Steve Volsen

Distribution of the voltage‐dependent calcium channel α1G subunit mRNA and protein throughout the mature rat brain

The molecular identity of a gene which encodes the pore‐forming subunit (α1G) of a member of the family of low‐voltage‐activated, T‐type, voltage‐dependent calcium channels has been described recently. Although northern mRNA analyses have shown α1G to be expressed predominantly in the brain, the detailed cellular distribution of this protein in the central nervous system (CNS) has not yet been reported. The current study describes the preparation of a subunit specific α1G riboprobe and antiserum which have been used in parallel in situ mRNA hybridization and immunohistochemical studies to localize α1G in the mature rat brain. Both α1G mRNA and protein were widely distributed throughout the brain, but variations were observed in the relative level of expression in discrete nuclei. Immunoreactivity for α1G was typically localized in both the soma and dendrites of many neurons. Whilst α1G protein and mRNA expression were often observed in cells known to exhibit T‐type current activity, some was also noted in regions, e.g. cerebellar granule cells, in which T‐type activity has not been described. These observations may reflect differences between the subcellular distribution of channels that can be identified by immunohistochemical methods compared with electrophysiological techniques.

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.

Distribution ofα1A, α1B andα1E voltage-dependent calcium channel subunits in the human hippocampus and parahippocampal gyrus

Abstract 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,α 1A , α 1B andα 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,α 1A , α 1B andα 1E subunits were differentially localized. In general,α 1A -immunoreactivity was most intense in cell bodies and dendritic processes, including dentate granule cells, CA3 pyramidal cells and entorhinal cortex pre-a and pri-a cells. Theα 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. Theα 1E staining pattern shared features in common with bothα 1A andα 1B with strong immunoreactivity in dentate granule, CA3 pyramidal and entorhinal cortex pri-α 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 ofα 1B andα 1E staining in the CA3 stratum lucidum with calbindin-immunoreactivity 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.

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.

Polyethylenimine-Mediated DNA Transfection of Peripheral and Central Neurons in Primary Culture: Probing Ca2+Channel Structure and Function with Antisense Oligonucleotides

To study neuronal ion channel function with antisense oligonucleotides, a reliable method is needed which allows different neuronal cell types to be transfected without artifactual disruptive effects on their electrical properties. Here we report that use of the recently introduced transfecting agent, polyethylenimine, fulfills this requirement. Four days after transfection, in both central and peripheral neurons, an antisense designed to block the synthesis of the Ca2+ channel beta subunits induced a maximal decrease of the Ca2- current amplitude and modification of their kinetics and voltage-dependence. Controls with scrambled oligonucleotides, as well as Na+ current recordings of antisense transfected neurons, confirm both that the transfecting agent does not modify the electrophysiological properties of the neurons and that the effect of the antisense is sequence specific.

Immunohistochemical Detection of α1E Voltage-gated Ca2+ 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 α1E isoforms of voltage-gated Ca2+ 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 α1E splice variant with a longer carboxy terminus, the so-called α1Ee 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 α1E 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 α1Ee isoform is expressed in neuroendocrine cells of the digestive system and that, in pancreas, α1Ee expression is restricted to the neuroendocrine part, the islets of Langerhans. α1E therefore appears to be a common voltage-gated Ca2+ channel linked to neuroendocrine and related systems of the body. (J Histochem Cytochem 47:981–993, 1999)

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 Ammons 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 Ammons horn sclerosis and of the lesion group showed a similar beta subunit distribution, except for beta1 and beta2 staining in the Ammons 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 Ammons horn sclerosis. Altered expression of these beta subunits may lead to increased currents carried by voltage-dependent calcium channels and to enhanced synaptic excitability.

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.

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.

Identification and cellular localisation of voltage-operated calcium channels in immature rat testis.

Sertoli cells regulate the spermatogenic process mainly through the secretion of a complex fluid into the lumen of the seminiferous tubules behind the blood-testis barrier, containing many of the essential proteins necessary for maintenance and maturation of male germ cells. Thus, the study of Sertoli cell secretory processes is strictly correlated with the understanding of the regulatory mechanisms of spermatogenesis. In this work the authors have explored the voltage-sensitive calcium channel variety in the immature rat testis, their localisation and distribution within the seminiferous epithelium and peritubular and interstitial tissues as well as the possible role in the control of Sertoli cell secretion. The results reported in this paper, obtained by in situ hybridisation, immunohistology of rat testicular sections and Western blot analysis of Sertoli cell plasma membranes, show that mammalian Sertoli cells express mRNA encoding for several voltage-operated calcium channel subunits and express such proteins on their surface. Experiments performed on Sertoli cell monolayers cultured in the presence of specific toxins indicate that both N and P/Q-type Ca(2+) channels are involved in the regulation of protein secretion.