Cholinergic activity in autism: abnormalities in the cerebral cortex and basal forebrain.

OBJECTIVE: Measures of cholinergic transmitter activity were investigated in patients with autism because of reported neuropathological abnormalities in cholinergic nuclei in the basal forebrain. METHOD: Levels of cholinergic enzyme and receptor activity were measured in the frontal and parietal cerebral cortex of deceased autistic adults, similarly aged normal adults without mental retardation, and nonautistic mentally retarded adults. The immunoreactivity levels of brain-derived neurotrophic factor and nerve growth factor were measured in the basal forebrain. RESULTS: There were no differences between the autistic and comparison groups in choline acetyltransferase or acetylcholinesterase activity in the cerebral cortex and basal forebrain or in muscarinic M(2) receptor or alpha-bungarotoxin binding within the cortex. Cortical M(1) receptor binding was up to 30% lower than normal in the autistic subjects, and the difference reached significance in the parietal cortex. In both the parietal and frontal cortices, differences in nicotinic receptors assessed by [(3)H]epibatidine binding were significant and extensive (65%-73% lower in the autistic group than in the normal subjects); there were no differences in nicotine binding in the basal forebrain. Immunochemical analysis indicated lower levels of both the alpha(4) and beta(2) nicotinic receptor subunits in the parietal cortex. The M(1) receptor abnormality was not evident in the nonautistic group with mental retardation, although the lower [(3)H]epibatidine binding was apparent. In the basal forebrain, the level of brain-derived neurotrophic factor in the autistic group was three times as high as the level of the normal group. CONCLUSIONS: These neurochemical abnormalities implicate the cholinergic system in developmental disorders such as autism and suggest the potential for intervention based on cholinergic receptor modulation.

Distribution of the voltage-dependent calcium channel alpha(1A) subunit throughout the mature rat brain and its relationship to neurotransmitter pathways.

The alpha(1) subunit provides both the voltage-sensing mechanism and the ion pore of voltage-dependent calcium channels. Of the six classes of alpha(1) subunit cloned to date, alpha)1A) is the subject of debate in terms of its functional correlate, although it is generally thought to encode voltage-dependent calcium channels of the omega-agatoxin IVA-sensitive, P/Q type. In the present study, an alpha(1A)-specific riboprobe and antibody were used with in situ hybridisation and immunohistochemical techniques to localise alpha(1A) messenger ribonucleic acid transcripts and subunit protein throughout the mature rat brain. Dual localisation of alpha(1A) protein and markers for acetylcholine, catecholamines, and 5-hydroxytryptamine have also been performed in a number of discrete areas. Abundant and widespread distribution of alpha(1A) protein was found, with immunoreactivity occurring both in cell bodies and as punctate staining in areas of neuronal processes. Several associations were noted across alpha(1A) localisation, defined neuroanatomical regions, and neurotransmitter systems. However, alpha(1A) expression was not confined to loci corresponding to any one neurotransmitter type, although a high level of expression was observed in cholinergic neurones. The distribution of the alpha(1A) subunit in the rat corresponded well with the limited human mapping data that are available.

A biotin-avidin technique for the localisation of membrane-bound monoclonal antibodies by low power transmission electron microscopy.

A biotin-avidin horseradish peroxidase system is described for the localisation of membrane-bound monoclonal antibodies by low power transmission electron microscopy. An indirect bridge technique was used, with a biotin-labelled goat anti-mouse immunoglobulin antibody and horseradish peroxidase-conjugated avidin D. The technique provided preparations of high morphological and ultrastructural quality which facilitated accurate cellular identification. Positive immunostaining was specific and precisely defined.

Identification of pore-forming subunit of P-type calcium channels: an antisense study on rat cerebellar Purkinje cells in culture.

Treatment of cerebellar neurones in culture with an antisense oligonucleotide (ODN) against alpha1A, reduced the whole-cell P-type calcium channel current relative to mismatch ODN treated controls (p < 0.001). Therefore, AgaIVA (50 nM) reduced whole-cell calcium current in mismatch and antisense treated cells by 70 +/- 4 and 19 +/- 3%, respectively.

Distribution of voltage-dependent calcium channel beta subunits in the hippocampus of patients with temporal lobe epilepsy.

Voltage-dependent Ca2+ channels constitute a major class of plasma membrane channels through which a significant amount of extracellular Ca2+ enters neuronal cells. Their pore-forming alpha1 subunits are associated with cytoplasmic regulatory beta subunits, which modify the distinct biophysical and pharmacological properties of the alpha1 subunits. Studies in animal models indicate altered expression of alpha1 and/or beta subunits in epilepsy. We have focused on the regulatory beta subunits and have analysed the immunoreactivity patterns of the beta1, beta2, beta3 and beta4 subunits in the hippocampus of patients with temporal lobe epilepsy (n = 18) compared to control specimens (n = 2). Temporal lobe epilepsy specimens were classified as Ammon's horn sclerosis (n = 9) or focal lesions without alteration of hippocampal cytoarchitecture (n = 9). Immunoreactivity for the beta subunits was observed in neuronal cell bodies, dendrites and neuropil. The beta1, beta2 and beta3 subunits were found mainly in cell bodies while the beta4 subunit was primarily localized to dendrites. Compared to the control specimens, epilepsy specimens of the Ammon's horn sclerosis and of the lesion group showed a similar beta subunit distribution, except for beta1 and beta2 staining in the Ammon's horn sclerosis group: in the severely sclerotic hippocampal subfields of these specimens, beta1 and beta2 immunoreactivity was enhanced in some of the remaining neuronal cell bodies and, in addition, strongly marked dendrites. Thus, hippocampal neurons apparently express multiple classes of beta subunits which segregate into particular subcellular domains. In addition, the enhancement of beta1 and beta2 immunoreactivity in neuronal cell bodies and the additional shift of the beta1 and beta2 subunits into the dendritic compartment in severely sclerotic hippocampal regions indicate specific changes in Ammon's horn sclerosis. Altered expression of these beta subunits may lead to increased currents carried by voltage-dependent calcium channels and to enhanced synaptic excitability.

Distribution of alpha 1A, alpha 1B and alpha 1E voltage-dependent calcium channel subunits in the human hippocampus and parahippocampal gyrus.

The distribution of voltage-dependent calcium channel subunits in the central nervous system may provide information about the function of these channels. The present study examined the distribution of three alpha-1 subunits, alpha 1A, alpha 1B and alpha 1E, in the normal human hippocampal formation and parahippocampal gyrus using the techniques of in situ hybridization and immunocytochemistry. All three subunit mRNAs appeared to be similarly localized, with high levels of expression in the dentate granule and CA pyramidal layer. At the protein level, alpha 1A, alpha 1B and alpha 1E subunits were differentially localized. In general, alpha 1A-immunoreactivity was most intense in cell bodies and dendritic processes, including dentate granule cells, CA3 pyramidal cells and entorhinal cortex pre-alpha and pri-alpha cells. The alpha 1B antibody exhibited relatively weak staining of cell bodies but stronger staining of neuropil, especially in certain regions of high synaptic density such as the polymorphic layer of the dentate gyrus and the stratum lucidum and radiatum of the CA regions. The alpha 1E staining pattern shared features in common with both alpha 1A and alpha 1B, with strong immunoreactivity in dentate granule, CA3 pyramidal and entorhinal cortex pri-alpha cells, as well as staining of the CA3 stratum lucidum. These findings suggest regions in which particular subunits may be involved in synaptic communication. For example, comparison of alpha 1B and alpha 1E staining in the CA3 stratum lucidum with calbindin-immuno-reactivity suggested that these two calcium channels subunits may be localized presynaptically in mossy fibre terminals and therefore may be involved in neurotransmitter release from these terminals.

Immunohistochemical localisation of nicotinic acetylcholine receptor subunits in human cerebellum.

Neuronal nicotinic acetylcholine receptors are members of the ligand-gated ion channel superfamily composed of alpha and beta subunits with specific structural, functional and pharmacological properties. In this study we have used immunohistochemistry to investigate the presence of nicotinic acetylcholine receptor subunits in human cerebellum. Tissue was obtained at autopsy from eight adult individuals (aged 36-56 years). Histological sections were prepared from formalin-fixed paraffin-embedded material. alpha 3, alpha 4, alpha 6, alpha 7, beta 2, and beta 4 subunits were present in this brain area associated with both neuronal and non-neuronal cell types. Most Purkinje cells were immunoreactive for all the above subunits, but most strongly for alpha 4 and alpha 7. A proportion of granule cell somata were immunoreactive for all subunits except alpha 3. Punctate immunoreactivity in Purkinje cell and granule cell layers was evident with antibodies against alpha 3, alpha 4, alpha 6, and alpha 7 in parallel with synaptophysin immunoreactivity, suggesting the presence of these subunits on nerve terminals in the human cerebellum. All subunits were present in the dentate nucleus associated with neurones and cell processes. Strong immunoreactivity of neuropil in both the molecular and granule cell layers and within the dentate nucleus was noted with alpha 4, alpha 7 and beta 4 subunits. Astrocytes and astrocytic cell processes appeared to be immunoreactive for alpha 7 and cell processes observed in white matter, also possibly astrocytic, were immunoreactive for beta2. Immunoreactivity to all subunits was noted in association with blood vessels. We suggest that nicotinic acetylcholine receptor subunits may be involved in the modulation of cerebellar activity. Further investigations are warranted to evaluate the participation of nicotinic acetylcholine receptors in cerebellar pathology associated with both developmental and age-related disorders.

The expression of voltage-dependent calcium channel beta subunits in human cerebellum.

The beta subunits of voltage-dependent calcium channels, exert marked regulatory effects on the biophysical and pharmacological properties of this diverse group of ion channels. However, little is known about the comparative neuronal expression of the four classes of beta genes in the CNS. In the current investigation we have closely mapped the distribution of beta1, beta2, beta3 and beta4 subunits in the human cerebellum by both in situ messenger RNA hybridization and protein immunohistochemistry. To our knowledge, these studies represent the first experiments in any species in which the detailed localization of each beta protein has been comparatively mapped in a neuroanatomically-based investigation. The data indicate that all four classes of beta subunits are found in the cerebellum and suggest that in certain neuronal populations they may each be expressed within the same cell. Novel immunohistochemical results further exemplify that the beta voltage-dependent calcium channel subunits are regionally distributed in a highly specific manner and studies of Purkinje cells indicate that this may occur at the subcellular level. Preliminary indication of the subunit composition of certain native voltage-dependent calcium channels is suggested by the observation that the distribution of the beta3 subunit in the cerebellar cortex is identical to that of alpha(1E). Our cumulative data are consistent with the emerging view that different native alpha1/beta subunit associations occur in the CNS.

Immunohistochemical detection of alpha1E voltage-gated Ca(2+) channel isoforms in cerebellum, INS-1 cells, and neuroendocrine cells of the digestive system.

Polyclonal antibodies were raised against a common and a specific epitope present only in longer alpha1E isoforms of voltage-gated Ca(2+) channels, yielding an "anti-E-com" and an "anti-E-spec" serum, respectively. The specificity of both sera was established by immunocytochemistry and immunoblotting using stably transfected HEK-293 cells or membrane proteins derived from them. Cells from the insulinoma cell line INS-1, tissue sections from cerebellum, and representative regions of gastrointestinal tract were stained immunocytochemically. INS-1 cells expressed an alpha1E splice variant with a longer carboxy terminus, the so-called alpha1Ee isoform. Similarily, in rat cerebellum, which was used as a reference system, the anti-E-spec serum stained somata and dendrites of Purkinje cells. Only faint staining was seen throughout the cerebellar granule cell layer. After prolonged incubation times, neurons of the molecular layer were stained by anti-E-com, suggesting that a shorter alpha1E isoform is expressed at a lower protein density. In human gastrointestinal tract, endocrine cells of the antral mucosa (stomach), small and large intestine, and islets of Langerhans were stained by the anti-E-spec serum. In addition, staining by the anti-E-spec serum was observed in Paneth cells and in the smooth muscle cell layer of the lamina muscularis mucosae. We conclude that the longer alpha1Ee isoform is expressed in neuroendocrine cells of the digestive system and that, in pancreas, alpha1Ee expression is restricted to the neuroendocrine part, the islets of Langerhans. alpha1E therefore appears to be a common voltage-gated Ca(2+) channel linked to neuroendocrine and related systems of the body.

The expression of neuronal voltage-dependent calcium channels in human cerebellum.

Little is known about the comparative distribution of voltage-dependent calcium channel subtypes in normal human brain. Previous studies in experimental animals have predominantly focused on the regional expression of single alpha 1 genes. We describe the preparation of riboprobes and antisera specific for human alpha 1A, alpha 1B and alpha 1E subunits and their application in comprehensive mapping studies of the human cerebellum. Within the cerebellar cortex, these pore forming proteins were found to have differential localisations when examined in adjacent sections. The alpha 1A and alpha 1B subunits broadly colocalised and were both present, though at apparently different levels, in the molecular, Purkinje and granule cell layers whilst alpha 1E was predominantly expressed in Purkinje cells. In the dentate nucleus, an area which has received little attention in previous studies, alpha 1A was highly expressed in regions in which Purkinje cell nerve terminals form synapses with deep cerebellar neurones.

The expression of voltage-dependent calcium channel beta subunits in human hippocampus.

The beta subunits of voltage-dependent calcium channels (VDCC) modulate the electrophysiology and cell surface expression of pore-forming alpha1 subunits. In the present study we have investigated the distribution of beta1,beta2,beta3 and beta4 in the human hippocampus using in situ hybridization (ISH) and immunohistochemistry. ISH studies showed a similar distribution of expression of beta1,beta2 and beta3 subunit mRNAs, including labelling of the dentate granule cell layer, all CA pyramidal regions, and the subiculum. Relatively low levels of expression of beta1 and beta2 subunit mRNAs correlated with low protein expression in the immunocytochemical (ICC) studies. There was a relative lack of beta4 expression by both ISH and ICC in the CA1 region, compared with high levels of expression in the subiculum. Immunostaining for beta1 and beta2 subunits was weak and relatively homogeneous throughout the hippocampus. The beta3 and beta4 subunits appeared to be more discretely localized. In general, beta3-immunoreactivity was moderate both in cell bodies, and as diffuse staining in the surrounding neuropil. Strongest staining was observed in mossy fibres and their terminal region in the CA3 stratum lucidum. In contrast, beta4-immunoreactivity in the neuropil showed intense dendritic localisation. Unlike the other subunits, beta4-immunoreactivity was absent from CA1 pyramidal neurones but was present in a small population of interneurone-like cells. The localisation of beta3 and beta4 may represent presynaptic and postsynaptic compartments in some populations of hippocampal neurones. Comparison of beta subunit distribution with previously published data on alpha1 subunits indicates certain neuronal groups and subcellular compartments in which the subunit composition of native pre- and postsynaptic VDCC can be predicted.

Differential nicotinic acetylcholine receptor subunit expression in the human hippocampus.

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels composed of alpha and beta subunits with specific structural, functional and pharmacological properties. In this study the distribution of alpha3, alpha4, alpha7, beta2 and beta4 nAChR subunits in the human hippocampus was investigated using immunohistochemistry. Most pyramidal neurons, pre-alpha cells of the entorhinal cortex and dentate granule cells were immunoreactive for all subunits. Small islands of alpha7 immunoreactive cells were present in the outer presubiculum. alpha4 and beta2, and alpha3, alpha4 and beta2 immunoreactive fibre tracts were present in the stratum radiatum and subiculum, respectively, suggesting nAChRs may play a role in modulating inputs to the hippocampus via Schaffer collaterals and along the perforant pathway. Some astrocytes were immunoreactive for alpha3, alpha7 and beta4 subunits. Immunoreactivity to all subunits was noted in association with blood vessels. These results indicate the involvement of multiple nAChR subtypes in the modulation of both neuronal and non-neuronal functions in the human hippocampus.

Embryonic stem-cell derived neurones express a maturation dependent pattern of voltage-gated calcium channels and calcium-binding proteins.

There are remarkable changes of calcium binding proteins and voltage dependent Ca(2+) channel subtypes during in vitro differentiation of embryonic stem cell derived neurons. To observe these maturation dependent changes neurones were studied using combined immunohistochemical, patch clamp and videomicroscopic time lapse techniques. Embryonic stem cell derived neuronal maturation proceeds from apolar to bi- and multipolar neurones, expressing all Ca(2+) channel subtypes. There is, however, a clear shift in channel contribution to whole cell current from apolar neurones with mainly N- and L-type channel contribution in favour of P/Q- and R-type participation in bi- and multipolar cells. Expression of the calcium binding protein parvalbumin could be detected in bipolar, while calretinin and calbindin was preferentially found in multipolar neurones. Our data provides new insights into fundamental neurodevelopmental mechanisms related to Ca(2+) homeostasis, and clarifies contradictory reports on the development of Ca(2+) channel expression using primary cultures of neurones already committed to certain brain compartments.

Immunohistochemical and in situ mRNA hybridisation techniques to determine the distribution of ion channels in human brain: a study of neuronal voltage-dependent calcium channels.

The molecular, structural and functional characterisation of ion channels in the CNS forms an area of intense investigation in current brain research. For strategic and logistical reasons, rodents have historically been the species of choice for these studies. The examination of human CNS tissues generally presents the investigator with specific challenges that are often less problematic in animal studies, e.g. post-mortem delay/agonal status, and thus both the experimental design and techniques must be manipulated accordingly. Since much pharmaceutical interest is currently focused on neuronal ion channels, the examination of their expression in human brain material is of particular importance. We describe here the details of methods that we have developed and used successfully in the study of the expression of voltage-dependent calcium channels (VDCCs) in human CNS tissues. Presynaptic neuronal VDCCs control neurotransmitter release and are important new drug targets. They are composed of three subunits, alpha 1, beta and alpha 2/delta and multiple gene classes of each protein have been identified. Little is known, however, about the distribution of neuronal VDCCs in the human central nervous system, although initial studies have been performed in rat and rabbit.

Preparation and purification of antibodies specific to human neuronal voltage-dependent calcium channel subunits.

Neuronal voltage-dependent calcium channels (VDCCs) each comprising of alpha 1, alpha 2 delta, and beta subunits, are one mechanism by which excitable cells regulate the flux of calcium ions across the cell membrane following depolarisation Studies have shown the expression of several alpha 1 and beta subtypes within neuronal tissue. The comparative distribution of these in normal human brain is largely unknown. The aim of this work is to prepare antibodies directed specifically to selected subunits of human neuronal VDCCs for use in biochemical and mapping studies of calcium channel subtypes in the brain. Previous studies have defined DNA sequences specific for each subunit Comparison of these sequences allows the selection of unique amino acid sequences for use as immunogens which are prepared as glutathione-S-transferase (GST) fusion proteins in E. coli. Polyclonal antibodies raised against these fusion proteins are purified by Protein A chromatography, followed by immunoaffinity chromatography and extensive adsorptions using the appropriate fusion protein-GST Sepharose 4B columns. The resultant antibodies are analysed for specificity against the fusion proteins by ELISA, and by immunofluorescence and Western immunoblot analysis of recombinant HEK293 cells stably transfected with cDNAs encoding alpha 1, alpha 2 delta and beta subunits.

The reactivity of Reed-Sternberg cells with monoclonal antisera at thin section and ultrastructural levels.

In an attempt to improve the morphology of Reed-Sternberg cells after immunochemical procedures, the biotin-avidin technique has been used. Satisfactory morphology was obtained at the level of both light and electron microscopy. Reed-Sternberg cells failed to react with a monoclonal antimonocyte serum but gave positive results with anti-1a and FMC7, a monoclonal anti-B cell serum. Positive results were also obtained with FMC1 and B1 both of which are monoclonal B cell antisera.

Cellular and molecular effects of di-n-octyltin dichloride on the rat thymus.

Experiments have been performed to characterise early changes in the proliferative capacity and phenotypic makeup of thymocytes obtained from DOTC-treated PVG rats. The analysis of density gradient-separated thymocytes demonstrated that within 72 h of oral dosing, spontaneous in vitro thymocyte proliferation was markedly suppressed. A concomitant depletion of an MRC OX18 positive thymocyte population was also observed. These events occurred prior to both overt thymic weight loss and characteristic, treatment-induced histopathological changes. Since recent evidences suggest that the growth factor interleukin-2 (IL-2) may play an important role in thymocyte proliferation, additional molecular biological studies were performed to evaluate whether DOTC may exert its anti-proliferative effects by compromising IL-2 production. Using a mRNA cytoplasmic dot blot technique, the examination of thymocyte lysates from treated and control animals revealed that DOTC markedly down regulated and at high doses abolished, the normal expression of the IL-2 gene. The high turnover gene alpha-actin was, however, unaffected by its action, thus demonstrating the selective effects of DOTC.

Acidic motif responsible for plasma membrane association of the voltage-dependent calcium channel beta1b subunit.

Voltage-dependent calcium channels consist of a pore-forming transmembrane alpha1-subunit, which is known to associate with a number of accessory subunits, including alpha2-delta- and beta-subunits. The beta-subunits, of which four have been identified (beta1-4), are intracellular proteins that have marked effects on calcium channel trafficking and function. In a previous study, we observed that the beta1b-subunit showed selective plasma membrane association when expressed alone in COS7 cells, whereas beta3 and beta4 did not. In this study, we have examined the basis for this, and have identified, by making chimeric beta-subunits, that the C-terminal region, which shows most diversity between beta-subunits, of beta1b is responsible for its plasma membrane association. Furthermore we have identified, by deletion mutations, an 11-amino acid motif present in the C terminus of beta1b but not in beta3 (amino acids 547-556 of beta1b, WEEEEDYEEE), which when deleted, reduces membrane association of beta1b. Future research aims to identify what is binding to this sequence in beta1b to promote membrane association of this calcium channel subunit. It is possible that such membrane association is important for the selective localization or clustering of particular calcium channels with which beta1b is associated.

Dendro-somatic distribution of calcium-mediated electrogenesis in purkinje cells from rat cerebellar slice cultures.

The role of Ca2+ entry in determining the electrical properties of cerebellar Purkinje cell (PC) dendrites and somata was investigated in cerebellar slice cultures. Immunohistofluorescence demonstrated the presence of at least three distinct types of Ca2+ channel proteins in PCs: the alpha1A subunit (P/Q type Ca2+ channel), the alpha1G subunit (T type) and the alpha1E subunit (R type). In PC dendrites, the response started in 66 % of cases with a slow depolarization (50 +/- 15 ms) triggering one or two fast (approximately 1 ms) action potentials (APs). The slow depolarization was identified as a low-threshold non-P/Q Ca2+ AP initiated, most probably, in the dendrites. In 16 % of cases, this response propagated to the soma to elicit an initial burst of fast APs. Somatic recordings revealed three modes of discharge. In mode 1, PCs display a single or a short burst of fast APs. In contrast, PCs fire repetitively in mode 2 and 3, with a sustained discharge of APs in mode 2, and bursts of APs in mode 3. Removal of external Ca2+ or bath applications of a membrane-permeable Ca2+ chelator abolished repetitive firing. Tetraethylammonium (TEA) prolonged dendritic and somatic fast APs by a depolarizing plateau sensitive to Cd2+ and to omega-conotoxin MVII C or omega-agatoxin TK. Therefore, the role of Ca2+ channels in determining somatic PC firing has been investigated. Cd2+ or P/Q type Ca2+ channel-specific toxins reduced the duration of the discharge and occasionallyinduced the appearance of oscillations in the membrane potential associated with bursts of APs. In summary, we demonstrate that Ca2+ entry through low-voltage gated Ca2+ channels, not yet identified, underlies a dendritic AP rarelyeliciting a somatic burst of APs whereas Ca2+ entry through P/Q type Ca2+ channels allowed a repetitive firing mainly by inducing a Ca2+-dependent hyperpolarization.