Peter J. Craig

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.

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.

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.

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.

Dendro‐somatic distribution of calcium‐mediated electrogenesis in Purkinje cells from rat cerebellar slice cultures

1 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 α1A subunit (P/Q type Ca2+ channel), the α1G subunit (T type) and the α1E subunit (R type). 2 In PC dendrites, the response started in 66 % of cases with a slow depolarization (50 ± 15 ms) triggering one or two fast (∼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. 3 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. 4 Tetraethylammonium (TEA) prolonged dendritic and somatic fast APs by a depolarizing plateau sensitive to Cd2+ and to ω‐conotoxin MVII C or ω‐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. 5 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.

Expression and functional characterisation of a human chimeric nicotinic receptor with α6β4 properties

Abstract Despite being cloned several years ago, the expression of functional nicotinic acetylcholine receptors containing the human α6 subunit in recombinant mammalian cell lines has yet to be demonstrated. The resulting lack of selective ligands has hindered the evaluation of the role of α6-containing nicotinic receptors. We report that functional channels were recorded following co-transfection of human embryonic kidney (HEK-293) cells with a chimeric α6/α4 subunit and the β4 nicotinic receptor subunit. They displayed an agonist rank order potency of epibatidine≫1,1-dimethyl-4-phenylpiperazinium (DMPP)≥cytisine>acetylcholine>nicotine measured in a fluorescent imaging plate reader assay. Nicotine, cytisine, DMPP and epibatidine displayed partial agonist properties whilst α-conotoxin MII and methyllycaconitine blocked the functional responses elicited by acetylcholine stimulation. Co-transfection of the α6/α4 chimera with the β2 nicotinic receptor subunit did not result in functional receptors. The human α6/α4β4 chimeric nicotinic receptor expressed in HEK-293 cells may provide a valuable tool for the generation of subtype specific ligands.

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.

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.