion within the nuclei of the human thalamus. They provide further support for the concept recently proposed by Jones (Jones, E.G., 1998., Viewpoint: the core and matrix of thalamic organization. Neuroscience 85, 331-345) that the primate thalamus comprises of a matrix of calbindin immunoreactive cells and a superimposed core of parvalbumin immunoreactive cells which may have differential patterns of cortical projections.

The distribution of mRNAs encoding voltage-gated sodium channel alpha subunits (I, II, III, and VI) and beta subunits (beta1 and beta2) was studied in selected regions of the human brain by Northern blot and in situ hybridisation experiments. Northern blot analysis showed that all regions studied exhibited heterogenous expression of sodium channel transcripts. In situ hybridisation experiments confirmed these findings and revealed a predominantly neuronal distribution. In the parahippocampal gyrus, subtypes II and VI and the beta-subunit mRNAs exhibited robust expression in the granule cells of the dentate gyrus and pyramidal cell layer of the hippocampus. Subtypes I and III showed moderate expression in granule cells and low expression in the pyramidal cell layer. Distinct expression patterns were also observed in the cortical layers of the middle frontal gyrus and in the entorhinal cortex. In particular, all subtypes exhibited higher levels of expression in cortical layers III, V, and VI compared with layers I and II. All subtypes were expressed in the granular layer of the cerebellum, whereas specific expression of subtypes I, VI, beta1, and beta2 mRNAs was observed in Purkinje cells. Subtypes I, VI, and beta1 mRNAs were expressed, at varying levels, in the pyramidal cells of the deep cerebellar nuclei. These data indicate that, as in rat, human brain sodium channel mRNAs have a distinct regional distribution, with individual cell types expressing different compliments of sodium channels. The differential distribution of sodium channel subtypes suggest that they have distinct roles that are likely to be of paramount importance in maintaining the functional heterogeneity of central nervous system neurons., (Copyright 2000 Wiley-Liss, Inc.)

In recombinant cell lines, functional GABA(B) receptors are only formed by the heterodimerisation between two related G-protein coupled receptor proteins GABA(B)R1 (GBR1) and GABA(B)R2 (GBR2), whilst the individual GBR1 or GBR2 do not produce fully functional receptors. To determine whether the heterodimerisation occurs in vivo, novel polyclonal antibodies targeting the C termini of GBR1 and GBR2, were raised in different species, characterised, and used to determine the relative localisation of the reported heterodimer components in human brain tissue, using immunohistochemistry. The use of different species for the raising of the antisera allowed double immunofluorescent labelling of the receptors as an indication of GBR1/GBR2 receptor co-localisation in human brain. The presence of both proteins is reported in cerebellum, hippocampus, cortex, thalamus and basal ganglia. Regions of the brainstem including pons and medulla, also express GBR1 and GBR2 protein. The double immunofluorescence demonstrated that GBR1 and GBR2 are co-localised in the human cerebellar cortex. Together these results suggest the widespread distribution of GABA(B) receptors in human brain, and that GABA(B) receptors GBR1 and GBR2 can exist in the same cell, and therefore may function as a heterodimer in the human brain.

There are three main mechanisms of neuronal cell death which may act separately or cooperatively to cause neurodegeneration. This lethal triplet of metabolic compromise, excitotoxicity, and oxidative stress causes neuronal cell death that is both necrotic and apoptotic in nature. Aspects of each of these three mechanisms are believed to play a role in the neurodegeneration that occurs in both Parkinson's and Huntington's diseases. Strategies to rescue or protect injured neurons usually involve promoting neuronal growth and function or interfering with neurotoxic processes. Considerable research has been done on testing a large array of neuroprotective agents using animal models which mimic these disorders. Some of these approaches have progressed to the clinical arena. Here, we review neuroprotective strategies which have been found to successfully ameliorate the neurodegeneration associated with Parkinson's and Huntington's diseases. First, we will give an overview of the mechanisms of cell death and the background of Parkinson's and Huntington's diseases. Then we will elaborate on a range of neuroprotective strategies, including neurotrophic factors, anti-excitotoxins, antioxidants, bioenergetic supplements, anti-apoptotics, immunosuppressants, and cell transplantation techniques. Most of these approaches hold promise as potential therapies in the treatment of these disorders.

The effect of the N-terminal tripeptide of insulin-like growth factor (IGF)-1, glycine-proline-glutamate (GPE), as a neuroprotective agent for nigro-striatal dopaminergic neurons was examined in the present study using a rat model of Parkinson's disease. A unilateral nigro-striatal lesion was induced in rats by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle (MFB). GPE (3 microgram) or its vehicle was administered intracerebroventricularly (i.c.v.) 2 h after the 6-OHDA lesion. Tyrosine-hydroxylase (TH) immunohistochemistry in the substantia nigra compacta (SNc) and the striatum were examined 2 weeks after the lesion. Following 6-OHDA injection, the number of TH immunopositive neurons in the ipsilateral SNc was reduced. The density of TH immunostaining was also reduced in the ipsilateral SNc and the striatum. Treatment with a single dose of GPE (n=9) significantly prevented the loss of TH immunopositive neurons (p<0. 001) and restored the TH immunoreactivity in both the SNc and the striatum compared with the vehicle control group (n=9, p<0.001). The results suggest that GPE showed promise as a potential treatment for Parkinson's disease.

Genetic polymorphism of human bleomycin hydrolase (hBH) has been reported to be associated with the risk of sporadic Alzheimer's disease (AD). The regional and cellular distribution of mRNA encoding hBH in the brain from controls and patients with AD was examined using in situ hybridization. A hybridization signal, in the form of clusters of single cells, was observed in the white matter. Our results indicate a predominantly astrocytic expression of hBH in the investigated human brain regions. Although the signal intensity was generally reduced in AD brains, the large variability among controls rendered this trend statistically insignificant.

Severe perinatal asphyxia can lead to injury and dysfunction of the basal ganglia. Post insult administration of insulin-like growth factor-1 is neuroprotective, particularly in the striatum. Insulin-like growth factor-1 is also known to be a neuromodulator of several types of striatal neurons. The striatum comprises various phenotypic neurons with a complex neurochemical anatomy and physiology. In the present study, we examined the specificity of neuronal rescue with insulin-like growth factor-1 on different striatal neurons. Bilateral brain injury was induced in near term fetal sheep by 30 min of reversible carotid artery occlusion. A single dose of 3 microg of insulin-like growth factor-1 was infused over 1 h into the lateral ventricle 90 min following ischemia. The histological and immunohistochemical outcome were examined after 4 days recovery using paraffin tissue preparations. Insulin-like growth factor-1 treatment (n = 11) significantly reduced the percentage of neuronal loss in the striatum compared with the vehicle treated group (n = 10, 28.3+/-5.1% vs 55.5+/-17.3%, P < 0.005). Immunohistochemical studies showed that ischemia resulted in a significant loss of calbindin-28kd, choline acetyltransferase, parvalbumin, glutamate acid decarboxylase, neuronal nitric oxide synthase and neuropeptide Y immunopositive neurons, compared with sham controls. Insulin-like growth factor-1 markedly prevented the loss of calbindin-28kd (n = 7, P < 0.05), choline acetyltransferase (n = 7, P < 0.05), neuropeptide Y (n = 7, P < 0.05), neuronal nitric oxide synthase (n = 8, P < 0.05) and glutamate acid decarboxylase (n = 9, P < 0.05) immunopositive neurons, but failed to protect parvalbumin (n = 6) immunopositive neurons. The present study indicates that the therapeutic effect of insulin-like growth factor-1 in the basal ganglia is selectively associated with cholinergic and some phenotypic GABAergic neurons. These data suggest a potential role for insulin-like growth factor-1 in preventing cerebral palsy due to perinatal asphyxia.

In order to investigate the sequence and pattern of neurodegeneration in Huntington's disease, the distribution and density of cannabinoid CB(1), dopamine D(1) and D(2), adenosine A(2a) and GABA(A) receptor changes were studied in the basal ganglia in early (grade 0), intermediate (grades 1, 2) and advanced (grade 3) neuropathological grades of Huntington's disease. The results showed a sequential pattern of receptor changes in the basal ganglia with increasing neuropathological grades of Huntington's disease. First, the very early stages of the disease (grade 0) were characterized by a major loss of cannabinoid CB(1), dopamine D(2) and adenosine A(2a) receptor binding in the caudate nucleus, putamen and globus pallidus externus and an increase in GABA(A) receptor binding in the globus pallidus externus. Second, intermediate neuropathological grades (grades 1, 2) showed a further marked decrease of CB(1) receptor binding in the caudate nucleus and putamen; this was associated with a loss of D(1) receptors in the caudate nucleus and putamen and a loss of both CB(1) and D(1) receptors in the substantia nigra. Finally, advanced grades of Huntington's disease showed an almost total loss of CB(1) receptors and the further depletion of D(1) receptors in the caudate nucleus, putamen and globus pallidus internus, and an increase in GABA(A) receptor binding in the globus pallidus internus. These findings suggest that there is a sequential but overlapping pattern of neurodegeneration of GABAergic striatal efferent projection neurons in increasing neuropathological grades of Huntington's disease. First, GABA/enkephalin striatopallidal neurons projecting to the globus pallidus externus are affected in the very early grades of the disease. Second, GABA/substance P striatonigral neurons projecting to the substantia nigra are involved at intermediate neuropathological grades. Finally, GABA/substance P striatopallidal neurons projecting to the globus pallidus internus are affected in the late grades of the disease. In addition, the finding that cannabinoid receptors are dramatically reduced in all regions of the basal ganglia in advance of other receptor changes in Huntington's disease suggests a possible role for cannabinoids in the progression of neurodegeneration in Huntington's disease.

PCR amplification of the CAG repeat in exon 1 of the IT15 gene is routinely undertaken to confirm a clinical diagnosis of Huntington disease (HD) and to provide predictive testing for at-risk relatives of affected individuals. Our studies have detected null alleles on the chromosome carrying the expanded repeat in three of 91 apparently unrelated HD families. Sequence analysis of these alleles has revealed the same mutation event, leading to the juxtaposition of uninterrupted CAG and CCG repeats. These data suggest that a mutation-prone region exists in the IT15 gene bounded by the CAG and CCG repeats and that caution should be exercised in designing primers that anneal to the region bounded by these repeats. Two of the HD families segregated null alleles with expanded uninterrupted CAG repeats at the lower end of the zone of reduced penetrance. The expanded repeats are meiotically unstable in these families, although this instability is within a small range of repeat lengths. The haplotypes of the disease-causing chromosomes in these two families differ, only one of which is similar to that reported previously as being specific for new HD mutations. Finally, no apparent mitotic instability of the uninterrupted CAG repeat was observed in the brain of one of the HD individuals.

Huntington's disease is a progressive, autosomal dominantly inherited, neurodegenerative disease that is characterized by involuntary movements (chorea), cognitive decline and psychiatric manifestations. This is one of a number of late-onset neurodegenerative disorders caused by expanded glutamine repeats, with a likely similar biochemical basis. Immunohistochemical studies on Huntington's disease tissue, using antibodies raised to the N-terminal region of huntingtin (adjacent to the repeat) and ubiquitin, have recently identified neuronal inclusions within densely stained neuronal nuclei, peri-nuclear and within dystrophic neuritic processes. However, the functional significance of inclusions is unknown. It has been suggested that the disease-causing mechanism in Huntington's disease (and the other polyglutamine disorders) is the ability of polyglutamine to undergo a conformational change that can lead to the formation of very stable anti-parallel beta-sheets; more specifically, amyloid structures. We examined, using Congo Red staining and both polarizing and confocal microscopy, post mortem human brain tissue from five Huntington's disease cases, two Alzheimer's disease cases and two normal controls. Brains from five transgenic mice (R6/2)(12) expressing exon 1 of the human huntingtin gene with expanded polyglutamine, and five littermate controls, were also examined by the same techniques. We have shown that some inclusions in Huntington's disease brain tissue possess an amyloid-like structure, suggesting parallels with other amyloid-associated diseases such as Alzheimer's and prion diseases.

Comparative brain proteome analysis is a new strategy to discover proteins and therefore genes whose altered expression may underlie schizophrenia. This strategy does not require an a priori theory of the pathogenesis or the mode of inheritance of schizophrenia. Using proteome analysis we previously compared the hippocampal proteome, that is, those proteins expressed by the hippocampal genome, of seven schizophrenic individuals with the hippocampal proteome of seven control individuals, matched for age and post mortem delay.1 We found 18 proteins that were significantly altered in concentration in the schizophrenic hippocampus (P < 0.05), when compared to control tissue. One of these proteins was characterised, by N-terminal sequencing, as diazepam binding inhibitor whose gene maps to 6q12-q21. Here we characterise a further three of the 18 proteins as: manganese superoxide dismutase, 6q25.3, T-complex protein 1, 6q25.3-q26 and collapsin response mediator protein 2, 8p21. That three of these four characterised proteins should map to the long arm of the same chromosome is significant (P < 0.002) and suggests the importance of chromosome 6q in schizophrenia. These results indicate that antioxidant defence is altered in the schizophrenic hippocampus and suggest that segregation distortion, of schizophrenia susceptibility genes, may be a possible causative factor in the high incidence of schizophrenia. Molecular Psychiatry (2000) 5, 85-90.

The regional and cellular localisation of gamma-aminobutyric acid(A) (GABA(A)) receptors was investigated in the human basal ganglia using receptor autoradiography and immunohistochemical staining for five GABA(A) receptor subunits (alpha(1), alpha(2), alpha(3), beta(2, 3), and gamma(2)) and other neurochemical markers. The results demonstrated that GABA(A) receptors in the striatum showed considerable subunit heterogeneity in their regional distribution and cellular localisation. High densities of GABA(A) receptors in the striosome compartment contained the alpha(2), alpha(3), beta(2, 3), and gamma(2) subunits, and lower densities of receptors in the matrix compartment contained the alpha(1), alpha(2), alpha(3), beta(2,3), and gamma(2) subunits. Also, six different types of neurons were identified in the striatum on the basis of GABA(A) receptor subunit configuration, cellular and dendritic morphology, and chemical neuroanatomy. Three types of alpha(1) subunit immunoreactive neurons were identified: type 1, the most numerous (60%), were medium-sized aspiny neurons that were immunoreactive for parvalbumin and alpha(1), beta(2,3), and gamma(2) subunits; type 2 (38%) were medium-sized to large aspiny neurons immunoreactive for calretinin and alpha(1), alpha(3), beta(2,3), and gamma(2) subunits; and type 3 (2%) were large sparsely spiny neurons immunoreactive for alpha(1), alpha(3), beta(2,3), and gamma(2) subunits. Type 4 neurons were calbindin-positive and immunoreactive for alpha(2), alpha(3), beta(2,3), and gamma(2) subunits. The remaining neurons were immunoreactive for choline acetyltransferase (ChAT) and alpha(3) subunit (type 5) or were neuropeptide Y-positive with no GABA(A) receptor subunit immunoreactivity (type 6). The globus pallidus contained three types of neurons: types 1 and 2 were large neurons and were immunoreactive for alpha(1), alpha(3), beta(2,3), and gamma(2) subunits and for parvalbumin alone (type 1) or for both parvalbumin and calretinin (type 2); type 3 neurons were medium-sized and immunoreactive for calretinin and alpha(1), beta(2, 3), and gamma(2) subunits. These results show that the subunit composition of GABA(A) receptors displays considerable regional and cellular variation in the human striatum but are more homogeneous in the globus pallidus., (Copyright 1999 Wiley-Liss, Inc.)

The pattern and time-course of cellular, neurochemical and receptor changes in the striatum and substantia nigra were investigated following unilateral quinolinic acid lesions of the striatum in rats. The results showed that in the central region of the striatal lesion there was a major loss of Nissl staining of the small to medium sized cells within 2 h and a substantial loss of neuronal staining within 24 h after lesioning. Immunohistochemical studies showed a total loss of calbindin immunoreactivity, a known marker of GABAergic striatal projection neurons, throughout the full extent of the quinolinic acid lesion within 24 h. Similarly, within 24 h, there was a total loss of somatostatin/neuropeptide Y cells in the centre of the lesion but in the periphery of the lesion these cells remained unaltered at all survival times. Striatal GABA(A) receptors remained unchanged in the lesion for 7 days, and then declined in density over the remainder of the time course. Glial fibrillary acidic protein immunoreactive astrocytes were present in the periphery of the lesion at 7 days, occupied the full extent of the lesion by 4 weeks, and remained elevated for up to 2 months. In the substantia nigra, following placement of a striatal quinolinic acid lesion, there was: a loss of substance P immunoreactivity within 24 h; a marked astrocytosis evident from 1-4 weeks postlesion; and, a major increase in GABA(A) receptors in the substantia nigra which occurred within 2 h postlesion and was sustained for the remainder of the time course (15 months). This study shows that following quinolinic acid lesions of the striatum there is a major loss of calbindin and somatostatin/neuropeptide Y immunoreactive cells in the striatum within 24 h, and a marked increase in GABA(A) receptors in the substantia nigra within 2 h. These findings are similar to the changes in the basal ganglia in Huntington's disease and provide further evidence supporting the use of the quinolinic acid lesioned rat as an animal model of Huntington's disease.

Huntington's disease is an incurable genetic neurological disorder characterized by the relatively selective degeneration of the striatum. Lesioning of the striatum in rodents using the excitatory amino acid agonist, quinolinic acid (QA), effectively mimics the human neuropathology seen in Huntington's disease. Using this animal model of Huntington's disease, we investigated the ability of the insulin-like growth factor-I (IGF-I) amino-terminal tripeptide glycine-proline-glutamate (GPE) to protect striatal neurons from degeneration. Adult rats received a single unilateral intrastriatal injection of QA (100 nmol) and then daily injection of either vehicle or GPE (0.3 microgram/microliter/day) into the striatum for 7 days. QA at this dose resulted in a partial lesioning of the striatum after 7 days to approximately 50% of cells of unlesioned levels in vehicle-treated animals. The major striatal neuronal phenotype, GABAergic projection neurons, were identified by immunocytochemical labeling of either glutamate decarboxylase 67 (GAD(67)) or the calcium binding protein calbindin in alternate sections. Treatment with GPE for 7 days reversed the loss in projection neurons when assessed by counts of calbindin-stained cells; however, these rescued cells did not regain immunologically detectable levels of GAD(67). GPE also significantly reversed the phenotypic degeneration of cholinergic interneurons identified by immunolabeling for choline acetyltransferase (ChAT) and NADPH diaphorase interneurons identified histochemically. GPE treatment failed to rescue the calcium binding protein interneuron populations of parvalbumin and calretinin neurons. These findings reveal that exogenous administration of GPE selectively prevents excitotoxin induced phenotypic degeneration of striatal projection neurons and cholinergic and NADPH diaphorase interneurons in an animal model of Huntington's disease., (Copyright 1999 Academic Press.)

Immunohistochemical studies have shown that the three calcium-binding proteins (calbindin-D28k, calretinin and parvalbumin) are heterogeneously distributed in the mammalian brain and are useful for delineating nuclear boundaries. We have investigated the distribution of the three calcium-binding proteins in the human thalamus in order to assist in the delineation of the equivocal nuclear boundaries of the intralaminar nuclei of the thalamus. The results show that each of the "functional" nuclear complexes in the human thalamus demonstrates a characteristic pattern of calcium-binding protein immunoreactivity. In particular, the intralaminar nuclei are characterized by a unique combination of calcium-binding protein staining which clearly delineates the component nuclei in this complex from the other nuclei of the human thalamus. The anterior group of intralaminar nuclei (central lateral nucleus, paracentral nucleus and central medial nucleus) showed intense staining for both calbindin-D28k and calretinin. By contrast, the posterior group of intralaminar nuclei (centre median nucleus and parafascicular nucleus) showed a complementary pattern of staining; the centre median nucleus showed immunoreactivity only for one calcium-binding protein, parvalbumin, while the parafascicular nucleus showed immunoreactivity for both calbindin-D28k and calretinin. No other nucleus in the human thalamus showed these particular combinations of calcium-binding protein staining. Since the intralaminar nuclei also have unique topographically organized connectional affiliations with both the cerebral cortex and the basal ganglia, these results suggest that the calcium-binding proteins may play an important role in the influence of the intralaminar nuclei on interactions between the cerebral cortex and the basal ganglia.

Two new spliced variants of the human metabotropic glutamate receptor 8 (HmGluR8), designated HmGluR8b and HmGluR8c, were identified in a human fetal brain cDNA library. The HmGluR8b and c differ from previously reported HmGluR8a by the out-of-frame insertions of 55-bp and 74-bp, respectively. The 55-bp insertion which contains a stop codon resulted in substitution of the last 16 amino acids in the C-terminus of HmGluR8a with 16 different amino acids in HmGluR8b. The 74-bp insertion introduces a frame shift in the predicted translation resulting in termination of the polypeptide before the putative seven transmembrane domains. Thus, the predicted HmGluR8c protein is 501 amino acids long and could represent a secreted isoform of the receptor. The pattern of mRNA expression of mGluR8 variants in human brain were analyzed by RT-PCR, Northern blot and in situ hybridization. Both HmGluR8a and b are expressed with similar abundance in fetal and adult brains. The in situ hybridization results indicate a predominantly glial cell expression of HmGluR8c in human brain. The three isoforms were transiently expressed in CHO cells from Semliki Forest Virus vectors. [3H]l-AP4 binding was performed on the cell membranes and the saturation curves showed the presence of a binding site with KD values of 249 and 182 nM and Bmax values of 13.6 and 10.5 pmoles/mg protein for HmGluR8a and b, respectively. For the six mGluR ligands studied, a similar rank order of potency was observed on both HmGluRa and b: l-AP4>l-SOP=l-CCG I>l-glutamate>DCG IV>LY 354740., (Copyright 1999 Elsevier Science B.V.)

The proteins expressed by a genome have been termed the proteome. Comparative proteome analysis of brain tissue offers a novel means to identify biologically significant gene products that underlie psychopathology. In this study we collected post mortem hippocampal tissue from the brains of seven schizophrenic, seven Alzheimer's disease (AD) and seven control individuals. Hippocampal proteomes were visualised by two-dimensional gel electrophoresis of homogenised tissue. A mean of 549 (s.d. 35) proteins were successfully matched between each disease group and the control group. In comparison with the control hippocampal proteome, eight proteins in the schizophrenic hippocampal proteome were found to be decreased and eight increased in concentration, whereas, in the AD hippocampal proteome, 35 proteins were decreased and 73 were increased in concentration (P<0.05). One protein, which was decreased in concentration in both diseases, was characterised as diazepam binding inhibitor (DBI) by N-terminal sequence analysis. DBI can regulate the action of the GABA(A) receptor. Protein changes involved 6% of the assessed AD hippocampal proteome, whereas, in schizophrenia protein changes involved less than 1% of the assessed hippocampal proteome. We conclude that schizophrenia has a subtle neuropathological presentation and comparative proteome analysis is a viable means by which to investigate diseases of the brain at the molecular level.

Insulin-like growth factor-1 has pleiotropic effects in the central nervous system and can act both as a survival and a differentiation factor. Insulin-like growth factor-1 can be proteolytically cleaved into des-N-(1-3)-insulin-like growth factor-1 and a N-terminal tripeptide fragment, glycine-proline-glutamate. Both insulin-like growth factor-1 and des-N-(1-3)-insulin-like growth factor-1 can improve neuronal survival after hypoxic-ischemic brain injury in vivo. The present study investigates the effects of glycine-proline-glutamate on different brain regions and neuronal populations after hypoxic-ischemic injury. Unilateral hypoxic-ischemic injury was induced in adult rats. Glycine-proline-glutamate (3 microg) was administered centrally 2 h after the injury and the extent of brain damage determined five days later. In a separate trial immunohistochemical techniques were used to determine the effects of glycine-proline-glutamate on specific populations of neurons in the striatum after the injury. Compared to the vehicle treatment, glycine-proline-glutamate (n=19) treatment reduced the extent of cortical damage and neuronal loss in the CA1-2 subregions of the hippocampus (P<0.05). In the striatum, there was a trend towards a reduction in neuronal loss after glycine-proline-glutamate treatment (P=0.053) compared to the vehicle (n=21)-treated animals. In a separate study, glycine-proline-glutamate (n=8) treatment prevented the loss of choline acetyltransferase (P<0.05), glutamate acid decarboxylase (P<0.05) and somatostatin (P<0.05) containing neurons in the ipsilateral striatum following hypoxic-ischemic brain injury and also increased the numbers of neuronal nitric oxide synthase (P<0.05) containing neurons in the contralateral side. These studies suggest that in addition to neuroprotective effects, glycine-proline-glutamate can influence neuronal activity after hypoxic-ischemic injury.

In the present study, we have isolated a cDNA encoding a novel member of the family of zinc metallopeptidases that includes neutral endopeptidase and endothelin-converting enzyme. The predicted amino-acid sequence of this enzyme, termed XCE, consists of 775 amino-acids with a single putative membrane-spanning region, an N-terminal cytoplasmic domain of 59 residues, and a large luminal domain that contains a characteristic zinc-binding motif. Western blot analysis of cells stably expressing this new metallopeptidase revealed a glycosylated protein of approximately 95 kDa. XCE mRNA was found to be predominantly expressed in the central nervous system, sympathetic ganglia and in uterine subepithelial cells. In the rat and human CNS, a very specific pattern of neuronal labelling (in presumptive cholinergic interneurons of basal ganglia, basal forebrain neurons, as well as brainstem and spinal cord motoneurons) was detected by in situ hybridization histochemistry. The enzyme substrate, as yet unidentified, might be found among the numerous neuropeptide transmitters which are colocalized with acetylcholine in these neurons., (Copyright 1999 Elsevier Science B.V.)

Insulin-like growth factor 1 (IGF-1) plays a critical role in CNS development. IGF-1 can block neuronal apoptosis in vitro and in vivo. IGF-1 is thought to be cleaved into des-N-(1-3)-IGF-1 and an amino terminal glycine-proline-glutamate (GPE tripeptide). Here we report a neuroprotective role for GPE tripeptide, with enhanced survival of the CA1-2 hippocampal neurons following an excitotoxic insult in vitro. Binding and displacement studies suggest uniquely distributed sites of action within the rat including the hippocampal CA1-2, pyriform cortex, amygdala, choroid plexus, blood vessels and to a lesser extent in the cortical regions. A similar pattern of binding was seen in the human. This finding could lead to new strategies to reduce neuronal death after injury and in disease.

Glutamate is the major excitatory neurotransmitter in the central nervous system and is implicated in the pathogenesis of neurodegenerative diseases. Five human glutamate transporters have been cloned and are responsible for the removal of potentially excitotoxic excess glutamate from the extracellular space. In this study we consider whether there are selective changes in the expression of the glutamate transporters in the medial temporal cortex and hippocampus from temporal lobe epilepsy patients, which might contribute to the development or maintenance of seizures. Since disruption of the glial transporter excitatory amino acid transporter 2 in mice results in lethal spontaneous seizures, we were interested primarily in studying changes in this transporter. Using in situ hybridization we show that there was no reduction in the level of excitatory amino acid transporter 2 encoding messenger RNA in the temporal lobe epilepsy cases compared to post mortem controls and indeed there was a relative increase in content of excitatory amino acid transporter 2 messenger RNA per cell in temporal lobe epilepsy cases. Western blotting showed that there was no change in the excitatory amino acid transporter 2 protein content in temporal lobe epilepsy cases as compared to post mortem controls. A small reduction in the level of the second astroglial transporter protein, excitatory amino acid transporter 1, was observed in temporal lobe epilepsy cases. Surprisingly, immunohistochemical experiments using a polyclonal antiexcitatory amino acid transporter 2 antibody, showed a different localization of this protein in epilepsy derived tissue as compared to post mortem controls although glial markers such as glial fibrillary acidic protein and glutamine synthase showed similar patterns of staining. However, repeating this experiment using control tissue from non-temporal lobe epilepsy biopsies demonstrated that this change in the excitatory amino acid transporter 2 transporter localization occurred post mortem. These data suggest that major changes in the level of expression of the glutamate transporters do not play an important role in the development of human temporal lobe epilepsy but may be implicated the aetiology of other types of epilepsy.

The distribution of messenger RNA encoding the human GAT-1 (a high-affinity GABA transporter) was investigated in the subthalamic nucleus of 10 neurologically normal human post mortem cases. Further, the distribution of messenger RNA and protein encoding the three neuronally expressed calcium-binding proteins (calbindin D28k, parvalbumin and calretinin) was similarly investigated using in situ hybridization and immunohistochemical techniques. Cellular sites of calbindin D28k, parvalbumin, calretinin and GAT-1 messenger RNA expression were localized using human-specific oligonucleotide probes radiolabelled with [35S]dATP. Sites of protein localization were visualized using specific anti-calbindin D28k, anti-parvalbumin and anti-calretinin antisera. Examination of emulsion-coated tissue sections processed for in situ hybridization revealed an intense signal for GAT-1 messenger RNA within the human subthalamic nucleus, indeed the majority of Methylene Blue-counterstained cells were enriched in this transcript. Further, a marked heterogeneity was noted with regard to the expression of the messenger RNA's encoding the three calcium-binding proteins; this elliptical nucleus was highly enriched in parvalbumin messenger RNA-positive neurons and calretinin mRNA-positive cells but not calbindin messenger RNA-positive cells. Indeed, only an occasional calbindin messenger RNA-positive cell was detected within the mediolateral extent of the nucleus. In marked contrast, numerous parvalbumin messenger RNA-positive cells and calretinin messenger RNA-positive cells were detected and they were topographically distributed; parvalbumin messenger RNA-positive cells were highly enriched in the dorsal subthalamic nucleus extending mediolaterally; calretinin messenger RNA-positive cells were more enriched ventrally although some degree of overlap was apparent. Computer-assisted analysis of the average cross-sectional somatic area of parvalbumin, calretinin and GAT-1 messenger RNA-positive neurons revealed them all to be in the range of 300 microm2. The unique patterns of calcium-binding protein gene expression were similarly reflected at the protein level; an abundance of parvalbumin- and calretinin-immunopositive neurons were observed whereas only occasional intensely-labelled calbindin-immunopositive fibres were seen, no calbindin-immunopositive cells were detected. Single and double labelling studies show that parvalbumin-immunopositive neurons were mainly localized in the dorsal region of the nucleus, and calretinin-immunopositive neurons were mainly localized in the ventral region although there was overlap with double-labelled neurons located in the middle and dorsal regions. The significance of these findings, in particular the expression of GAT-1, a high-affinity GABA uptake protein, for basal ganglia signalling is discussed.

The proteins expressed by a genome have been termed the proteome. By comparing the proteome of a disease-affected tissue with the proteome of an unaffected tissue it is possible to identify proteins that play a role in a disease process. The hippocampus is involved in the processing of short-term memory and is affected in Alzheimer's disease. Any comparative proteome analysis that can identify proteins important in a disease affecting the hippocampus requires the characterization of the normal hippocampal proteome. Therefore, we homogenised normal hippocampal tissue and separated the proteins by two-dimensional polyacrylamide gel electrophoresis (2DE). Seventy-two unique protein spots were collected from Coomassie blue-stained 2DE gels and subjected to in-gel digestion with trypsin, reversed-phase high-pressure liquid chromatography peptide separation, and N-terminal protein sequencing. Sufficient protein sequence was obtained to successfully characterize 66 of the 72 protein spots chosen (92%). Three of the 66 proteins were not present in any database (4.5%). The characterized proteins comprised two dominant functional groups, i.e., enzymes involved in intermediary cellular metabolism (40%), and proteins associated with the cytoskeleton (15%). The identity, molecular mass, isoelectric point, and relative concentration of the characterized proteins are described and constitute a partial proteome map of the normal human hippocampus.

Recent studies using DNA fragmentation assays suggest a role for apoptosis in cell death in Huntington's disease. In this study, we investigated the relationship between the degree of DNA fragmentation and the number of trinucleotide (CAG) repeats of the Huntington's disease gene in striatal tissue from Huntington's disease brains. We used frozen striatal tissue from 27 post mortem Huntington's disease brains (graded 0-4 on the Vonsattel classification, post mortem delay ranging from 4 to 41 h), plus control sections which were age, sex and post mortem delay matched from neurologically normal and Alzheimer's diseased striatal tissue. Our results show a significant positive correlation between the number of CAG repeats in the Huntington's disease gene and the degree of DNA fragmentation in Huntington's disease striatum. These results suggest that expanded CAG repeats in the Huntington's disease gene may lead to neuronal degeneration in Huntington's disease through an apoptotic mechanism.

In Huntington's diseased human brain, it is in the caudate nucleus (CN) and globus pallidus (GP) of the basal ganglia where nerve cell death is seen most dramatically. The distribution of five gap junction proteins (connexins 26, 32, 40, 43 and 50) has been examined in these areas in normal and Huntington's diseased human brain using immunohistochemical techniques. There was no Cx50 expression observed and Cx40 was localized in the endothelial cells of blood vessels, with the Huntington's diseased brains having more numerous and smaller blood vessels than normal tissue. Cx26 and Cx32 revealed a similar distribution pattern to each other in both normal and diseased brains with little labelling in the CN but clear labelling in the GP. Cx43, expressed by astrocytes, was the most abundant connexin type of those studied. In both normal and diseased brains Cx43 in the GP was homogeneously distributed in the neuropil. In the CN, however, Cx43 density was both increased with Huntington's disease and became located in patches. Glial fibrillary acidic protein(GFAP) staining of astrocytes was also highly increased in the CN compared with normal brains. These labelling patterns indicate a reactive astrocytosis around degenerating neurons with an increased expression of astrocytic gap junctions. The enhanced coupling state between astrocytes, assuming the junctions are functional, could provide an increased spatial buffering capacity by the astrocytes in an attempt to maintain a proper environment for the neurons, helping promote neuronal survival in this neurodegenerative disorder., (Copyright 1998 Academic Press.)

3-Nitropropionic acid (3-NP) is a mitochondrial toxin which interferes with ATP synthesis. Accidental ingestion of 3-NP by humans as well as other mammals results in neuronal degeneration within the basal ganglia and movement dysfunction characterized by dystonia, chorea, and hypokinesia. The selective degeneration of structures of the basal ganglia occurs despite the non-selective impairment of energy metabolism throughout the brain and body. These effects of 3-NP are shared with the genetic disorder Huntington's disease (HD), which is characterized by progressive neurodegeneration of the basal ganglia and choreic motor dysfunction. These similarities have prompted further investigation of 3-NP as an animal model of HD. Metabolic compromise with 3-NP causes neurodegeneration that involves three interacting processes: energy impairment, excitotoxicity, and oxidative stress. This triplet of cooperative pathways of neurodegeneration helps to explain 3-NP's regional selectivity of neurotoxicity to the basal ganglia. This mini-review will focus on the actions of 3-NP and the related compound, malonic acid (MA), in the central nervous system, with an emphasis on the more current findings regarding their mechanisms of action.

The distribution of gamma-aminobutyric acid(A) (GABA(A)) receptors was investigated in the basal ganglia in the baboon brain by using receptor autoradiography and the immunohistochemical localisation of the alpha1 and beta2,3 subunits of the GABA(A) receptor by light and electron microscopy. In the caudate-putamen, the alpha1 subunit was distributed in high densities in the matrix compartment, and the beta2,3 subunits were more homogeneously distributed; the globus pallidus showed lower levels of the alpha1 and beta2,3 subunits. Four types of alpha1 subunit immunoreactive neurons were identified in the baboon striatum: the most numerous (75%) were type 1 medium-sized aspiny neurons; type 2 (2%) were large aspiny neurons with an indented nuclear membrane located in the ventral striatum; type 3 neurons were the least numerous (1%) and were comprised of large neurons in the ventromedial regions of the striatum; and type 4 (22%) neurons were medium to large aspiny neurons located in striosomes. At the ultrastructural level, alpha1 and beta2,3 subunit immunoreactivity was localised in the neuropil of the striatum in both symmetrical and asymmetrical synaptic contacts. In the globus pallidus, alpha1 and beta2,3 subunits were localised on large neurons and were found in three types of synaptic terminals: type 1 terminals were small and established symmetrical synapses; type 2 terminals were large; and type 3 terminals formed small synaptic terminals with subjunctional dense bodies. These results show that the subunit composition of GABA(A) receptors varies between the striosome and the matrix compartments in the striatum and that there is receptor subunit homogeneity in the globus pallidus.

The anticonvulsant compound felbamate (2-phenyl-1,3-propanediol dicarbamate; FBM) appears to inhibit the function of the N-methyl-D-aspartate (NMDA) receptor complex through an interaction with the strychnine-insensitive glycine recognition site. Since we have demonstrated previously that FBM inhibits the binding of [3H]5, 7-dichlorokynurenic acid (DCKA), a competitive antagonist at the glycine site, we assessed the ability of FBM to modulate the binding of an agonist, [3H]glycine, to rat forebrain membranes and human brain sections. In contrast to its ability to inhibit [3H]5,7-DCKA binding, FBM increased [3H]glycine binding (20 nM; EC50 = 485 microM; Emax = 211% of control; nH = 1.8). FBM, but not carbamazepine, phenytoin, valproic acid or phenobarbital, also increased [3H]glycine binding (50 nM; EC50 = 142 microM; Emax = 157% of control; nH = 1.6) in human cortex sections. Autoradiographic analysis of human brain slices demonstrated that FBM produced the largest increases in [3H]glycine binding in the cortex, hippocampus and the parahippocampal gyrus. Because various ions can influence the binding of glycine-site ligands, we assessed their effects on FBM-modulation of [3H]glycine binding. FBM-enhanced [3H]glycine binding was attenuated by Zn++ and not inhibited by Mg++ in human brain. These results suggest that FBM increases [3H]glycine binding in a manner sensitive to ions which modulate the NMDA receptor. These data support the hypothesis that FBM produces anticonvulsant and neuroprotective effects by inhibiting NMDA receptor function, likely through an allosteric modulation of the glycine site.

The distribution and cellular localization of the somatostatin sst2(a) receptor was investigated in selected human brain areas using an anti-peptide antibody raised against a carboxy-terminal portion of the receptor protein. The sst2(a) receptor was found to be present on neurones and processes in the deep layers of the cerebral cortex, in the subicular complex and the hippocampal formation. Further signals were obtained in the molecular and granular layer of the cerebellum, with occasional weakly stained Purkinje cells. The regional distribution of the receptor protein was compared with quantitative autoradiography using a sst2 receptor selective ligand [125I]BIM23027.

Alzheimer's disease may be due to a deficiency in neurotrophin protein or receptor expression. Consistent with this hypothesis, a reduction in BDNF mRNA expression has been observed in human post-mortem Alzheimer's disease hippocampi. To further investigate this observation, we examined whether the alteration in BDNF expression also occurred at the protein level in human post-mortem Alzheimer's disease hippocampi and temporal cortices using immunohistochemical techniques. We observed a reduction in the intensity and number of BDNF-immunoreactive cell bodies within both the Alzheimer's disease hippocampus and temporal cortex when compared to normal tissue. These results support and extend previous findings that BDNF mRNA is reduced in the human Alzheimer's disease hippocampus and temporal cortex, and suggest that a loss of BDNF may contribute to the progressive atrophy of neurons in Alzheimer's disease.

IGF-I has been shown to enhance neuronal survival and inhibit apoptosis. IGF-I immunoreactivity was examined in the Alzheimer's disease and normal post-mortem human hippocampus and temporal cortex to determine whether IGF-I protein levels are altered in response to neurodegeneration. IGF-I immunoreactivity was induced in a subpopulation of GFAP-immunopositive astroglia in the Alzheimer's disease temporal cortex. These observations raise the possibility that IGF-I has a neuroprotective role in the Alzheimer's disease brain.

Apoptosis is an active process of cell death characterized by distinct morphological features and is often the end result of a genetic program of events, i.e., programmed cell death (PCD). There is growing evidence supporting a role for apoptosis and/or PCD in Alzheimer's disease (AD), based on DNA fragmentation studies and recent findings of increased levels of inducible transcription factors (ITFs) such as c-Jun in AD brains. We have characterized the expression of a large range of ITFs (c-Fos, Fos B, Fos-related antigens, c-Jun, Jun B, Jun D, Krox20, and Krox24) using multiple antisera in AD postmortem hippocampi and compared this with human control hippocampi as well as Huntington's disease hippocampi and human epilepsy biopsy tissue. We found little evidence of nuclear expression of any ITF except c-Jun in the human postmortem tissue, compared with nuclear staining in biopsy tissue. We found some evidence for increased levels of c-Jun and Krox24 protein and krox24 mRNA in the CA1 region of AD hippocampi, suggesting that PCD may be involved in the pathogenesis of AD. In general, staining characteristics of ITFs varied with different antisera directed against the same protein, indicating the need for caution when interpreting results.

The distribution, morphology and chemical characteristics of neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the striatum of the basal ganglia in the rat brain were investigated at the light, confocal and electron microscope levels using single, double and triple immunohistochemical labelling techniques. The results showed that alpha1-subunit immunoreactive neurons were sparsely distributed throughout the rat striatum. Double and triple labelling results showed that all the alpha1-subunit-immunoreactive neurons were positive for glutamate decarboxylase and immunoreactive for the beta2,3 and gamma2 subunits of the GABA(A) receptor. Three types of alpha1-subunit-immunoreactive neurons were identified in the striatum on the basis of cellular morphology and chemical characteristics. The most numerous alpha1-subunit-immunoreactive neurons were medium-sized, aspiny neurons with a widely branching dendritic tree. They were parvalbumin-negative and were located mainly in the dorsolateral regions of the striatum. Electron microscopy showed that these neurons had an indented nuclear membrane, typical of striatal interneurons, and were surrounded by small numbers of axon terminals which established alpha1-subunit-immunoreactive synaptic contacts with the soma and dendrites. These cells were classified as type 1 alpha1-subunit-immunoreactive neurons and comprised 75% of the total population of alpha1-subunit-immunoreactive neurons in the striatum. The remaining alpha1-subunit-immunoreactive neurons comprised of a heterogeneous population of large-sized neurons localized in the ventral and medial regions of the striatum. The most numerous large-sized cells were parvalbumin-negative, had two to three relatively short branching dendrites and were designated type 2 alpha1-subunit-immunoreactive neurons. Electron microscopy showed that the type 2 neurons were characterized by a highly convoluted nuclear membrane and were sparsely covered with small axon terminals. The type 2 neurons comprised 20% of the total population of alpha1-subunit-immunoreactive neurons. The remaining large-sized alpha1-immunoreactive cells were designated type 3 cells; they were positive for parvalbumin and were distinguished by long branching dendrites extending dorsally for 600-800 microm into the striatum. These neurons comprised 5% of the total population of alpha1-subunit-immunoreactive neurons and were surrounded by enkephalin-immunoreactive terminals. Electron microscopy showed that the alpha1-subunit type 3 neurons had an indented nuclear membrane and were densely covered with small axon terminals which established alpha1-subunit-immunoreactive symmetrical synaptic contacts with the soma and dendrites. These results provide a detailed characterization of the distribution, morphology and chemical characteristics of the alpha1-subunit-immunoreactive neurons in the rat striatum and suggest that the type 1 and type 2 neurons comprise of separate populations of striatal interneurons while the type 3 neurons may represent the large striatonigral projection neurons described by Bolam et al. [Bolam J. P., Somogyi P., Totterdell S. and Smith A. D. (1981) Neuroscience 6, 2141-2157.].

The cellular expression of dopamine D1 and D2 receptor mRNAs was investigated in the postmortem human caudate nucleus of control cases and genetically and pathologically confirmed cases of Huntington's disease (HD) by using quantitative in situ hybridization. The HD cases were categorized (0-4) by severity of striatal neuropathology according to the Vonsattel scale. For the HD grade 0 case, a pronounced reduction in the number of D1 and D2 mRNA-positive cells was observed compared with controls; however, the abundance of both receptor mRNAs per remaining cell was within the control range. For D2 receptor mRNA, the number of detectable D2-positive medium-sized cells decreased with increasing pathology; this decrease was accompanied by a gradual reduction in the intensity of D2 signal per cell. By contrast, for D1 receptor mRNA, despite a decrease in the number of D1 mRNA-positive cells detected, the average cellular expression of D1 mRNA was markedly reduced in the HD grade 1 case and then increased (relative to the grade 1 case) with increasing pathology, presumably reflecting the relative survival of D1-expressing striatal interneurons. The implications of these findings for providing further information on the neurodegenerative process in HD are discussed.

Apoptosis is an active process of cell death characterized by distinct morphological features, and is often the end result of a genetic programme of events, i.e. programmed cell death (PCD). There is growing evidence supporting a role for apoptosis in some neurodegenerative diseases. This conclusion is based on DNA fragmentation studies and findings of increased levels of pro-apoptotic genes in human brain and in in vivo and in vitro model systems. Additionally, there is some evidence for a loss of neurotrophin support in neurodegenerative diseases. In Alzheimer's disease, in particular, there is strong evidence from human brain studies, transgenic models and in vitro models to suggest that the mode of nerve cell death is apoptotic. In this review we describe the evidence implicating apoptosis in neurodegenerative diseases with a particular emphasis on Alzheimer's disease.

Recent studies indicate that the proto-oncogene Bax, and other related proteins (eg Bcl-2) may play a major role in determining whether cells will undergo apoptosis under conditions which promote cell death. Increased expression of Bax has been found to promote apoptosis, while over-expression of Bcl-2 can inhibit apoptosis. To investigate the role of Bax in nerve cell death in the rat brain we examined the level of Bax expression in cells undergoing apoptosis, using a hypoxic-ischemic stroke model. We found that Bax was expressed at high levels in the nuclei of neurons in the hippocampus, cortex, cerebellum, and striatum on the control side, and that Bax levels increased in hippocampal neurons undergoing apoptosis on the stroke side, and then declined (correlating with cell loss). In the Alzheimer's disease hippocampi we found a concentrated localisation of Bax in senile plaques, which correlated with the localisation of beta-amyloid protein in adjacent sections from the same brains. beta-Amyloid positive plaques are thought to contribute to the Alzheimer's disease process, possibly via an apoptotic mechanism, and this may occur via an increase in Bax in these areas. Bax was also strongly stained in tau-positive tangles in Alzheimer's disease hippocampi, suggesting Bax may play a role in tangle formation. In addition, we observed a loss of Bax expression in the dentate granule cells of Alzheimer's disease hippocampi compared with moderate Bax expression in control hippocampi, and this loss may be related to the survival of these neurons in Alzheimer's disease. Finally, we observed substantially different staining patterns of Bax using three different commercially available antisera to Bax, indicating the need for caution when interpreting results in this area.