Cell Tissue Res (2003) 313:177–186DOI 10.1007/s00441-003-0759-4

R E G U L A R A R T I C L E

Ivan Lopez · Gail Ishiyama · Dora Acuna ·Akira Ishiyama · Robert W. Baloh

Immunolocalization of voltage-gated calcium channel a1 subunitsin the chinchilla cochlea

Received: 26 March 2003 / Accepted: 2 June 2003 / Published online: 4 July 2003� Springer-Verlag 2003

Abstract The immunohistochemical localization of a1A,a1B, a1C, a1D, and a1E voltage-gated calcium channelsubunits was investigated in the chinchilla organ of Cortiand spiral ganglia with the use of specific antipeptideantibodies. The inner and outer hair cells were immuno-reactive for a1A and a1D subunit antibodies. a1Cimmunoreactivity localized to the nerve terminals inner-vating inner hair cells and the basal pole of the outer haircell. There was only non-specific staining to a1B anda1E. Supporting cells were non-immunoreactive. Spiralganglia neurons were a1B, a1C, and a1D immunoreac-tive. A few spiral ganglia neurons were a1E immunore-active. The importance of a1D, the pore-forming subunitof the L-type channel, in outer and inner hair cell functionhas been clearly demonstrated in electrophysiological,molecular biological, and knockout models. The presenceof a1A, the pore-forming subunit of the P/Q typechannels, has not previously been demonstrated in innerand outer hair cells, and its function in the cochlear haircell is unknown.

Keywords Immunohistochemistry · Inner ear · Inner andouter cochlear hair cells · Chinchilla laniger (Rodentia)

Introduction

Cochlear inner hair cells (IHC) are specialized mechano-electrical transducers that transform the mechanical

deflection of the sensory cilia into an intracellularpotential. Sound stimuli are transmitted via the ear canaland the middle ear ossicles to the cochlea, where theresulting pressure differences across the hearing organelicit a complex vibratory motion. The resultant bendingof the sensory hairs in the direction of the talleststereocilia causes the opening of non-selective cationchannels located near the top of the hair bundle (Hudspeth1985, 1989, 1997). The resultant depolarization increasesthe opening probability of voltage-gated calcium channels(VGCCs) in the IHC. The increase in calcium influxaccelerates vesicle fusion and the release of neurochem-ical transmitters from the IHC (Parsons et al. 1994).Additionally, VGCCs are closely linked with calcium-activated potassium channels that determine the frequen-cy selectivity of the IHC (Fuchs 1996; Ramanathan et al.1999; Roberts et al. 1990; Tucker and Fettiplace 1996;Wu et al. 1995). The outer hair cells (OHC) areresponsible for the exquisite sensitivity, frequency selec-tivity, and dynamic range of the cochlea. These cells arepart of a mechanical feedback system involving thetectorial membrane and basilar membrane (see review byGummer et al. 2002). The VGCCs likely play a role inOHC, as well as IHC function.

The VGCCs are classified by electrophysiological andpharmacological properties into types P/Q, L, N, R, and T(Triggle 1999). Membrane-bound VGCCs are hetero-oligomers composed of four different subunits: thea1 subunit, and the regulatory subunits a2-d, b, and d(Hofmann et al. 1994). The a1 subunit, the pore-formingunit, is an integral membrane protein organized in fourhomologous domains, each containing six alpha-helicalmembrane-spanning segments (Fletcher et al. 1998). Thebiophysical and pharmacological properties of VGCCsare primarily determined by the a1 subunit, for which tengenes have been identified (A, B, C, D, E, F, G, H, I, andS; Catterall 1998; see review by Fisher and Bourque2001; Perez-Reyes and Schneider 1995; Tsien 1998).

Molecular biological studies have demonstrated theexistence of several VGCC subunits in the auditory andvestibular organs (Beisel et al. 1998; Fuchs 1996; Greenet al. 1996; Kollmar et al. 1997a, b; Lopez et al. 1999;

The National Institutes of Health grants AG09693-10, DC005224,00140-02, and DC05187-01 supported this work.

I. Lopez ()) · D. Acuna · A. IshiyamaDepartment of Surgery, Division of Head and Neck, 31-25Rehabilitation Center,UCLA School of Medicine,1000 Veteran Avenue, Los Angeles, CA 90095, USAe-mail: ivan@hnsurg.medsch.ucla.eduTel.: +1-310-8255331

G. Ishiyama · R. W. BalohNeurology Department,UCLA School of Medicine,Los Angeles, California, USA

Platzer et al. 2000; Su et al. 1995; Zidanic and Fuchs1995). A critical role of the a1D VGCC subunit inauditory function has been demonstrated, in that a1D-deficient mice are deaf (Engel et al. 2002; Platzer et al.2000). There are no prior studies investigating theimmunolocalization of the subunits of the VGCCs inthe auditory periphery. Therefore, we conducted immu-nohistochemical localization of a1A, a1B, a1C, a1D, anda1E in the organ of Corti and spiral ganglia in thechinchilla model.

Materials and methods

Animals

Young male chinchillas (Chinchilla laniger; n=12) were obtainedfrom PSK Ranch (Los Angeles, Calif., USA). Ages ranged from 6to 24 months, and weight ranged from 450 to 550 g. The protocoladheres to the NIH Guide for the Care and Use of LaboratoryAnimals and was approved by the UCLA Chancellor’s AnimalResearch Committee.

Antibodies

The development of specific peptide antibodies against the fiveclasses of neuronal calcium channel a1 subunits has been described(Hell et al. 1993; Volsen et al. 1995; Westenbroek et al. 1992,1995; Williams et al. 1992, 1994; Yokoyama et al. 1995). Theknown splice variants, which are recognized by these antibodies,are summarized in Table 1.

Tissue processing

To obtain the inner ear specimens, animals were deeply anesthe-tized with intramuscular administration of 20 mg ketamine/3 mgxylazine per kilogram, and then perfused transcardially with salinesolution, followed with 4% paraformaldehyde in sodium phosphatebuffer solution (0.12 M, pH 7.4). The temporal bones wereremoved from the skull, immersed in the same fixative for 3 h, andthen decalcified in 3% EDTA buffered phosphate solution for7 days. The whole cochlea was microdissected to obtain surfacepreparations of the organ of Corti.

Immunofluorescence staining

The tissue was incubated at room temperature for 30 min with ablocking solution containing 5% normal goat serum (NGS; VectorLaboratories, Burlingame, Calif., USA), 5% normal horse serum(NHS; Vector Laboratories), and 1% bovine serum albumin in0.25% Triton X-100 (Sigma, St. Louis, Mo., USA) in phosphate-buffered saline solution (PBS). The tissue was incubated with

primary antibodies against a1A, a1B, or a1D (1:200 in PBS;Alomone Research, Israel) or a1E subunits (kindly donated bySIBIA, La Jolla, Calif., USA; diluted 1:200 in 1% NGS and NHS inPBS) for 16 h in the shaker at 4–8�C. Sections were rinsed in PBS(3�15 min) between each step. The tissue was then incubated with aTexas red-conjugated anti-rabbit secondary antibody (1:800 in PBScontaining 1% NGS and 1% NHS; Vector Laboratories) for 1 h atroom temperature in the dark. Following incubation, the nucleardye DAPI was applied together with aqueous mounting solution(Vectashield-DAPI; Vector Laboratories). The immunoreactedspecimens were viewed and imaged in a Nikon Eclipse E800microscope equipped with an RTSlider spot digital camera andImage Pro Plus software.

Immunohistochemistry using free-floating sections

Tissue was fixed as described above, then the auditory bullae weredecalcified in 5% EDTA in PBS for 7 days. The otic capsule wasseparated from the temporal bone, and the membranous portion ofthe cochlea was dissected and embedded in 5% agar (FisherScientific). Thick sections (150 mm) throughout the mid-modioluswere obtained using a vibratome, and kept in ice-cold PBS. Theimmunohistochemical technique was performed according toprevious protocol for the chinchilla vestibular sensory periphery(Lopez et al. 1999). Free-floating sections were incubated for10 min in 3% hydrogen peroxide (Fisher Scientific) diluted in100% methanol, followed by incubation in sodium borohydride0.25% (Sigma) in PBS, for 20 min. Sections were rinsed in PBS(3�15 min) between each step. The sections were preincubated for30 min in PBS containing 10% NGS and 0.1% Triton X-100(Sigma). This solution was removed and the tissue was incubated atroom temperature for 16 h with rabbit polyclonal antibodiesdirected against peptides specific to a1C (1:200 in PBS). Tissuesections were then incubated with the biotinylated goat anti-rabbitsecondary antibody (1:50) for 1 h at room temperature (Elite kit;Vector Laboratories), and then incubated with the avidin-biotin-peroxidase solution (Elite kit) for 1 h at room temperature. Theimmunohistochemical reaction was visualized by incubating thetissue sections with a diaminobenzidine solution (DAB kit; VectorLaboratories) for 5 min at room temperature. The reaction washalted with distilled water (3�15 min). The immunoreacted tissuesections were placed in a solution of 1% OsO4 (EMS, FortWashington, Pa., USA) in phosphate buffer for 5 min, dehydratedin ascending ethyl alcohols, and flat embedded in Epon–Araldite(Fluka). Polymerization of the Epon–Araldite mixture containingthe tissue sections was accomplished in an oven at 65�C for 48 h.The plastic-embedded sections were mounted in the properorientation, on polymerized plastic blocks, using cyanoacrylate(Polysciences). One-�m-thin serial sections were made with adiamond knife (Polysciences) on a Sorvall MT-2 ultramicrotomeuntil the immunoreaction product was visible. The sections weremounted on glass slides and counterstained with toluidine blue(0.05% in borate buffer) for light-microscopic examination.

Controls

Non-specific staining was tested by replacement of the primaryantibody with NGS. Tissue sections were processed for immuno-histochemistry as described above. No immunoreactivity wasdetected in any of these controls. For absorption control of eachantibody, 1 mg of specific antigen (Alomone Research) wasincubated with 1 mg of antibody for 3 h at 37�C, and the mixturewas applied to the tissue sections. There was no immunoreactionproduct in the antibody absorption control sections (Figs. 2D, 3D,4D). The cerebellar cortex of the experimental animals was used asthe positive control (Lopez et al. 1999).

Table 1 Characteristics of the a1 subunits antipeptide antibodiesused in the present study. Sources: a1A to a1D from Alomone, a1Efrom SIBIA

Subunit Antigen Residues Subunit specificity

a1A CNA1 865–881 190 and 210 kDaa1B CNB1 851–867 210 and 240 kDaa1C CNC1 818–835 190 and 210 kDaa1D CND1 809–825 All formsa1E CND3 984–1099 All forms

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Results

There was a differential pattern of immunoreactivity foreach of the a1 subunits in the chinchilla auditoryperiphery. The pattern of immunoreactivity was similarthroughout the entire organ of Corti, from basal to apicalend. Within the organ of Corti, a1 subunit immunoreac-tivity was seen within the sensory hair cells or theinnervating terminals beneath the sensory hair cells. Thesupporting cells were non-immunoreactive to all a1 sub-units. The normal cytoarchitecture of the mammaliancochlea is schematically diagrammed in Fig. 1. The spiralganglion neurons send processes (primary afferents) thatinnervate predominantly IHC.

Localization of a1A

The a1A subunit was immunoreactive within the IHC andall three rows of the OHC in the organ of Corti(Fig. 2A, C, immunostaining in red color). a1A immu-noreactivity was seen throughout the cell bodies of IHCand OHC. There was no apparent differential degree ofimmunoreaction between IHC and OHC, and all threerows of OHC appeared to be equally immunoreactive. Allof the supporting cells of the organ of Corti were a1Anon-immunoreactive. The innervating terminals andnerve fibers of the IHC and OHC were also non-

immunoreactive. No immunoreactivity was observedwhen the primary antibody was absorbed with thecorresponding antigen (Fig. 2D).

Consistent with the lack of a1A immunoreactivity inthe nerve terminals and nerve fibers beneath the IHC, thespiral ganglia neurons were also a1A non-immunoreac-tive (Fig. 6A).

Localization of a1D

The a1D subunit was immunoreactive within the IHCcytoplasm (Fig. 3A). All three rows of the OHCdemonstrated OHC cytoplasm a1D immunoreactivity(Fig. 3C). There was no apparent differential degree ofimmunoreaction between IHC and OHC, and all threerows of OHC appeared to be equally immunoreactive.a1D immunoreactivity was also observed in some of thenerve terminals beneath the IHC (Fig. 3A arrowheads).All of the supporting cells were non-immunoreactive toa1D. No immunoreactivity was observed when theprimary antibody was absorbed with the correspondingantigen (Fig. 3D).

Consistent with the a1D immunoreactivity in the nerveterminals innervating the IHC, most of the spiral ganglianeurons were a1D immunoreactive (Fig. 6D).

Fig. 1 Schematic diagram of the mammalian organ of Corti. Mid-modiolar cross-section (left) and surface preparation (right). Theorgan of Corti sits upon the basilar membrane within the fluid-filledduct of the cochlea. The different cell types in the organ of Cortiinclude the inner and outer hair cells, inner and outer pillar cells,Deiter’s cells, inner and outer phalangeal cells, inner sulcus cells,interdental cells, inner border cells, and Hensen, Claudius, and

Boettcher cells (Lim and Kalineck 1998; Slepecky 1996). It isgenerally believed that the pillar and Deiter’s cells providemechanical support for the organ of Corti, but the biologicalfunction of non-sensory supporting cells is not well understood.The inner hair cells are round in shape whereas the outer hair cellsare elongated and outnumber the inner hair cells in a ratio 3:1.Figure modified from Hawkins and Johnsson 1976

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Localization of a1C

The a1C subunit was immunoreactive in the inner spiralbundle beneath the IHC, the spiral tunnel bundle, and theouter spiral bundle at the basal pole of the OHC (Fig. 4A).All three rows of the outer spiral bundle were a1Cimmunoreactive (Fig. 4A, C). Figure 4C shows punctuateimmunoreactivity at the basal pole of the OHC. Theradiating tunnel fibers were also a1C immunoreactive(Fig. 4A). The OHC were a1C immunoreactive, andimmunoreactive punctae were evident up to the neck of theOHC (Fig. 4A arrows). The IHC appeared to be non-immunoreactive. The afferent nerve terminals underneaththe IHC were a1C immunoreactive (Fig. 4A, B). Thetunnel-crossing fibers were also immunoreactive (Fig. 4A).No immunoreactivity was observed when the primaryantibody was absorbed with the corresponding antigen(Fig. 4D).

Consistent with the a1C immunoreactivity in theinnervating terminals and fibers under IHC and OHC, themost of the spiral ganglia neurons were strongly a1Cimmunoreactive (Fig. 6C).

Localization of a1B and a1E

There was no specific immunoreactivity to a1B (Fig. 5A)and a1E (Fig. 5B) noted in the OHC, IHC, supportingcells, or nerve fibers within the organ of Corti. The spiralganglia neurons were strongly immunoreactive to a1B(Fig. 6D), but only a few spiral ganglia neurons were a1Eimmunoreactive (Fig. 6E). No immunoreactivity for thesesubunits was observed in the OHC, IHC, nerve fibers, andterminals. No immunoreactivity was observed when theprimary antibody was absorbed with the correspondingantigen.

Discussion

Differential distribution of a1A, a1B, a1C, a1D,and a1E in the organ of Corti and spiral gangliaof the chinchilla

We present the first immunohistochemical demonstrationfor the differential distribution of the VGCC a1 subunits

Fig. 2A–D a1A immunoreactivity in the chinchilla organ of Corti(surface preparation). A Inner hair cells (IHC) and outer hair cells(OHC) were a1A immunoreactive, with immunoreaction seenthroughout the cell body (immunostaining in red color). BBrightfield image from A. Pillar cells (PC) can be appreciated. CMerged panels A and B to illustrate the specific localization of a1A

immunoreactivity within the IHC and OHC. DAPI (blue) showscell nuclei. Supporting cells are non-immunoreactive. D Absorptioncontrol. No specific immunoreactivity was observed when theantibody against the a1A subunit was preabsorbed with itscorresponding antigen. Magnification bar 25 mm

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Fig. 3A–D a1D immunoreac-tivity in the chinchilla organ ofCorti (surface preparation). AThe IHC and OHC cytoplasmwas strongly a1D immunoreac-tive. Nerve terminals innervat-ing the IHC at the basal portionof the hair cell were also a1Dimmunoreactive (arrowheads).B Brightfield image from APillar cells (PC) can be appre-ciated. C Merged panels A andB to illustrate the specific lo-calization of the a1D subunit inthe IHC and OHC. DAPI (blue)shows cell nuclei. D Absorptioncontrol. No specific immunore-activity was observed when theantibody against the a1D sub-unit was preabsorbed with itscorresponding antigen. Magni-fication bar 20 mm

Fig. 4A–D a1C immunoreactivity in the organ of Corti. A Mid-modiolar cross section using light microscopy and DAB immuno-histochemistry, counterstaining with toluidine blue. IHC and pillarcells were non-immunoreactive. OHC (arrows) and the nerveterminals underneath IHC (arrows) and crossing fibers (arrows)were immunoreactive. B Higher magnification view of A to

illustrate the immunoreactive terminals (arrows) innervating theIHC. C Higher magnification view of A to demonstrate immuno-reactivity at the basal pole (arrows) of OHC. D Absorption control.No specific immunoreactivity was observed when the antibodyagainst the a1C subunit was preabsorbed with its correspondingantigen. Magnification bars 6 mm in A; 12 mm in B, C; 40 mm in D

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in the organ of Corti and spiral ganglia. The supportingcells in the organ of Corti were non-immunoreactive to allof the a1 subunits. In general, the IHC and the three rowsof OHC had similar immunoreactivity patterns. Theresults from the present study are in accordance withelectrophysiological studies.

a1D immunolocalizes to the IHC and OHC

The IHC and OHC expressed the a1D subunit, which isthe pore-forming subunit of the L-type VGCC. There isstrong electrophysiological evidence for involvement ofan L-type VGCC that couples IHC receptor potential toneurotransmitter release at the afferent synapses in the

Fig. 5A, B a1B and a1E and immunoreactivity were absent in the organ of Corti (surface preparation). A No specific reaction wasobserved for a1B subunit. B In a similar fashion no a1E was detected. Magnification bars 20 mm

Fig. 6A–E a1A–E immunoreactivity in the chinchilla spiral gan-glia. A a1A immunoreactivity. There was no a1A immunoreac-tivity in the spiral ganglia neurons. Non-immunoreactive spiralganglia neurons are shown (arrows). B a1B immunoreactivity.Most of the spiral ganglia neurons were a1B immunoreactive. Ca1C immunoreactivity. The majority of the spiral ganglia neuronswere a1C immunoreactive (arrows). There was a subset of a1C

non-immunoreactive neurons. D a1D immunoreactivity. Nearly allof the spiral ganglia neurons were strongly a1D immunoreactive. Ea1E immunoreactivity. A few of the spiral ganglia neurons werea1E immunoreactive (arrows). Most of the spiral ganglia neuronswere a1E non-immunoreactive. Magnification bars 30 mm in A;20 mm in B–D

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mammalian cochlea (Engel et al. 2002; Robertson andPaki 2002). Exocytosis from the chick basilar papilla talland short hair cells is dihydropyridine-sensitive (Spassovaet al. 2001; Zidanic and Fuchs 1995). The VGCCs arealso involved in the electrical tuning of hair cells byactivating large-conductance, calcium-activated potassi-um channels (Art et al. 1995; Fuchs and Evans 1988;Fuchs et al. 1988). Quantitative PCR demonstrated a 100-to 500-fold abundance of a1D versus a1C in chickcochlear hair cells (Kollmar et al. 1997a, b). Studies usingelectron microscopy and three-dimensional reconstructionhave demonstrated that L-type VGCCs are expressedpreferentially at release sites, where they likely mediateneurotransmitter release from the hair cell to primaryafferent nerve (Martinez-Dunst et al. 1997). In the chickbasilar papilla in both tall and short hair cells, at least95% of the calcium conductance is sensitive to dihy-dropyridine, and can be described by a single activationprocess sensitive to dihydropyridines (Zidanic and Fuchs1995). In the mouse cochlea, the relative contribution ofa1D (L-type) VGCCs to whole-cell calcium channelcurrents in IHC in the semi-intact organ of Corti was atleast 90%, calculated by comparing the calcium currentdensities in wild type against current densities from a1D�/� mice (Engel et al. 2002; Platzer et al. 2000). L-typeVGCCs formed by a1D exhibit unusual electrophysio-logical characteristics with rapid activation, rapid deac-tivation, and very little deactivation (Koschak et al. 2001;Platzer et al. 2000).

There is ample electrophysiological evidence for L-type VGCCs in mammalian OHC. In dissociated guineapig OHC, whole-cell patch-clamp recordings are indica-tive of L-type VGCC activity (Nakagawa et al. 1994), andnimodipine blocks calcium currents in guinea pig OHC.Of clinical relevance, there is evidence that L-type VGCCblockers, such as nimodipine, verapamil, and flunarizine,may have a protective effect on OHC in noise exposure(Heinrich et al. 1999) and calcium toxicity (Nilles 1995).Similarly, it has been demonstrated that diltiazem, an L-type VGCC blocker, protects the IHC from noise-inducedincrease in intracellular calcium ion levels (Maurer et al.1999). The critical importance of a1D-containing VGCCsin OHC and IHC function has been proven in the a1D-deficient model. Platzer et al. (2000) demonstrated thatmice lacking the a1D subunit are deaf due to the completeabsence of L-type currents in cochlear IHC, and thesubsequent degeneration of both IHC and OHC. The roleof L-type VGCCs in OHC function is unknown. Thedemonstration of a1D subunits in the OHC raises thepossibility that L-type VGCCs, at the basolateral mem-brane, are involved in the OHC modulation, a calciumion-dependent event (Heinrich et al. 1997).

a1D immunolocalizes to the nerve terminalsinnervating IHC and OHC

a1D immunoreactivity was present in nerve terminals atthe base of the IHC, as well as in the spiral ganglia

neurons. The nerve terminals beneath the OHC appearedto be a1D immunoreactive. However, a precise subcel-lular location of this subunit in the IHC, OHC, and nerveterminals can only be determined with transmissionelectron-microscopic techniques. The role of a1D-con-taining VGCCs in the primary afferent auditory neuron isunknown. There is electrophysiological evidence for L-type VGCCs in the spiral ganglia neurons, and the L-typeVGCC blocker, nifedipine, inhibited an increase incalcium induced by the neurotoxin trimethyltin (Fechterand Liu 1995). Thus, L-type VGCC blockers may have aclinical role in the neuroprotection of the spiral ganglionprimary afferent neurons against ototoxicity.

a1C immunolocalizes to the OHC at the basal pole,and the tunnel-crossing fibers

The pore-forming subunit of L-type VGCCs, a1C, hasbeen demonstrated in RT-PCR studies on chick cochlearhair cells (Kollmar et al. 1997b), as well as postnatalcochlear tissue from mouse (Green et al. 1996). Quanti-tative RT-PCR has demonstrated a 100- to 500-foldabundance of a1D versus a1C (Kollmar et al. 1997b).Furthermore, as noted above, there is strong evidence thata1D-containing VGCCs control neurotransmitter release(Engel et al. 2002; Platzer et al. 2000). Thus, the role ofthe a1C subunit is unknown. The localization to the basalpole is consistent with studies demonstrating the alter-ation of calcium ion in the guinea pig organ of Corti byapplication of diltiazem, an L-type VGCC blocker(Heinrich et al. 1997). The localization of a1C to thetunnel-crossing fibers and the radiating tunnel fibers isconsistent with a1C-containing VGCCs in the efferentpathways that innervate the OHC. These a1C-containingVGCCs may be involved in the neurotransmitter releasefrom efferent terminals synapsing onto OHC.

a1A immunolocalizes to the IHC and OHC

The a1A subunit constitutes the main pore-forming andvoltage-sensing subunit for the P- and Q-type calciumchannels. P/Q channels are heavily expressed in neuronsthroughout the brain, and are involved in the regulation ofneurotransmitter release from synaptic vesicles (Catterall1998). With regard to the cochlea, the regulation ofneurotransmitter release from the hair cells is apparentlymediated via L-type VGCCs, a1D-containing subtype(Engel et al. 2002; Fuchs 1996; Lenzi and Roberts 1994;Platzer et al. 2000; Roberts et al. 1991; Zhang et al. 1999).The presence of a1A subunit immunoreactivity on thestereocilia of IHC and OHC of the rat cochlea has beenreported (Hillman et al. 1995). However, there is noelectrophysiological evidence for P- or Q-type VGCCs inthe neurotransmitter release of IHC or OHC of thecochlea. In the chick cochlear hair cells, both tall andshort hair cells are insensitive to peptide toxins specific toP-type VGCCs (omega-agatoxin IVa), and Q-type

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VGCCs (omega-conotoxin MVIIC) (Zidanic and Fuchs1995), which would argue against the presence of a1Asubtype, which encodes the P/Q-type VGCCs, in the IHCand OHC. However, it is possible that there are species-specific differences between avian and mammalian sys-tems. Further studies are needed to evaluate the role ofa1A-containing VGCCs in IHC and OHC function.

Spiral ganglia neurons are a1B, a1C, a1D,and a1E immunoreactive

The primary afferent spiral ganglia neurons were immu-noreactive for all a1 VGCC subunits except a1A. N- andL-type VGCC currents have been detected in dissociatedspiral ganglia neurons (Jimenez et al. 1997). a1B is thepore-forming subunit for N-type VGCCs, and thus thepresence of a1B in the spiral ganglia is expected. Becausea1B was not present in the auditory periphery, it may beinvolved in release of neurotransmitter from primaryafferent nerve to cochlear nucleus. Nerve terminals withinthe organ of Corti were immunoreactive for the a1C anda1D subunits, and thus it is expected that the spiralganglia is a1C and a1D immunoreactive. The role ofVGCCs in the postsynaptic nerve terminal at thecytoneural junction is unknown.

No immunoreactivity for the a1B and a1E subunitsin the mammalian auditory periphery

There was no a1B (pore-forming subunit for N-typeVGCCs) or a1E immunoreactivity in the IHC or OHC.These findings are consistent with electrophysiologicalevidence for a lack of effect of conotoxin GVIA, an N-type VGCC blocker, when perfused into the cochlea(Robertson and Paki 2002; Zidanic and Fuchs 1995). Incontrast, Green et al. (1996) used RT-PCR to demonstratea1E mRNA expression in postnatal mouse cochleartissue, and Beisel et al. (1998) used amplified PCRproducts from rat whole cochlear library to demonstratea1E, as well as a1B and a1D products. It is possible thata1E is expressed in very low amounts in the organ ofCorti, but was not able to be detected using immunohis-tochemistry.

Differential distribution of VGCC a1 subunitsin the cochlear versus vestibular periphery

The pattern of distribution of the a1 subunits in thechinchilla auditory periphery differed greatly from that inthe chinchilla vestibular periphery (Lopez et al. 1999).a1B was highly expressed in vestibular hair cells, thestereocilia, and the afferent vestibular nerve terminals. Incontrast, a1B was not expressed in the cochlear hair cellsor nerve terminals. a1A was expressed in the IHC andOHC of the cochlea, but was not expressed in thevestibular hair cells. The a1D subunit was weakly

expressed in the vestibular hair cells, but was stronglyexpressed in cochlear IHC and OHC. L-type channelsformed by the a1D subunit possess unique characteristics,activating at low voltage and failing to inactivate (Platzeret al. 2000), and the differential expression of VGCCa1 subunits undoubtedly accounts for some of thedifferential electrophysiological properties of the vestib-ular and auditory periphery. Vestibular ganglia neuronswere a1B and a1C immunoreactive, whereas nearly allspiral ganglia neurons were a1B, a1C, and a1D immu-noreactive. A few spiral ganglia neurons were a1Eimmunoreactive. Both the vestibular and the auditoryprimary afferent ganglia neurons were non-immunoreac-tive to a1A subunit.

To date, the only known human a1 subunit mutation isin the a1A subunit. Point and missense mutations in theCACNA1A gene, coding for the a1A subunit result in arange of phenotypes, from familial hemiplegic migraineto progressive and episodic ataxia (Ophoff et al. 1996;Yue et al. 1998). Hearing symptoms have not beendescribed in patients with mutations in CACNA1A, butdetailed auditory testing has not been reported. Changesin auditory function may be subtle, and only identifiedwith specialized testing, such as otoacoustic emissions.CACNA1D, located on chromosome 3p14.3 (Lory et al.1997), codes for the a1D subunit, and is an excellentcandidate gene for mutations in inherited sensorineuralhearing loss (Platzer et al. 2000).

Acknowledgements We thank Dr. Richard Altschuler from theKresge Hearing Research Institute, Ann Arbor Michigan, for hisassistance in the manuscript.

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