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

Distribution of the Voltage-Dependent

Calcium Channel a1A Subunit Throughout

the Mature Rat Brain and its Relationship

to Neurotransmitter Pathways

PETER J. CRAIG,1* ANDREW D. MCAINSH,1 ALISON L. MCCORMACK,1 WILLIAM

SMITH,1 RUTH E. BEATTIE,1 JOHN V. PRIESTLEY,2 JENNIFER LAI YEE YIP,3

SHARON AVERILL,2 E. REBECCA LONGBOTTOM,1 AND STEPHEN G. VOLSEN1

1CNS Research, Eli Lilly & Company, Lilly Research Centre,Windlesham, Surrey, GU20 6PH, United Kingdom

2Department of Anatomy, Faculty of Basic Medical Sciences,Queen Mary & Westerfield College, London, E1 4NS, United Kingdom

3Department of Physiology, UMDS, St Thomas’s Hospital,London, SE1 7EH, United Kingdom

ABSTRACTThe a1 subunit provides both the voltage-sensing mechanism and the ion pore of

voltage-dependent calcium channels. Of the six classes of a1 subunit cloned to date, a1A is thesubject of debate in terms of its functional correlate, although it is generally thought to encodevoltage-dependent calcium channels of the v-agatoxin IVA-sensitive, P/Q type. In the presentstudy, an a1A-specific riboprobe and antibody were used with in situ hybridisation andimmunohistochemical techniques to localise a1A messenger ribonucleic acid transcripts andsubunit protein throughout the mature rat brain. Dual localisation of a1A protein and markersfor acetylcholine, catecholamines, and 5-hydroxytryptamine have also been performed in anumber of discrete areas. Abundant and widespread distribution of a1A protein was found,with immunoreactivity occurring both in cell bodies and as punctate staining in areas ofneuronal processes. Several associations were noted across a1A localisation, defined neuroana-tomical regions, and neurotransmitter systems. However, a1A expression was not confined toloci corresponding to any one neurotransmitter type, although a high level of expression wasobserved in cholinergic neurones. The distribution of the a1A subunit in the rat correspondedwell with the limited human mapping data that are available. J. Comp. Neurol. 397:251–267,1998. r 1998 Wiley-Liss, Inc.

Indexing terms: in situ hybridisation; immunohistochemistry; ion channel; central nervous system

The role of calcium as a trigger for neurotransmitterrelease was initially demonstrated in studies by Katz andMiledi (1967). More recently, calcium influx via voltage-dependent Ca21 channels (VDCCs) has been shown to beresponsible for this process, the evidence coming fromexperiments in which neurotransmitter release has beensuppressed after the blocking of VDCCs by specific ligands(Hirning et al., 1988; Dunlap et al., 1995). Historically,VDCCs have been classified according to different func-tional characteristics such as response to pharmacologicalagents, biophysical properties, and cellular distribution.By using these parameters, six main classes of VDCCshave been identified, namely T, L, N, P, Q, and R types

(Bean, 1989; Llinas et al., 1989; Hess, 1990; Tsien et al.,1991; Sather et al., 1993; Wheeler et al., 1994). Analyses ofthe activity and spatial distribution of VDCCs have demon-strated both pre- and postsynaptic functions within theneurone. The neuronal N-, P-, and Q-type channels have

Grant sponsor: Woolfson Foundation.*Correspondence to: P.J. Craig, CNS Research, Eli Lilly & Company, Lilly

Research Centre, Erl Wood Manor, Windlesham, Surrey, UK.E-mail: [email protected]

Received 11 March 1997; Revised 24 March 1998; Accepted 27 March1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 397:251–267 (1998)

r 1998 WILEY-LISS, INC.

well-established roles in the presynaptic regulation ofneurotransmitter release (Olivera et al., 1994; Wheeler etal., 1994; Huang et al., 1996), whereas postsynapticallythey are involved in integration of dendritic inputs (Tanket al., 1988).

Biochemical and molecular biological analyses haveshown that neuronal VDCCs are composed of at least threegene products: the pore-forming a1 subunit and the struc-tural/regulatory a2d and b subunits. Multiple isoforms ofthe a1 and b subunits have been identified, with furtherdiversification arising due to the existence of splice vari-ants. Of the six classes of the a1 subunit cloned thus far(a1A, a1B, a1C, a1D, a1E, a1S), the a1B gene product has beenshown to constitute the pore-forming subunit of the N-typecalcium channel (Williams et al., 1992); the a1C, a1D, anda1S gene products correspond to the pore-forming subunitof dihydropyridine-sensitive L-type currents (Liu et al.,1996); and there is evidence to suggest that the a1E subunitis a strong candidate as the carrier of R-type currents(Schneider et al., 1994). However, the nature of the nativechannel encoded by a1A remains the subject of debate(Starr et al., 1991; Stea et al., 1994).

The identification of a1A messenger ribonucleic acid(mRNA) in the cerebellar Purkinje cell (Mori et al., 1991)suggested that this subunit may encode the P-type chan-nel because more than 90% of the whole cell calciumcurrent of these cells is of that type (Mintz et al., 1992).However, it has been shown more recently (Stea et al.,1994) that the electrophysiological characteristics of thea1A subunit depend on which class of the b subunit it iscoexpressed with. Thus, in Xenopus oocytes, coexpressionof the a1A subunit with a b2a subunit resulted in a channelwith P-like biophysical characteristics, whereas coexpres-sion with either the b1b or b3 subunit produced a channelwith Q-like currents.

Because both the P- and Q-type channels are involved inthe modulation of neurotransmitter release, we haveundertaken a mapping study into the distribution of a1AmRNA transcripts and protein in normal rat brain byusing the techniques of in situ hybridisation (ISH) andimmunohistochemistry (IHC). In selected areas, dual im-munolocalisation has been performed with a number ofneurotransmitter markers to determine the correlationbetween a1A distribution and neurotransmitter systems.The present study extends those data currently availablein the literature (Mori et al., 1991; Tanaka et al., 1995;Westenbroek et al., 1995; Sakurai et al., 1996) in terms ofthe number of neuroanatomical areas examined, by inves-tigating both mRNA and protein expression and by corre-lating the results with neurotransmitter distribution. Ourobservations on the abundant and diverse distribution ofthe a1A subunit are considered in relation to neurotransmit-ter pathways. Increased understanding of the role ofindividual VDCC types in the control of the release ofspecific neurotransmitters may provide a potential fortherapeutic intervention in diseases associated with synap-tic dysfunction, e.g., Parkinson’s disease, manic depres-sion, and schizophrenia (Miljanich and Ramachandran,1995). The implications of our findings in terms of usingVDCCs as potential therapeutic targets for the regulationof neurotransmitter release are discussed.

MATERIALS AND METHODS

Preparation of paraffin-processed tissue

Male Lister rats (Harlan UK Ltd., Bicester, Oxford, UK)weighing 200–300 g were anaesthetised with halothaneand transcardially perfused with 1% glutaraldehyde/1%paraformaldehyde in 0.1 M phosphate buffered saline, pH 7.4.

Abbreviations

7 facial nucleus10 dorsal motor nucleus vagus12 hypoglossal nucleusAmb ambiguus nucleusAmy amygdaloid nucleusAOP anterior olfactory nucleus, posteriorB basal nucleus of MeynertBf basal forebrainBIC nucleus brachium inferior colliculusCA1–3 fields CA1–3 of Ammon’s hornCPu caudate putamenDC dorsal cochlear nucleusDG dentate gyrusDM dorsomedial hypothalamic nucleusDEn dorsal endopiriform cortexDLG dorsal lateral geniculate nucleusDR dorsal raphe nucleusECu external cuneate nucleusEW Edinger-Westphal nucleusGi gigantocellular reticular nucleusGP globus pallidusHDB nucleus horizontal limb—diagonal bandICj islands of Callejaicp inferior cerebellar peduncleIO inferior oliveIOB inferior olive, subnucleus B of medial nucleusLat lateral cerebellar nucleusLC locus coeruleuslfp longitudinal fasciculus ponsll lateral lemniscusLM lateral mammillary nucleusLPMC lateral posterior thalamic nucleus, mediocaudal

LRt lateral reticular nucleusMCPO magnocellular preoptic nucleusmcp middle cerebellar peduncleMd medulary reticular fieldMed medial cerebellar nucleusMe5 mesencephalic trigeminal nucleusMnR median raphe nucleusMo5 motor trigeminal nucleusPir piriform cortexPn pontine nucleusPnC pontine reticular nucleus, caudalPnV pontine reticular nucleus, ventralPr5 principal sensory trigeminal nucleuspy pyramidal tractRMg raphe magnus nucleusSNC substantia nigra, pars compactaSNR substantia nigra, pars reticulataSO supraoptic nucleusSol nucleus of the solitary tractsp5 spinal trigeminal tractSp5 spinal trigeminal nucleusSp5C spinal trigeminal nucleus, caudalSuG superficial grey layerTh thalamic nucleusTz nucleus trapezoid bodyVCA ventral cochlear nucleus, anteriorVCP ventral cochlear nucleus, posteriorVDB nucleus vertical limb—diagonal bandVLL ventral nucleus lateral lemniscusVM ventromedial thalamic nucleusVTA ventral tegmental area

252 P.J. CRAIG ET AL.

The brains were rapidly removed and postfixed by immer-sion for 48 hours in 4% neutral buffered formalin. Brainswere dissected either coronally into 2-mm blocks by usinga rat brain matrix (ASI Instruments, Waren, MI) orsagittally along the mid-line and then processed for conven-tional paraffin histology. Sections were cut at 4 µm,mounted onto silane-coated slides (3-aminopropyltriethox-ysilane; Aldrich, Gillingham, Dorset, UK), dried overnightat 37°C, and stored at room temperature before ISH orIHC.

Riboprobe generation

Polymerase chain reaction (PCR) primers were designedfor the generation of a template for a rat a1A riboprobe. Asequence analysis software package (MacDNASIS Pro,Hitachi Software Engineering Co. Ltd., San Bruno, CA)was used to select a locus from the published rat a1A mRNAsequence (Starr et al., 1991), which is shared betweenknown splice variants but has minimum homology withother rat a1 sequences. The intracellular loop regionbetween the transmembrane domains IIS6–IIIS1 waschosen as an area of suitable diversity. Sense and anti-sense oligonucleotide primers, corresponding to nucleotidepositions 3200–3220 and 3366–3386, were synthesised.The primer for the antisense strand was modified byaddition to the 58 end of a 20-nucleotide extension contain-ing the promoter sequence for T7 DNA-dependent RNApolymerase (Milligan et al., 1987). This was used with anunmodified sense primer to generate by PCR a templatefor an antisense riboprobe. Similar reactions were per-formed with a sense primer containing the T7 promoterand an unmodified antisense primer to generate a tem-plate for a sense control riboprobe.

Restriction digest analyses were performed on the PCRproducts to confirm that the desired sequence had beengenerated. Further confirmation that an appropriate se-quence had been generated for use as a probe for the a1Asubunit was obtained by Northern hybridisation.

A commercially available kit (RNA colour kit, AmershamInternational, Buckinghamshire, UK) was used to gener-ate fluorescein-labelled sense and antisense cRNA probesfrom the appropriate template by using a standard in vitrotranscription method (Krieg et al., 1987). Briefly, 1 µg oftemplate was used in a 20-µl reaction containing 20 mMdithiothreitol, 6 mM MgCl2, 2 mM spermidine, 0.01%bovine serum albumin (BSA), 20 units human placentalribonuclease inhibitor, 0.125 mM fluorescein-11-UTP, 0.5mM ATP, 0.5 mM CTP, 0.5 mM GTP, 0.375 mM UTP, 25units T7 RNA polymerase, and 40 mM Tris-HCl, pH 7.6;reactions were performed at 37°C for 4 hours. Probes wereanalysed by blotting an aliquot of the reaction product ontoa nitrocellulose membrane (Hybond N,Amersham Interna-tional) and comparing the fluorescent intensity observedwhen illuminated with a 254-nm ultraviolet (UV) lightsource with that obtained from a known standard. Probeswere stored at 220°C and used in hybridisation experi-ments without further purification.

Northern hybridisation

The PCR product corresponding to a 186-bp sequencefrom the IIS6–IIIS1 cytoplasmic loop was labelled byincorporation of deoxycytidine 58-triphosphate a-[32P] byusing the random primed method (Feinberg and Vogel-stein, 1983). This probe was then used in the analysis of acommercially prepared Northern blot of rat poly(A1) RNA

(MTNy, Clontech, Palo Alto, CA). Hybridisation was per-formed overnight at 65°C in the ExpressHyby solutionsupplied by the manufacturers and by following theirprotocol. The blot was washed for 1.5 hours at roomtemperature in two changes of 23 standard saline citrate(SSC; 13 SSC 5 0.15 M NaCl, 0.015 M sodium citrate,pH 7.4)/0.05% sodium dodecyl sulphate (SDS) followed by0.13 SSC/0.1% SDS (30 minutes at 50°C). The blot wasthen sealed in plastic wrap and exposed to X-ray film for 2days at 270°C.

Antibody production

A polyclonal antibody specific for the human a1A VDCCsubunit was produced and characterised as describedpreviously (Volsen et al., 1995). Briefly, antisera wereraised in rabbits by immunisation with a glutathione-S-transferase (GST) fusion protein containing a segment ofthe IIS6–IIIS1 intracellular loop from the a1A subunit(amino acids 1048–1208). The immunoglobulin fractionwas purified from the antiserum by protein A chromatogra-phy, and anti-GST antibodies were removed by adsorptionon a GST-Sepharose 4B column. Further immunoadsorp-tion was used to remove residual cross reactivity withother classes of the a1 subunit. The specificity of theantiserum was confirmed by an enzyme-linked immunosor-bant assay, immunofluorescent analysis of a1A-transfectedHEK 293 cells, and Western blotting. Sequence analysisshowed a greater than 85% homology between the regionof the human sequence to which the anti-human a1Aantibody was raised and the corresponding rat sequence.

Western blot analysis

The specificity of the anti-human a1A antibody and itsapplication for immunolocalisation in the rat were con-firmed by immunoblotting. Membrane fractions were pre-pared by following a standard protocol (Beattie et al.,1997) from recombinant HEK 293 cells stably transfectedwith the cDNA for the a1A subunit, nontransfected HEK293 cells, and whole rat brain. Briefly, the cell pelletderived from 108 HEK 293 cells or rat brain tissue that hadbeen dissected into approximately 1-mm cubes was resus-pended in 10 ml of lysis buffer (0.1 M e-aminocaproic acid,450 µM benzamidine, 1.0 µM leupeptin, 3.5 mM4-[2-aminoethyl]-benzenesulphonyl fluoride, 5 mM N-ethyl maleimide in 50 mM Tris-HCl/0.1 mM EDTA, pH 7.2)and homogenised by hand. The suspension was centri-fuged (5 minutes, 400g, 4°C), and the supernatant wasremoved and retained on ice. Another volume of lysisbuffer was added to the pellet, which was then subjected toanother round of homogenisation and centrifugation. Thesupernatants were pooled and then centrifuged at 35,000gfor 15 minutes; the resulting pellet was resuspended inLaemmli buffer then boiled for 4 minutes. Aliquots of themembrane preparation were run under reducing condi-tions on a Novex precast 4–12% gel (Novex, San Diego,CA). Proteins were transferred onto a nitrocellulose mem-brane (0.45 µm; Pharmacia, St Albans, Herts., UK) with aNovex Blot Module by following the manufacturer’s recom-mendations.

The membrane was incubated for 1 hour in 5% (w/v)skimmed milk in 10 mM sodium phosphate buffered 0.9%(w/v) saline, pH 7.5 (PBS) to block nonspecific bindingsites. After washing (3 3 5 minutes in PBS), the rabbitanti-a1A antibody was applied for 30 minutes at 50 ng/mlin 2% (w/v) skimmed milk/PBS. Washing was performed as

a1A VDCC SUBUNIT DISTRIBUTION IN THE RAT BRAIN 253

before, and then a goat anti-rabbit–horseradish peroxi-dase conjugated secondary antibody (Europath, Bude,Cornwall, UK) was applied at a 1:4,000 dilution in 2%(w/v) skimmed milk/PBS.After another washing, a commer-cially available enhanced chemiluminescence kit (ECLKit, Amersham International) was used to demonstrateimmunolocalised bands.

In situ mRNA hybridisation

In situ hybridisation was performed by using a standardtechnique (Pringle et al., 1990). Briefly, sections weredewaxed in xylene, rehydrated in graded alcohols, andtreated with 25 µg/ml proteinase K (Boehringer Mann-heim, Lewes, East Sussex, UK). Sections were prehybrid-ised for 2 hours at 55°C in 25 µl of a solution containing 23SSC, 13 Denhardt’s solution (0.02% [w/v] Ficoll, 0.02%[w/v] polyvinylpyrrolidone, 0.02% [w/v] BSA), 300 µg/mlherring testes DNA, and 50% (v/v) deionised formamide.The prehybridisation solution was then replaced with asolution of the same composition but to which fluorescein-labelled riboprobe was added to a final concentration of 0.5ng/µl, and hybridisation allowed to proceed overnight at55°C. The sections were washed sequentially at 55°C for2 3 5 minutes in 43 SSC, 23 SSC, and 0.23 SSC; eachwash solution containing 30% (v/v) formamide. Probe/RNAhybrids were localised with a sheep anti-fluorescein alka-line phosphatase–conjugated antibody (Amersham Inter-national). Alkaline phosphatase activity was demon-strated with the nitroblue tetrazolium/bromochloroindolylphosphate technique (Bland et al., 1991). Nonspecifichybridisation was evaluated by using a fluorescein-labelledsense riboprobe or by pretreatment of sections with RNase.Omission of the riboprobe allowed the specificity of thedetection system to be confirmed.

Immunolocalisation of the a1A

subunit protein

For the general mapping study, a1A subunit protein waslocalised in paraffin sections by a streptavidin/biotin immu-noperoxidase method with a commercially available kit(Vectastain Elite ABC Kit, Vector Laboratories, Bretton,Peterborough, UK). All manipulations were performed atroom temperature, and, unless stated otherwise, washescomprised 2 3 5 minutes in PBS. Briefly, after dewaxingand rehydration through graded alcohols, sections weretreated for 30 minutes with 0.3% (v/v) hydrogen peroxideto remove endogenous peroxidase activity. After washing,nonspecific binding sites were blocked by incubation in1.5% (v/v) goat serum in PBS. The blocking solution wasdrained from the slides, and the sections were coveredwith 200 µl anti-a1A serum (2.5 µg IgG/ml in PBS) for 30minutes. After washing, sections were incubated for 30minutes in biotinylated goat anti-rabbit secondary anti-

body (5 µg/ml in PBS/1.5% [v/v] goat serum). After wash-ing, sections were incubated with streptavidin/biotin com-plex for 30 minutes. Sections were washed, and then thediaminobenzidine tetrahydrochloride/nickel chloride sub-strate supplied with the kit was added. Colour develop-ment was allowed to proceed for 7 minutes, and thensections were washed in tap water. After dehydration ingraded alcohols, sections were cleared in xylene amd thenmounted in DPX (BDH, Eastleigh, Hants., UK).

Nonspecific staining was determined by replacement ofthe primary antibody with a solution of 2.5 µg/ml normalrabbit immunoglobulin. The specificity of the antibody wasconfirmed by incubating control sections with primaryantibody that had been preincubated for 30 minutes with a50-fold molar excess of the immunising a1A fusion protein.The remainder of the staining procedure was then per-formed as described above.

Dual localisation of a1A subunit proteinand neurotransmitter or glial markers:

Tissue preparation

Wistar rats (Harlan UK Ltd) weighing 200–300g wereperfused transcardially with 4% paraformaldehyde in 0.1M phosphate buffer. Brains were removed and postfixed inthe same fixative and then cryoprotected in 15% sucroseovernight. Free-floating 30-µm sections were cut on aVibratome. Some blocks were snap-frozen in O.C.T. tissueembedding medium (Miles Laboratories, Slough, Berks.,UK), and sections were cut on a sliding microtome.

Dual localisation experiments were performed by usingan immunofluorescent technique on cryopreserved tissue.Sections were incubated for 1 hour in 10% serum homolo-gous to the species in which the secondary antibody wasraised (normal animal serum in 0.2% Triton X-100/PBS). A1:10 dilution of the rabbit anti-a1A primary antibody (in0.2% Triton X-100/PBS) was then applied, and the sectionswere incubated for 48 hours at room temperature. Afterwashing in PBS (3 3 10 minutes), tetramethylrhodamineB isothiocyanate-conjugated secondary antibody was ap-plied at a 1:100 dilution, and the sections were incubatedfor 2–4 hours.

This staining procedure was then repeated on the samesection by using a primary antibody to the appropriateneurotransmitter or glial marker that had been raised in aspecies other than rabbit. The antibody to choline acetyl-transferase was obtained commercially (Chemicon, Har-low, Essex, UK); the remainder, which was kindly providedby the developers, had previously been demonstrated to bespecific. A fluorosceine isothiocyanate-conjugated second-ary antibody was used for detection. The combinations ofantibodies and references to their previous use are sum-marised in Table 1. The results of the immunofluorescence

TABLE 1. Blocking Sera and Antibodies Used for Dual Localisation1

Antigen Reference Blocking serum Primary antibody Secondary antibody

a1A Subunit protein Normal goat serum Rabbit anti-a1A (1:10) Goat anti-rabbit TRITC (1:100)Normal donkey serum2 Donkey anti-rabbit TRITC (1:100)

ChAT Michael and Priestley (1996) Normal donkey serum Goat anti-ChAT (1:50) Biotinylated horse anti-goat (1:400)/avidin FITCTH Semenenko et al. (1986) Normal sheep serum Mouse anti-TH (1:20) Sheep anti-mouse FITC (1:100)5-HT Lopez Costa et al. (1991) Normal goat serum Rat anti-5-HT (1:100) Goat anti rat-FITC (1:100)GFAP Curtis et al. (1991) Normal sheep serum Mouse anti-GFAP (1:500) Sheep anti-mouse FITC (1:100)

1ChAT, choline acetyltransferase; TH, tyrosine hydroxylase; 5-HT, 5-hydroxytryptamine; GFAP, glial fibrillary acidic protein; TRITC, tetramethylrhodamine isothiocyanate; FITC,fluorescein isothiocyanate.2Donkey anti rabbit secondary antibody was used for dual localisation with ChAT in order to prevent cross reaction.


experiments were viewed and documented by photographyby using the appropriate filters on a Leitz UV microscope.

Policy on the use of animals

All of the procedures used in the current study thatinvolved live animals were performed in accordance withthe appropriate UK regulations governing the use ofanimals in scientific research.

Photography, digital imaging,and preparation of distribution maps

Sections were photographed by using Kodak T-Maxblack-and-white negative film and were either hand ordigitally processed. In the latter case, negatives werescanned at 900 pixels/inch by using a Nikon LS-1000 filmscanner. Figure 10 was assembled by using Adobe Photo-shop. Grey levels were stretched to optimise contrast, andthe final plate was printed on a Sony UP-D8800 digitalprinter.

The maps illustrating the distribution of mRNA andprotein were redrawn from The Rat Brain in StereotacticCoordinates (Paxinos and Watson, 1996) with permissionof the publishers.

RESULTS

A radiolabelled probe, generated to a region of the rata1A calcium channel subunit, was used in Northern hybridi-sation analysis of mRNA prepared from different rattissues. This probe detected two abundant transcripts ofapproximately 9 and 7.4 kb (Fig. 1a) in rat whole brainmRNA; no hybridisation was observed to mRNA fromskeletal muscle or lung.

The antibody raised against the human a1A calciumchannel subunit was used in Western blot analysis ofmembrane preparations from HEK 293 cells transfectedwith the a1A subunit and from whole rat brain. Severalimmunoreactive bands were seen in the membrane prepa-rations from both transfected HEK cells and rat wholebrain, with prominent bands of approximately 260, 190,and 130 kD (Fig. 1b). No immunoreactivity was observedin membrane preparations from nontransfected HEK 293cells.

This probe and antibody were used for subsequent ISHand immunolocalisation studies into the distribution of thea1A subunit. Sections that had undergone the entire hy-bridisation procedure but in the absence of any probe weredevoid of staining, whereas sections hybridised in thepresence of the sense riboprobe showed a small amount ofnonspecific background signal, often associated with theedge of the section (Fig. 2a). In contrast, sections hybrid-ised with the antisense riboprobe for the a1A subunitshowed abundant, widely distributed staining (Fig. 2b),the details of which are described below. Control studies inwhich the primary antibody was preabsorbed with a50-fold molar excess of the a1A fusion protein were devoidof staining (Fig. 2c). Sections incubated with the rabbitanti-human a1A antibody showed abundant staining thatcomplemented the ISH signal (Fig. 2d).

The distribution and intensity of a1A subunit expressionwas determined by visual assessment of staining intensity.In the following description and the accompanying dia-grams, the intensity of staining is described as being‘‘weak,’’ ‘‘moderate,’’ or ‘‘strong.’’ Although such a scoringsystem provides some indication of relative subunit expres-

sion, caution must be exercised because other factors, inparticular relative cell density, can cause apparent differ-ences in staining intensity between neuroanatomical ar-eas. The majority of a1A mRNA expression and immunore-activity was neuronal, although staining was also observedin certain white matter tracts, suggesting possible localisa-tion in glial cells.

Cerebellum

Within the cerebellum the most intense staining for a1Aat both the mRNA and protein levels was observed in thesoma of Purkinje cells, with weaker signal in cells of boththe molecular and granule layers (Fig. 2e,f). Immunoreac-tivity in the Purkinje cells was seen in the cell body andextended into the apical dendrite (Fig. 2f). In addition tothe staining in basket and stellate cells, punctate immuno-reactivity was observed throughout the molecular layer.No a1A protein or mRNA was detected in the fibre tracts.

The a1A mRNA expression and immunoreactivity (IR)were also observed in the cerebellar nuclei. The large cellbodies of cerebellar output neurones in the nucleus media-lis (Fig. 2g) and interpositus displayed strong a1A-IR andmRNA expression. A similar pattern of staining was foundin the nucleus lateralis, although here, apart from the cellbodies, a1A-IR was observed in the surrounding neuropil,the region where Purkinje cell axons terminate (Fig. 2h).

Hindbrain

In the medulla (Figs. 3a–c, 6a,b), the following displayedstrong neuronal staining for both a1A protein and mRNA:posterior ventral cochlear, hypoglossal, lateral reticular,inferior olivary, raphe magnus, raphe obscurus (Fig. 6c),and facial nuclei. Strong a1A-IR but moderate mRNAexpression was seen in the nucleus of the solitary tract(Fig. 6d) and the dorsal motor nucleus of the vagus. Thenucleus ambiguus also contained abundant a1A-IR, but theassociated mRNA expression in this area was weak.Strong a1A-IR and mRNA expression was found in a smallnumber of cells in the spinal trigeminal nucleus (Fig. 6e).

Moderate a1A-IR was found in the medullary reticularformation and the gigantocellular reticular nucleus, al-though mRNA expression in these areas was weak. Withinthe medullary reticular field, moderate a1A-IR and weakmRNA expression were seen in a high percentage of cells;in the external cuneate nucleus, a1A-IR was also seen at a

Fig. 1. Expression of a1A subunit mRNA transcripts (a) and protein(b) in rat tissues. Autoradiograph of hybridisation of [32P]-labelled rata1A subunit probe to a Northern blot of rat tissue RNA. Brain (lane 1),skeletal muscle (lane 2), and lung (lane 3). The migration of molecu-lar weight markers and their sizes (Kb) are shown to the left.Immunodetection of the a1A subunit protein by using the anti-humanpolyclonal antibody on a Western blot of membrane preparations of a1Atransfected HEK 293 cells (lane 1), untransfected HEK 293 cells(lane 2), and whole rat brain (lane 3). The migration of molecularweight markers and their sizes (kD) are shown to the left.


Figure 2


moderate level, although the associated mRNA expressionwas high. The dorsal cochlear nucleus displayed moderatea1A mRNA expression and weak immunoreactivity.

Within the pons (Fig. 3d), strong neuronal staining forboth a1A protein and mRNA was observed in the pontinenuclei, the anterior ventral cochlear nucleus, nucleus of

the trapezoid body (Fig. 6f,g), the ventral part of thepontine reticular nucleus, and the dorsal raphe nucleus.Strong a1A-IR was seen in the motor trigeminal nucleus,although a1A mRNA was expressed at only a moderatelevel in this area. Strong a1A-IR was seen in the ventralnucleus of the lateral lemniscus, with weak staining in thesurrounding lemniscal area: mRNA expression was moder-ate in both of these regions. The principle sensory trigemi-nal nucleus displayed moderate a1A-IR but with strong a1AmRNA expression.

Weak a1A-IR and mRNA expression were seen both inthe sensory neurones of the mesencephalic trigeminalnucleus and the locus coeruleus, the major noradrenergiccell group of the brain.

Midbrain

The a1A mRNA expression and protein distribution areillustrated in Figure 4. The mammillary nuclei displayedstrong a1A mRNA expression and immunoreactivity (Fig.7a,b), as did the cholinergic Edinger-Westphal nucleus.Strong a1A mRNA expression coincident with high levels ofimmunoreactivity were also seen in the the ventral tegmen-tal area and the substantia nigra pars compacta (Fig.7c,d). The substantia nigra pars reticulata, however,showed no detectable a1A expression or immunoreactivity.High levels of a1A mRNA expression with moderate a1A-IRwere observed in the dorsal lateral geniculate nucleus andthe lateral posterior thalamic nucleus.

Weak a1A-IR with associated strong mRNA expressionwas detected in the superficial grey layer of the superior

Fig. 2. Localisation of the a1A subunit in the cerebellum. a,b: Insitu mRNA hybridisation in the cerebellum with sense (a) andantisense (b) probes. b: Strong mRNA expression is observed in thePurkinje cell (P) and granule cell (G) layers, together with weakerexpression in the cells of the molecular layer (M). c: Immunohistochem-istry control section in which the primary antibody was preabsorbedwith a 50-fold molar excess of the a1A fusion protein. d: Immunohisto-chemical localisation of a1A in the cerebellum. Strong immunoreactiv-ity is observed in the Purkinje cell (P) and granule cell (G) layers.Weaker staining is seen in the stellate and basket cells of themolecular layer (M). e: High power photomicrograph of a1A mRNAexpression in the cerebellar cortex showing intensely stained Purkinjecells (arrowheads), together with staining in the granule cell layer (G)and cells of the molecular layer (M). f: High power photomicrograph ofa1A immunolocalisation in the cerebellar cortex. Intense staining isobserved in Purkinje cell bodies (arrowheads), which extends into theapical dendrites (hollow arrows) and in granule cells (G). Slightlyweaker staining is observed in the cells of the molecular layer (M)together with punctate immunoreactivity in the surrounding neuropil.g,h: Immunolocalisation in the cerebellar nuclei. Note staining in thecell bodies of the large output neurones (arrowheads) in both thenucleus medialis (g) and the nucleus lateralis (h). Immunoreactivity isalso seen in the neuropil surrounding the output neurone cell bodies inthe nucleus lateralis (hollow arrows). Scale bar 5 165 µm in a–d,75 µm in e, 250 µm in g, 40 µm in f,h.

Fig. 3. a–d: Distribution of mRNA (left) and protein (right) for thea1A voltage-dependent calcium channel subunit in the hindbrain.Figures in parentheses indicate distance from the interaural line(Paxinos and Watson, 1996). Filled areas indicate intense staining;heavily stippled areas indicate moderate staining; lightly stippled

areas indicate weak staining. Dashes indicate areas of white matterstaining. Staining in the cerebellar folium is indicated by an asterisk(see Results). The protein distribution represents staining in cellbodies; staining in the neuropil is commented upon in the text.


colliculus; in the nucleus brachium of the inferior collicu-lus, strong a1A-IR was observed but with only weak mRNAexpression.

Forebrain

Within the hippocampal formation, abundant a1A mRNAtranscripts and immunoreactivity were observed (Fig.8a,b). Moderately strong a1A expression and immunolocali-sation were seen in the granule cells of the dentate gyrus,with a weak general immunoreactivity in the molecularlayer. The pyramidal cells of the CA3 and CA2 regionsdisplayed stronger immunoreactivity than did the dentategranule cells. Staining in CA1 was slightly weaker than inthe other CA regions. A weak general immunoreactivitywas observed in the stratum radiatum and stratum oriens.

Strong a1A-IR was seen in the thalamic (mediodorsaland ventromedial) and hypothalamic nuclei together withstrong mRNA expression in the former nucleus and weakexpression in the latter. Strong a1A-IR and mRNA expres-sion were also observed in the lateral amygdala, whereasstrong a1A-IR but only weak mRNA expression wereobserved in the basal nucleus of Meynert (Fig. 5a).

High levels of a1A-IR and mRNA were expressed in thebasal forebrain hypothalamic areas, the nuclei of thehorizontal and vertical diagonal bands (Fig. 8c,d), themedial septal nucleus, and in the supraoptic nucleus (Fig.5b,c).

Strong a1A-IR and mRNA expression were present inscattered large cells within the caudate putamen, withlighter staining being seen in other smaller cells of thisregion (Fig. 5b,c). The anterior olfactory nucleus alsodisplayed strong a1A-IR and mRNA expression, whereasmoderate immunoreactivity and mRNA expression wereobserved in the ventral pallidum and islands of Calleja(Fig. 5d).

Olfactory bulb

Differential staining was observed in the various layersof the olfactory bulb. Strong a1A-IR was seen in the mitrallayer together with staining in isolated cells of the externalplexiform layer and the periglomerular cells. Negligibleimmunoreactivity was observed in the granule cell layer(Fig. 8e).

Fig. 4. a,b: Distribution of mRNA (left) and protein (right) for thea1A voltage-dependent calcium channel subunit in the midbrain.Figures in parentheses indicate distance from the interaural line.Filled areas indicate intense staining; heavily stippled areas indicatemoderate staining; lightly stippled areas indicate weak staining.

Dashes indicate areas of white matter staining. Staining in the cortexis indicated by a dagger (see Results). The protein distribution repre-sents staining in cell bodies; staining in the neuropil is commented uponin the text.


Cortex

The a1A mRNA expression and immunoreactivity withincells of the cortex occurred in a distinct pattern (Fig. 9a–e).Strong staining was seen in the densely packed cells of theexternal granular layer (layer II), which, under highmagnification, was seen to occur in both the cell bodies andprimary and secondary dendrites (Fig. 9c). Staining inten-sity then decreased until the inner pyramidal cell layerswere reached (layers IV and V), where a1A mRNA expres-sion and immunoreactivity were again intense. Closeexamination of the a1A-IR demonstrated immunoreactivityextending into some of the processes of the pyramidal cells.Another band of intense immunoreactivity was observedin the stellate cells at the base of layer VI, immediatelyadjoining the white matter. An a1A-IR of an amorphousnature was observed in the cortex, presumably represent-ing staining of nerve fibres; such staining was particularlynoticeable in the molecular layer (layer I), a region ofdensely packed fibres. No a1A mRNA expression was seenin these areas of amorphous a1A-IR.

Abundant a1A mRNA expression and immunoreactivitywere observed in the pyriform cortex. In the frontal cortex,

a moderate, relatively uniform, level of mRNA expressionand immunoreactivity was seen.

Dual localisation of a1A andneurotransmitter markers

The single labelling studies demonstrated high levels ofa1A expression in neuroanatomical areas associated withparticular neurotransmitters. Thus, a1A-IR was observedin several regions known to contain cholinergic cell groups(e.g., striatum, nucleus of the diagonal band), in serotoner-gic neurones (e.g., raphe magnus and dorsal raphe), and indopaminergic and noradrenergic neurones (e.g., substan-tia nigra and locus coeruleus). The association between a1Aand individual neurotransmitters was therefore analysedby double-labelling immunofluorescence for a1A and eithercholine acetyltransferase (ChAT), serotonin (5-hydroxytryp-tamine; 5-HT), or tyrosine hydroxylase (TH). In addition,expression of a1A in glial cells was examined by doublelabelling with glial fibrillary acidic protein (GFAP).

Parts of the basal forebrain (medial septum, olfactorytubercle, horizontal and vertical limbs of the diagonalband), striatum, and potine reticular formation were exam-

Fig. 5. a–d: Distribution of mRNA (left) and protein (right) for thea1A voltage-dependent calcium channel subunit in the forebrain.Figures in parentheses indicate distance from the interaural line.Filled areas indicate intense staining; heavily stippled areas indicate

moderate staining; lightly stippled areas indicate weak staining.Staining in the cortex is indicated by a dagger (see Results). Theprotein distribution represents staining in cell bodies; staining in theneuropil is commented upon in the text.


Figure 6


ined for ChAT double labelling. In all regions ChAT-IRneurones were seen to have high levels of a1A-IR. Forexample, extensive double labelling was seen in the medialseptum (Fig. 10c,d) and the large strongly a1A-IR neuronesof the striatum were confirmed to be cholinergic. However,the relationship between a1A and ChAT was not exclusive.Most regions contained strongly a1A-IR neurones that

were not ChAT-IR and a few cholinergic neurones that hadrelatively light immunoreactivity (Fig. 10a,b).

The dorsal raphe nucleus was examined for colocalisa-tion of 5-HT and a1A. Many 5-HT-IR neurones showedmoderate levels of a1A-IR, but there were also 5-HT-IRneurones with negligible a1A immunostaining (Fig. 10e,f).

Tyrosine hydroxylase and a1A-IR were examined in thedopaminergic neurones of the substantia nigra and ven-tral tegmental region and in the noradrenergic locuscoeruleus. Neurones in all regions showed low-level a1A-IRthat was comparable to surrounding nonmonoaminergicneurones (Fig. 10g,h).

The relationship between GFAP and a1A was examinedin the pons and cerebellum. In no region was any evidenceseen of a1A-IR in GFAP-IR processes (Fig. 10i,j).

DISCUSSION

In the present study, the distribution of mRNA andprotein for the a1A VDCC subunit has been mappedthroughout the normal mature rat brain in a neuroanatomi-cally detailed study. Although there have been previousreports in the literature on the localisation of a1A mRNA(Stea et al., 1994; Tanaka et al., 1995), protein (Westen-

Fig. 6. Localisation of the a1A subunit in the hindbrain.a,b: Localisation of a1A mRNA expression (a) and immunoreactivity (b)in a coronal section approximately 2.6 mm behind the interaural line.Messenger RNA expression is observed to be distinctly cellular, withan absence of reactivity in the neuropil. In contrast, a1A immunoreac-tivity is seen as a more diffusely cellular stain against the moregeneral reactivity observed in the neuropil. c: The a1A immunoreactiv-ity in the raphe obscurus (arrowheads) demonstrates the characteris-tic intense staining seen in the mid-line structures. d: The a1Aimmunoreactivity in the nucleus of the solitary tract shows intensestaining of both the cell bodies (arrowheads) and processes (hollowarrows). e: The a1A mRNA expression is visible in the spinal trigeminalnucleus (filled star) and tract (hollow star). f,g: The a1A mRNAexpression (f) and immunoreactivity (g) are visible in the trapezoidbody nucleus (Tz) and cells of the pyramidal tract (py). Scale bar 5600 µm in a,b, 75 µm in c,d, 250 µm in e–g.

Fig. 7. Localisation of the a1A subunit in the midbrain. a,b: The a1AmRNA expression in the mammillary body (M), low and mediumpower. c,d: The a1A mRNA expression (c) and immunoreactivity (d) in

the substantia nigra. Note intense staining in the pars compacta(hollow star) and lack of staining in the pars reticulata (filled star).Scale bar 5 650 µm in a, 250 µm in b–d.


broek et al., 1995; Sakurai et al., 1996), and the P-channelprotein (Hillman et al., 1991), no comparative studies onthe mRNA and protein distribution have been described.Unlike previous studies in which isotopic ISH has beenperformed, we have employed nonradioactive detectionprocedures, thus allowing a higher degree of cellularresolution. In addition to the more detailed neuroanatomi-cal nature of our mapping study, we have interpreted theobserved distribution of the a1A subunit in terms of neuro-logical pathways and neurotransmitter associations.

The riboprobe and antibody used in this study werecharacterised by Northern hybridisation and Western blotanalysis, respectively. Northern analysis showed that theprobe hybridised to transcripts corresponding to 9 and 7.4 kb

in whole brain mRNA. The apparent sizes of the tran-scripts obtained with this probe were similar to thosereported in previous studies (Starr et al., 1991). Nohybridisation was obtained to mRNA derived from skeletalmuscle or lung, suggesting that the probe was recognisinga channel found predominantly in the brain.

Immunoblotting experiments demonstrated that theanti-a1A subunit antibody recognised specific bands inmembrane preparations from HEK 293 cells transfectedwith the a1A subunit and from whole rat brain. A band ofapparent molecular weight 260 kD was obtained with ourantibody: this corresponds closely to the 252-kD molecularmass predicted from the cDNA sequence (Starr et al.,1991). Other, smaller, bands were also observed: compara-

Fig. 8. Localisation of the a1A subunit in the forebrain. a,b: The a1AmRNA expression (a) and immunoreactivity (b) in the hippocampusshows abundant localisation in the granule cells of the dentate gyrus(arrowheads) and the pyramidal cells of the CA3 region (hollowarrows). c,d: The a1A mRNA expression (c) and immunoreactivity (d)

are visible in the vertical limb of the nucleus of the diagonal band. e:The a1A immunoreactivity in the olfactory bulb shows prominentstaining of the mitral cells (arrowheads), together with clusters ofpunctate staining in the glomeruli (hollow arrows). Scale bar 5140 µm in a,b, 125 µm in c–e.


tive bands of similar size have been reported by otherworkers using antibodies directed against different re-gions of the rat a1A subunit (Westenbroek et al., 1995;Sakurai et al., 1996).

In situ hybridisation and immunolocalisation studiesshowed good overall correlation between mRNA and pro-tein distribution. However, caution must be exercisedbefore comparing levels of expression. Staining intensity

Fig. 9. Localisation of the a1A subunit in the cortex. a,b: The a1Aimmunoreactivity (a) and mRNA expression (b) in the parietal cortexshow strong staining in the external granular layer (G) and the innerpyramidal cell layer (P). c: High power field of a1A immunoreactivity inlayer II of the cortex demonstrates strong staining in the cell bodies,

apical dendrites, and collaterals. d,e: a1A Immunoreactivity in thecingulate/frontal cortex. Under high power (e), staining can be seenextending into the pyramidal cell processes (arrowheads). Scale bar 5250 µm in a,b,d, 35 µm in c, 75 µm in e.


Figure 10


has been estimated by visual scoring, and no attempt hasbeen made to quantify the reaction product. Comparison ofstaining intensity within the IHC or ISH studies individu-ally can give a meaningful indication of distributionaldifferences in the level of protein or mRNA expression, butit is not relevant to compare directly the amount of proteinand mRNA staining. Some comment can, however, bemade regarding the relative expression of protein andmRNA in certain areas. For example, in the externalcuneate nucleus, spinal trigeminal nucleus, and inferiorolive, a1A mRNA expression is of a comparable high level,but a1A-IR was considerably weaker in the external cune-ate nucleus than in the other two areas.

Prominent staining for a1A protein and mRNA wasobserved in neuronal cell bodies together with punctate IRthroughout many regions of neuropil. For example, in thecerebellum, punctate IR was seen throughout the molecu-lar layer, where parallel and climbing fibres form excita-tory synapses with Purkinje cell dendrites, whereas in thenucleus lateralis, a distinct band of punctate IR wasobserved in the region where Purkinje cell axons forminhibitory GABAergic synapses with the cerebellar outputneurones. The observation of high levels of mRNA expres-sion in the cell body is to be expected because this is thelocation where translation occurs. However, variations inthe amount of cellular immunoreactivity have been re-ported in the literature.

Using an antibody raised to a peptide corresponding to a19-amino-acid sequence of the rat rbA isoform (Starr et al.,1991) within the intracellular loop between IIS6 and IIIS1,Westenbroek et al. (1995) described immunoreactivity aspredominantly punctate in dendrites and areas rich insynapses, with generally weak staining in cell bodies. In amore recent study by the same group (Sakurai et al., 1996),immunolocalisation in a limited number of regions of ratbrain, by using isoform-specific anti-peptide antibodies,produced differences in cellular versus dendritic staining.Thus, by using a second antibody specific for the rbAisoform (CNA3), generated to an amino acid sequence

adjacent to that used in their previous study, staining wasreported in the soma of Purkinje cells, cortical neurones,and certain hippocampal cells. However, when an antibodyfor the rabbit BI isoform (Mori et al., 1991) was used in thesame rat brain areas, predominantly punctate stainingwas observed, similar to that reported with their originalantibody to the rat rbA isoform, i.e., dendritic or synapticassociations. In the current study, the antibody was raisedagainst a GST-fusion protein containing a 160-amino-acidsequence of the intracellular loop between IIS6 and IIIS1 ofthe human a1A subunit, which is conserved among the knownsplice variants and has 85% homology with rbA. The promi-nent cell body staining seen with our antibody is most similarto that observed by Sakurai et al. (1996) when using theCNA3 antibody. Clearly, the pattern of staining observed isdependent on the epitope recognised by the antibody.

High levels of a1A-IR in the cell body may be due to thepresence of pools of protein awaiting transport and inser-tion into nerve terminals. Alternatively, the a1A subunitmay have a function in maintaining calcium homeostasisin the cell body. The presence of v-agatoxin IVA-sensitive(v-agatoxin fraction IVA from the venom of the spiderAgecenopsis aperta) channels in the cell bodies of Purkinjecells has been demonstrated electrophysiologically (Mintzet al., 1992), suggesting a functional role for these chan-nels in this location.

Besides neuronal expression, a1A mRNA and proteinwere also found in the present study to be localised withinwhite matter tracts, although in the areas examined thea1A protein did not colocalise with the astrocyte markerGFAP. This suggests that the a1A subunit is not expressedin all varieties of neuroglia. The observation of calciumchannel expression in neuroglia supports the developingliterature regarding ion channel expression in these cells.It has been shown that injection of mRNA extracted fromcorpus callosum, fornix, and optic nerve into Xenopusoocytes results in the expression of transcripts coding forneurotransmitter receptors and VDCCs (Matute andMiledi, 1993; Matute et al., 1994).

In keeping with previous reports of Northern blot analy-ses of brain mRNA preparations (Mori et al., 1991; Starr etal., 1991), the current study shows that the a1A subunit iswidely, although not uniformly, distributed throughout therat central nervous system (CNS). High levels of expres-sion were seen in areas such as the cerebellar Purkinjecells, the pontine nuclei, and the horizontal and verticallimbs of the diagonal band; elsewhere, for example in themedullary reticular formation and the caudate putamen,only moderate or weak expression was observed. Theabundant signal for both mRNA and protein detected inthe cerebellar Purkinje cell is consistent with the predomi-nant P-type current reported in this cell type (Llinas et al.,1989). Within the cerebellum, both a1A protein and mRNAwere also detectable in the granule cells and, to a lesserdegree, in the cells of the molecular layer. The latterobservation is in contradiction to certain previous reportsthat indicate that no mRNA for a1A is found in themolecular layer (Stea et al., 1994; Tanaka et al., 1995), butthis difference may reflect the limited cellular resolution ofisotopic ISH methods used in these studies. In addition,our observation parallels the hybridisation pattern seen inthe human when using a similar nonisotopic method(Volsen et al., 1995). Elsewhere, the distribution of mRNAtranscripts for a1A is in good agreement with the currentlyavailable literature. Although limited calcium channel

Fig. 10. Pairs of micrographs (a,b, c,d, e,f, g,h, i,j) showingimmunofluorescence double labelling for a1A (a,c,e,g,i) and neurotrans-mitter (b,d,f,h) or glial (j) markers in different regions of the rat brain.The primary antibody is indicated in each panel, with a1A identified as1A. a–d: Choline acetyltransferase (ChAT) immunoreactive (IR) neu-rones in the forebrain contain high levels of a1A-IR. a,b: Olfactorytubercle. c,d: Medial septum. Arrows indicate cells with high levels ofboth a1A (a,c) and ChAT (b,d). The cholinergic group illustrated in aand b also contains strongly immunoreactive a1A cells that lack ChAT(hollow arrowheads) and ChAT-IR cells with light a1A staining (arrow-heads). The asterisks indicate the principal cells of the olfactorytubercle. These cells are mainly not cholinergic and have low levels ofa1A-IR. e,f: Serotonin (5-hydroxytryptamine; 5-HT)-IR neurones (f) inthe median raphe show moderate or low levels of a1A-IR (e). Arrowsindicate double-labelled cells, arrowheads indicate serotonergic neu-rones with negligible a1A-IR, and asterisks indicate nonserotonergicneurones with a1A-IR. g,h: Tyrosine hydroxylase (TH)-IR noradrener-gic neurones (h) of the locus coeruleus show low levels of a1A-IR (g).Arrows indicate double-labelled cells. Many cells adjoining the locuscoeruleus show comparable levels of a1A-IR, including the large cells(asterisks) of the trigeminal mesencephalic nucleus (Me5). The Me5cells lack TH-IR. The star indicates the fourth ventricle. i,j: Glialfibrillary acidic protein (GFAP)-IR in the cerebellum. GFAP-IR pro-cesses (arrowheads in j) are visible in the molecular layer. a1A-IRPurkinje cells are present (asterisks in i), but there is little immunore-activity in the molecular layer and no double labelling of the glialprocesses. Scale bars 5 50 µm.


data are available for the human, our findings in the ratshow a good correlation with the distribution observed inthe human cerebellum (Volsen et al., 1995) and hippocam-pus (Day et al., 1996).

Two regions of interest in relation to Alzheimer’s diseasedisplayed abundant a1A localisation. The nucleus basalisof Meynert, which is believed to play an important role inintegrating subcortical function, suffers a profound loss ofneurones during disease, whereas the basal forebrainbundle/nucleus of the diagonal band provides a majorafferent pathway to the entorhinal cortex, an area whereneurofibrillary tangles are found. The involvement ofVDCCs in the pathology of Alzheimer’s disease is currentlyuncertain. However, calcium channel dysfunction couldcause impaired synaptic transmission or precipitate neuro-nal loss due to calcium-mediated cell death. Examinationof VDCC expression in tissue from Alzheimer’s patients orfrom one of the transgenic animal models of the diseasemay provide further illumination.

Considering the distribution of the a1A subunit in rela-tion to neurotransmitter involvement, it is apparent fromboth the dual-localisation experiments and examination ofthe observed distribution of a1A in relation to definedneurological pathways that no simple correlation exists.Dual-labelling experiments demonstrated that a1A coloca-lised with markers for cholinergic, noradrenergic, andserotonergic neurones. Although the dual-localisationanalyses are incomplete, particularly with regard to aminoacid neurotransmitters, further correlation is suggestedby examination of the neuroanatomical distribution of a1A.Thus, expression was observed in Purkinje cells that useg-aminobutyric acid (GABA) as their neurotransmitterand in the glutamatergic cells of the inferior olive.

These observations suggest that if selective modulationof neurotransmitter release at defined loci is to be achievedby pharmacological modulation of VDCCs, then factorsthat can confer a greater degree of diversity on thesechannels, other than the class of the a1 subunit which theycontain, must be considered. The first factor may be theexistence of alternatively spliced forms of the a1 subunit.In the present study, both the RNA probe and antibodywere raised to a region of the a1A subunit sequence that isconserved among the known splice variants. Obviously,the existence of splice variants provides a means ofincreasing the number of specific VDCC types that canexist by allowing for additional combinations of subunitcoexpression and potentially more discrete pharmacologi-cal targets.

A second means by which diversity could be increased isby variation in the overall subunit composition of thechannel, i.e., which b subunit is coexpressed with a1A. Thecurrent literature cites four classes of b subunit (Castel-lano and Perez-Reyes, 1992), each of which exists in anumber of splice variants. Some evidence has been putforward that certain a1 and b subunits are always found tocolocalise, e.g., it was initially reported that a1B associatedwith b3 (Witcher et al., 1993). However, more recentbiochemical studies have indicated that this may be anoversimplification and that the same class of a1 subunitmay associate with different b subunits (Snutch et al.,1990; Liu et al., 1996; Scott et al., 1996). Further diversityin the regulation of VDCC-mediated neurotransmitterrelease may be conferred by the existence of cooperativitybetween channel types at single release sites. By usingspecific calcium channel toxins to block neurotransmitter

release from rat striatal nerve terminals, it has beenshown that N- and P-type channels must coexist in thesame population of terminals, where they act synergisti-cally to regulate dopamine release (Turner et al., 1993).However, in the same preparation, it was shown thatglutamate release was sensitive only to the P-channelblocker v-agatoxin-IVA and not the N-channel blockerv-conotoxin-GVIA (v-conotoxin fraction VIA from the conesnail Conus geographus). Similar effects have been demon-strated in antagonist studies of rat hippocampal slices(Gaur et al., 1994). In that study, both glutamate andGABA release were found to be mediated by cooperativitybetween the P-type and a second (possibly Q-type) VDCC.Investigations by other workers (Takahashi and Momi-yama, 1993; Wheeler et al., 1994) suggest that cooperativ-ity between VDCC subtypes may be a widespread arrange-ment in the CNS as a mechanism to regulateneurotransmitter release that may confer several func-tional advantages (Wheeler et al., 1996).

Taken together, different mechanisms are available toprovide a means whereby a considerable diversity ofunique calcium channel types and cooperatively actingassociations can exist to regulate neurotransmitter releasein a specific manner. To understand further the regulatoryrole of calcium channels in specific neurological pathways,it will be necessary to investigate VDCC subunit associa-tions at the subcellular level by immunoelectron micros-copy and biochemical analyses. Such studies will be bothexacting and laborious to apply to widespread mapping.An alternative approach to determine the role of indi-vidual subunits in neurotransmitter release and hencetheir potential as therapeutic targets may be to perform invivo gene knockout studies. Two potential methods areavailable for such investigation: first, a transgenic animalbearing a deletion of a specific calcium channel gene couldbe produced; second, use could be made of antisensetechnology to interfere with subunit expression. The cur-rent mapping studies provide useful data for such futureinvestigations in suggesting potential targets for anti-sense injection and in setting baseline expression levels.

ACKNOWLEDGMENTS

We are grateful to Prof. A.C. Cuello, Dr. T. Gorcs, and Dr.G.P. Wilkin for their generous provision of antisera, Mr. T.Westcott for assistance in drawing the distributional maps,and the Woolfson Foundation for a studentship to J. Yip.

LITERATURE CITED

Bean, B.P. (1989) Neurotransmitter inhibition of neuronal calcium currentsby changes in channel voltage dependence. Nature 340:153–156.

Beattie, R.E., S.G. Volsen, D. Smith, A.L. McCormack, S.E. Gillard, J.P.Burnett, S.B. Ellis, A. Gillespie, M.M. Harpold, and W. Smith (1997)Preparation and purification of antibodies to human neuronal voltage-dependent calcium channel subunits. Brain Res. Prot. 1:307–319.

Bland, Y.S., M.A. Critchlow, and D.E. Ashurst (1991) Digoxigenin as a probelabel for in situ hybridisation of skeletal tissues. Histochemistry23:415–418.

Castellano, A. and E. Perez-Reyes (1992) Molecular diversity of Ca21

channel b subunits. Biochem. Soc. Trans. 22:483–487.Curtis, R., R. Hardy, R. Reynolds, B.A. Spruce, and G.P. Wilkin (1991) Down

regulation of GAP-43 during oligodendrocyte development and lack ofexpression by macroglial astrocytes in vivo: Implications for differentia-tion. Eur. J. Neurosci. 3:876–886.

Day, N.C., P.J. Shaw, A.L. McCormack, P.J. Craig, W. Smith, R. Beattie, T.L.Williams, S.B. Ellis, P.G. Ince, M.M. Harpold, D. Lodge, and S.G. Volsen(1996) Distribution of a1A, a1B, a1E voltage-dependent calcium channel


subunits in the human hippocampus and parahippocampal gyrus. J.Neurosci. 71:1013–1024.

Dunlap, K., J.I. Luebke, and T.J. Turner (1995) Exocytotic Ca21 channels inmammalian central neurons. Trends Neurosci. 18:89–98.

Feinberg, A.P. and B. Vogelstein (1983) A technique for radiolabeling DNArestriction endonuclease fragments to high specific activity. Anal.Biochem. 132:6–13.

Gaur, S., R. Newcomb, B. Rivnay, J.R. Bell, D. Yamashiro, J. Ramachan-dran, and G.P. Miljanich (1994) Calcium channel antagonist peptidesdefine several components of transmitter release in the hippocampus.Neuropharmacology 33:1211–1219.

Hess, P. (1990) Calcium channels in vertebrate cells. Annu. Rev. Neurosci.13:337–356.

Hillman, D., S. Chen, T.T. Aung, B. Cherksey, M. Sugimori, and R.R. Llinas(1991) Localisation of P-type calcium channels in the central nervoussystem. Proc. Natl. Acad. Sci. USA 88:7076–7080.

Hirning, L., A. Fox, E. McCleskey, B. Olivera, S. Thayer, R. Miller, and R.Tsien (1988) Dominant role of N-type calcium channels in evokedrelease of norepinephrine from sympathetic neurons. Science 239:57–61.

Huang, C.-C., K.-S. Hsu, and P.-W. Gean (1996) Isoproterenol potentiatessynaptic transmission primarily by enhancing presynaptic calciumchannel influx via P- and/or Q-type calcium channels in the ratamygdala. J. Neurosci. 16:1026–1033.

Katz, B. and R. Miledi (1967) The timing of calcium channel action duringneuromuscular transmission. J. Physiol. Lond. 189:535–544.

Krieg, P.A. and D.A. Melto (1987) In vitro RNA synthesis with SP6 RNApolymerase. Methods in Enzymology. 155: 397–415.

Liu, H.Y., M. DeWaard, V.E. Scott, C.A. Gurnett, V.A. Lennon, and K.P.Campbell (1996) Identification of three subunits of the high affinityv-conotoxin MVIIC-sensitive Ca21 channel. J. Biol. Chem. 271:13804–13810.

Llinas, R., M. Sugimori, J.-W. Lin, and B. Cherksey (1989) Blocking andisolation of a calcium channel from neurons in mammals and cephalo-pods utilising a toxin fraction (FTX) from funnel-web spider poison.Proc. Natl. Acad. Sci. USA 86:1689–1693.

Lopez Costa, J.J., S. Marter, T.J. Grocs, J. Pecci Saavedra, and J.V.Priestley (1991) Enkephalin and serotonin innervation of somatastatinimmunoreactive medullary formation neurones. Brain Res. 548:300–304.

Matute, C. and R. Miledi (1993) Neurotransmitter receptors and voltagedependent Ca21 channels encoded by mRNA from the adult corpuscallosum. Proc. Natl. Acad. Sci. USA 90:3270–3274.

Matute, C., J.S. Garcia-Barcina, and R. Miledi (1994) Expression ofneurotransmitter receptors and Ca21 channels in the adult fornix andoptic nerve. NeuroReport 5:1457–1460.

Michael, G.J. and J.V. Priestley (1996) Expression of trkA and p75 nervegrowth factor receptors in the adrenal gland. NeuroReport 7:1617–1622.

Miljanich, G.P. and J. Ramachandran (1995) Antagonists of neuronalcalcium channels: Structure, function and therapeutic implications.Annu. Rev. Pharmacol. Toxicol. 35:707–734.

Milligan, J.F., D.R. Groebe, G.W. Witherell, and O.C. Uhlenbeck (1987)Oligoribonucleotide synthesis using T7 RNA polymerase and syntheticDNA template. Nucleic Acids Res. 15:8783–8798.

Mintz, I.M., M.E. Adams, and B.P. Bean (1992) P-type calcium channels inrat central and peripheral neurones. Neuron 9:85–95.

Mori, Y., T. Friedrich, M.S. Kim, A. Mikami, J. Nakai, P. Ruth, E. Bosse, F.Hofmann, V. Flockerzi, and T. Furuichi (1991) Primary structure andfunctional expression from complementary DNA of a brain calciumchannel. Nature 350:398–402.

Olivera, B.M., G.P. Miljanich, J. Ramachandran, and M.E. Adams (1994)Calcium channel diversity and neurotransmitter release: The v-conotoxins and &ohrgr;-agatoxins. Annu. Rev. Biochem. 63:823–867.

Paxinos, G. and C. Watson (1996) The Rat Brain in Stereotactic Coordi-nates. San Diego: Academic Press.

Pringle, J.H., A.K. Ruprai, L. Primrose, J. Keyte, L. Potter, P. Close, and I.Lauder (1990) In situ hybridisation of immunoglobulin light chain

mRNA in paraffin sections using biotinylated or hapten labelledoligonucleotide probes. J. Pathol. 162:197–207.

Sakurai, T., R.E. Westenbroek, J. Rettig, J. Hell, and W.A. Catterall (1996)Biochemical properties and subcellular distribution of the BI and rbAisoforms of a1A subunits of brain calcium channels. J. Cell Biol.134:511–528.

Sather, W.A., T. Tanabe, J.F. Zhang, Y. Mori, M.E. Adams, and R.W. Tsien(1993) Distinctive biophysical and pharmacological properties of class A(BI) calcium channel alpha 1 subunits. J. Neurosci. 12:222–234.

Schneider, T., X.Y. Wei, R. Olcese, J.L. Costantin, A. Neely, P. Palade, E.Perez-Reyes, N. Qin, J.M. Zhou, G.D. Crawford, R.G. Smith, S.H. Appel,E. Stefani, and L. Birnbaumer (1994) Molecular analysis and functionalexpression of the human type-E neuronal Ca21 channel alpha 1 subunit.Recept. Channels 2:255–270.

Scott, V.E., M. DeWaard, H.Y. Liu, C.A. Gurnett, D.P. Venzke, V.A. Lennon,and K.P. Campbell (1996) b Subunit heterogeneity in N-type Ca21

channels. J. Biol. Chem. 271:3207–3212.Semenenko, F.M., A.C. Cuello, M. Goldstein, C.Y. Lee, and E. Sidebottom

(1986) A monoclonal antibody against tyrosine hydroxylase: Applicationin light and electron microscopy. J. Histochem. 34:817–821.

Snutch, T., J. Leonard, M. Gilbert, and H. Lester (1990) Rat brain expressesa heterogenous family of calcium channels. Proc. Natl. Acad. Sci. USA87:3391–3395.

Starr, T.V.B., W. Prystay, and T.P. Snutch (1991) Primary structure of acalcium channel that is highly expressed in the rat cerebellum. Proc.Natl. Acad. Sci. USA 88:5621–5625.

Stea, A., W.J. Tomlinson, T.W. Soong, E. Bourinet, S.J. Dubel, S.R. Vincent,and T.P. Snutch (1994) Localization and functional properties of a ratbrain alpha 1A calcium channel reflect similarities to neuronal Q- andP-type channels. Proc. Natl. Acad. Sci. USA 91:10576–10580.

Takahashi, T. and A. Momiyama (1993) Different types of calcium channelsmediate central synaptic transmission. Nature 366:156–158.

Tanaka, O., S. Hiroyuki, and H. Kondo (1995) Localisation of mRNAs ofvoltage-dependent Ca21 channels: Four subtypes of a1- and b-subunitsin developing and mature rat brain. Mol. Brain Res. 30:1–16.

Tank, D.W., M. Sugimori, J.A. Connor, R.R. Llinas (1988) Spatially resolvedcalcium dynamics of mammalian Purkinje cells in cerebellar slice.Science 242:773–777.

Tsien, R., P. Ellinor, and W. Horne (1991) Molecular diversity of voltagedependent Ca21 channels. Trends Pharmacol. Sci. 12:349–354.

Turner, T.J., M.E. Adams, and K. Dunlap (1993) Multiple Ca21 channeltypes coexist to regulate synaptosomal neurotransmitter release. Proc.Natl. Acad. Sci. USA 90:9518–9522.

Volsen, S.G., N.C. Day, A.L. McCormack, W. Smith, P.J. Craig, R. Beattie,P.G. Ince, P.J. Shaw, S.B. Ellis, A. Gillespie, M.M. Harpold, and D.Lodge (1995) The expression of neuronal voltage-dependent calciumchannels in human cerebellum. Mol. Brain Res. 34:271–282.

Westenbroek, R.E., T. Sakuari, E.M. Elliot, J.W. Hell, T.V.B. Starr, T.P.Snutch, and W.A. Catterall (1995) Immunochemical identification andsubcellular distribution of the a1A subunits of brain calcium channels.J. Neurosci. 15:6403–6418.

Wheeler, D.B., A. Randall, and R.W. Tsien (1994) Roles of N-type and Q-typeCa21 channels in supporting hippocampal synaptic transmission. Sci-ence 264:107–111.

Wheeler, D.B., A. Randall, and R.W. Tsien (1996) Changes in actionpotential duration alter reliance of excitatory synaptic transmission onmultiple types of Ca21 channels in rat hippocampus. J. Neurosci.16:2226–2237.

Williams, M.E., P.F. Brust, D.H. Feldman, S. Patthi, S. Simerson, A.Maroufi, A.F. McCue, G. Velicelebi, S.B. Ellis, and M.M. Harpold (1992)Structure and functional expression of an omega-conotoxin-sensitivehuman N-type calcium channel. Science 257:389–395.

Witcher, D.R., M. De Ward, J. Sakamoto, C. Franzini-Armstrong, M.Pragnell, S.D. Kahl, and K.P. Campbell (1993) Subunit identificationand reconstruction of the N-type Ca21 channel complex purified frombrain. Science 261:486–489.


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