Expression of � and � Parvalbumin Is DifferentiallyRegulated in the Rat Organ of Corti duringDevelopment

D. Yang,1 I. Thalmann,2 R. Thalmann,2 D. D. Simmons1,2,3

1 Siebens Hearing Research Center, Central Institute for the Deaf, Washington University School ofMedicine, St. Louis, Missouri 63110

2 Department of Otolaryngology, Washington University School of Medicine, St. Louis, Missouri 63110

3 Department of Anatomy and Neurobiology, Washington University School of Medicine,St. Louis, Missouri 63110

Received 14 April 2003; accepted 16 July 2003

ABSTRACT: The expression of two calcium-bindingproteins of the parvalbumin (PV) family, the � isoform(�PV) and the � isoform known as oncomodulin (OM),was investigated in the rat cochlea during postnatal devel-opment and related to cholinergic efferent innervation.Using RT-PCR analysis, we found that OM expressionbegins between postnatal day 2 (P2) and P4, and peaks asearly as P10, while �PV mRNA begins expression beforebirth and remains highly expressed into the adult period.Both in situ hybridization and immunoreactivity confirmthat OM is uniquely expressed by the outer hair cells(OHCs) in the rat cochlea and occurs after efferent inner-vation along the cochlear spiral between P2 and P4. Incontrast to OM expression, �PV immunoreactivity is ex-pressed in both inner hair cells (IHCs) and OHCs at birth.Following olivocochlear efferent innervation, OHCs dem-

onstrate weak OM immunoreactivity beginning at P5 anddiminished �PV immunoreactivity after P10. In organcultures isolated prior to the efferent innervation of OHCs,OM immunoreactivity failed to develop in OHCs, but �PVimmunoreactivity remained present in both IHCs andOHCs. In contrast, organ cultures isolated after efferentinnervation of OHCs show OHCs with low levels of OMimmunoreactivity and high levels of �PV immunoreactiv-ity. This study suggests that OM and �PV are differentiallyregulated in OHCs during cochlear development. Ourfindings further raise the possibility that the expression ofPV proteins in OHCs may be influenced by efferent inner-vation. © 2003 Wiley Periodicals, Inc. J Neurobiol 58: 479–492, 2004

Keywords: oncomodulin; calcium-binding proteins; haircell; efferent innervation; auditory periphery

INTRODUCTION

A large number of calcium binding proteins of the EFhand family play a role in buffering Ca2� levels in

inner and outer hair cells (IHCs and OHCs) of themammalian organ of Corti (Pack and Slepecky, 1995;Imamura and Adams, 1996; Thalmann et al., 1997;Sakaguchi et al., 1998; Harter et al., 1999). Theseproteins are all characterized by a common sequenceof around 30 residues forming a helix-loop-helix mo-tif, which when bound to Ca2� causes a conforma-tional change in the protein that exposes a hydropho-bic surface, allowing for interaction with downstreamproteins (Lewit-Bentley and Rety, 2000). Calmodulin,calbindin, and calretinin are present in both IHCs andOHCs (Pack and Slepecky, 1995; Harter et al., 1999;

Correspondence to: D. D. Simmons (dsimmons@cid.wustl.edu).Contract grant sponsor: NIH; contract grant numbers: R01 DC

04086 (D.D.S.), K02 DC 00136 (D.D.S.), and R01 DC 001414 (I.T.).Contract grant sponsor: HHMI (D.Y.).

© 2003 Wiley Periodicals, Inc.

DOI 10.1002/neu.10289

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Sage et al., 2000), while �-parvalbumin (�PV) isbelieved to be only in IHCs (Celio, 1990; Pack andSlepecky, 1995; Soto-Prior et al., 1995). In OHCs,calmodulin is believed to form a heteromeric complexwith small conductance K� (SK) channels that openwith Ca2� influx (Kong et al., 2000; Oliver et al.,2000). While most calcium binding proteins are alsofound elsewhere in the central and peripheral nervoussystems, the � isoform of PV, known as oncomodulin(OM) because of its original discovery in a rat hepa-toma (MacManus, 1979), is found only in the OHCs(Thalmann et al., 1995; Henzl et al., 1997; Thalmannet al., 1997; Sakaguchi et al., 1998). Although little isknown about the precise function of OM, it is reason-able to infer that OM plays a role in the function ofCa2�-sensitive OHC motility because of its exclusiveexpression in OHCs (Sakaguchi et al., 1998).

The OHCs contribute to the exquisite sensitivity ofthe auditory system by serving as a feedback mechanismcapable of rapid motile responses that tune the signalreceived by the IHCs (Robles and Ruggero, 2001). Cho-linergic efferent axons from the superior olivary com-plex in the brainstem modulate OHC motility by inhib-iting the Ca2�-sensitive motility found in these cells(Liberman et al., 1996; Simmons, 2002). This cholin-ergic inhibition is mediated by nicotinic acetylcholinereceptors (nAChRs) that contain �9 and �10 subunits(Elgoyhen et al., 2001; Sgard et al., 2002). Activation ofthe OHC nAChR by efferent neurons induces Ca2�

influx, which is followed immediately by a larger K�

efflux through nearby SK channels (Housley and Ash-more, 1991; Fuchs and Murrow, 1992a, b; Evans, 1996;Nenov et al., 1996). Thus, the regulation of Ca2� ions iscritical to the function of OHCs.

Because OHC motility is modulated by efferentinnervation, the expression of OM may be related toefferent innervation. During the period of efferentsynaptogenesis, the number of nAChRs and Ca2�-dependent K� channels in OHCs dramatically in-creases (Dulon and Lenoir, 1996; He and Dallos,1999; Klocker et al., 2001). The expression of �10nAChR subunits and the onset of acetylcholine-in-duced responses in OHCs occur during efferent syn-aptogenesis (Dulon and Lenoir, 1996; Morley andSimmons, 2002). In gerbils, the development of ace-tylcholine-induced responses coincides with the timeperiod when OHCs also develop motility (He et al.,1994; He and Dallos, 1997, 1999).

However, most developmental studies concludethat OHC maturation is independent of either afferentor efferent innervation (Zuo et al., 1999; Jagger et al.,2000; Beurg et al., 2001; He et al., 2001). For in-stance, several studies have suggested that onset ofexpression of the �9 nAChR subunit does not require

efferent innervation (Simmons and Morley, 1998;Zuo et al., 1999; He et al., 2001). Studies of OHCsisolated at birth show that they not only developacetylcholine-induced responses but also develop mo-tile responses independent of any efferent innervation(He et al., 1994; He and Dallos, 1997, 1999; Beurg etal., 2001). It should be noted that these studies do notpreclude a role for efferents modulating the levels ofmolecules necessary for OHC function.

To date, the only cochlear studies of the PV pro-teins have been in older postnatal and adult animals(Pack and Slepecky, 1995; Sakaguchi et al., 1998).During development, it is not known whether OM and�PV follow the same expression patterns as found inthe adult cochlea. This study examines the expressionof OM and �PV during development. Additionally, anisolated organ preparation is used to assess the auton-omy of PV expression in hair cells.

EXPERIMENTAL METHODS

Animals

A total of 102 Sprague-Dawley rats from birth through adultwere used in this experiment. The day of birth (roughlyembryonic day 21) represents postnatal day 0 (P0). For insitu hybridization and immunocytochemical studies, ani-mals were anesthetized with 2 �L/g of sodium pentobarbitaland perfused with 4% paraformaldehyde in 0.1 M phos-phate-buffered saline (PBS; pH 7.4). Temporal bones weredissected and placed in 4% paraformaldehyde solution over-night. The next day, the cochleae were dissected and theround and oval windows exposed. For protein and mRNAtranscript studies, animals were decapitated and the cochleaeither frozen in liquid nitrogen or stored in RNALater(Ambion). For quantitative analysis of OM, the cochleaewere rapidly frozen, freeze-dried in toto, and the organ ofCorti was dissected at room temperature at a relative hu-midity of less than 40% and weighed on quartz fiber bal-ances. All procedures used for this study followed NIHguidelines and were approved by the institutional animalcare committees at the Central Institute for the Deaf andWashington University.

PCR/Gel Electrophoresis

Semiquantitative reverse transcription polymerase chain re-action (RT-PCR) was performed on mRNA isolated fromE18, P1, P4, P7, P10, P20, and adult rat cochleae. Thereverse transcription (RT) reaction was done using a SigmaRT Kit on 200 ng of total RNA from each of the ages.Primers were designed to amplify a 292-bp fragment of OMsequence (GenBank Accession number J02705) from 75–366 bp (forward: 5�-TGAGCATCACGGACATCCTG-3�,reverse: 5�-ATCTTCCCGTCTCCGTCGTT-3�), a 105-bp

480 Yang et al.

fragment of �PV sequence (GenBank Accession numberM12725) from 223–327 bp (forward: 5�-CTGGACAAA-GACAAAAGTGG-3�, reverse: 5�-CGTCTTTGTTTCCT-TAGCAG-3�), and a 452-bp fragment of glyceraldehyde-3-phosophate dehydrogenase (G3PDH; Clontech) from 586–1037 bp (forward: 5�-ACCACAGTCCATGCCATCAC-3�,reverse: 5�-TCCACCACCCTGTTGCTGTA-3�). Primerswere generated using Vector NTI software (InforMax). ForPCR, 20 �M of G3PDH and 50 �M of �PV primers werediluted 1:25, and 50 �M of OM primers were diluted 1:10.The JumpStart Taq kit (Sigma) was used for the PCR, whichran for 31 cycles in a thermal cycler (MJ Research). ThePCR product was size fractionated on a 5% polyacrylamidegel, which was stained with SYBR Gold (Molecular Probes)and then visualized using a Kodak digital imaging system.Quantity One software (Bio-Rad) was used to compare therelative intensities of the OM and �PV bands to that of theG3PDH band for each age. All PCR products were verifiedby sequencing. A control was done using RNA from brain,heart, and liver, and a control with no RT was also per-formed for all reactions.

In Situ Hybridization

Rat cochlear sections were taken from P2, P5, P10, P22, andadult rats. Cochleae from rats aged P5 and up were decal-cified in 10% EDTA. For cryostat sectioning, cochleae werecryoprotected using a sucrose gradient, ending with anovernight wash in 30% sucrose in PBS at 4°C. Then, co-chleae were infiltrated overnight in Tissue-Tek O.C.T.�embedding medium (Sakura Finetek, Inc.) and frozen byimmersing the cryostat mold in an ethanol/dry ice bath.Cryostat sections were cut at 14 �m and thaw mounted ontoSuperfrost Plus slides (VWR).

The slides were dried at 35°C for at least 30 min and thenstored at �80°C for later use. DIG-labeled RNA probes (bothsense and antisense) for OM were generated for the sameregion amplified by PCR. Sections were washed in xylene andrehydrated in a graded series of ethanols (100–50%). Follow-ing proteinase K digestion (5 min at 37°C), sections weredehydrated and hybridized overnight at 55°C with DIG-labeledprobes in a mixture of 50% formamide, 4X SSC, 10% dextransulfate, 1X Denhardts, and 50 �g/mL yeast t-RNA. Immuno-detection was performed with anti-DIG antibody conjugated toalkaline phosphatase (1:600 dilution) for 45 min at room tem-perature. NBT (4-nitro blue tetrazolium chloride; 0.338 �g/mL) and BCIP (5-bromo-4-chloro-3-indolyl phosphate; 0.175�g/mL) were used as enzyme substrates in 0.1 M Tris pH 9.5,2 mM levamisole, 0.1 M NaCl, 0.05 M MgCl2 and 0.1%Tween-20. Sense controls demonstrated no specific labeling.

Nondenaturing Isoelectric Focusing

Protein from rat organ of Corti at P2, P5, P10, P20, andadult was quantified using nondenaturing isoelectric focus-ing (Thalmann et al., 1998). The dry weight obtained fororgan of Corti from one rat ear was approximately 4.5 �g.Samples were pooled to place the signal into the linear

range of the OM standard curve. Isoelectric focusing wasperformed under nondenaturing (native) conditions usingthe Hoefer SE200 Mighty Small Mini-Vertical Unit [gelsize: 10 � 8 cm; gel thickness: 0.75 mm; gel composition:11% polyacrylamide, 1% piperazine di-acrylamide, 10%glycerol, ampholytes (1.66% of pI 3.5-10 and 0.33% of pI2.5-4)] with several modifications to the protocol describedin the Hoefer Scientific Instruments Protein ElectrophoresisApplication Guide (1994).

The gels were prefocused for 15 min at 5 W at 15°Cusing 0.02 M acetic acid and 0.02 M NaOH as anolyte andcatholyte, respectively. After the samples were loaded, thegels were run at 5 W until the dye front reached the bottom(approximately 1.5 h), and the protein was visualized withthe silver staining procedure described by Oakley et al.(1980). On each gel an OM dilution curve was included.

Gels were scanned on a Pharmacia LKB Image Masterone-dimensional flatbed scanning densitometer in transmit-ted light mode. The concentration of the OM band, identi-fied previously with antibodies and confirmed by proteinsequencing (Thalmann et al., 1995; Henzl et al., 1997), wasdetermined by integration and comparison with the OMstandard curve using software from Biosentient.

Cochlear Cultures

Neonatal rats were anesthetized by hypothermia and then de-capitated. Temporal bones were quickly removed and placedin Medium 199 supplemented with Hank’s salts (GIBCO)containing 0.39% amphotericin and 1.5% penicillin. The organof Corti was further isolated and put into 30 mm culture dishescoated with Cell Tak (BD Biosciences) containing 10% FBS,0.39% amphotericin, and 1.5% penicillin in Medium 199 sup-plemented Earl’s salts (GIBCO). Organs were incubated at37°C and 5% CO2. Culture media was changed every 48 h.Organs isolated from P0, P4, and P6 animals were maintainedin culture between 2–6 days in vitro (DIV). At the end of theculture period, the organs were immediately fixed using 4%paraformaldehyde solution for 30 min at room temperature andthen stored in TBS (Tris-buffered saline; pH 7.4) at 4°C untilfurther processing. Control organs (0 DIV) were isolated fromP8 and younger animals, placed into culture media for up to6 h, and then fixed.

Immunocytochemistry

Cryostat sections and organ of Corti cultures were reactedwith monoclonal antibodies to OM (1:25–1:200 dilution;courtesy of the Henzl lab, University of Missouri - Colum-bia) and �PV (1:1000 dilution; Sigma; clone PARV-19).Whole mounts of the organ cultures were also reacted withAlexa Fluor 647 phalloidin (1:100 dilution; MolecularProbes) and rabbit polyclonal antibodies against synapsin(1:500 dilution; Zymed) or the high affinity choline trans-porter (1:500 dilution; Chemicon). All antibodies were di-luted using 1% bovine serum albumin (BSA) and 1% TritonX-100 in TBS. Nonspecific labeling was blocked by a 1 hincubation at 4°C in TBS containing 10% BSA and 1%

Parvalbumins in the Rat Organ of Corti 481

Triton X-100. Tissues were incubated in primary antibodyovernight at 4°C, and then incubated in a cy-3 donkeyantimouse secondary antibody (Jackson ImmunoresearchLaboratories) at 1:200 for 2 h. In addition, an Alexa 488goat antirabbit secondary antibody (Molecular Probes) at1:500 was used with cultured organs. Sectioned materialwas coverslipped with Vectashield (Vector Laboratories,Inc.), while whole organs were mounted between coverslips with Glycergel (Dako).

Data Analysis

An epifluorescence microscope (Nikon) and a laser scan-ning confocal microscope (Bio-Rad) were used to visualizeimmunocytochemical labeling. In situ hybridization label-ing was visualized using Nomarski optics (40X objective,N.A. 0.8). Images were digitized, and for confocal stacks,three-dimensional projections of labeled structures weremade with Volocity software (Improvision). Using imageprocessing software such as Photoshop 7.01 (Adobe) andCanvas 8.01 (Deneba), images were digitally processed byadjusting contrast and brightness, and in some cases byusing sharpness and noise removal routines.

RESULTS

Development of �PV and OM TranscriptExpression

To identify the temporal expression patterns of OMand �PV during development, RT-PCR analysis was

performed on RNA isolated from cochlear tissues atembryonic, postnatal, and adult ages. Using primersspecific for OM and �PV, the transcripts identifiedhad 100% identity with the published rat sequences.As seen in Figure 1A, G3PDH transcripts werepresent across all ages, while OM transcript expres-sion did not appear until P4. The relative intensity ofOM transcript expression varied greatly across ages,reaching peak intensities at P10 and P20. By adultages, the relative level of OM transcript expressionhad decreased. The expression of �PV transcriptscontrasted with the expression of OM transcripts. The�PV transcripts appeared between E18 and P1, anddemonstrated consistent levels of expression acrosspostnatal and adult ages. In control tissues, theG3PDH transcripts were amplified in P1 and P5cDNA from the brain, heart, and liver, while OMtranscripts were not present (data not shown). The�PV fragment was not amplified in the heart or livercDNA, but did show very weak expression in thebrain.

Although previous reports of OM immunoreactiv-ity suggested that OM is restricted to OHCs in theadult cochlea, we investigated whether OM wasuniquely expressed by OHCs during development.The early postnatal expression of OM mRNA waslocalized with in situ hybridization on cochlear tissuesusing the transcripts generated by RT-PCR. As seenin Figure 2, digoxigenin-labeled transcripts were lo-

Figure 1 The expression of oncomodulin (OM) and �-parvalbumin (�PV) using RT-PCR fromRNA isolated from developing and adult tissues in the rat. Oncomodulin was not detected on apolyacrylamide gel at E18 or P1, but was detected beginning at P4 and through adult ages. Although�PV was not detected at E18, it was present at all other ages examined. As indicated, the top rowis the 452 bp G3PDH fragment, the middle row is the 292 bp OM fragment, and the bottom row isthe 105 bp �PV fragment.

482 Yang et al.

calized only over OHCs. At P2, OM mRNA waseither nonexistent or too weak to be detected. By P5,weak transcript labeling was observed in OHCs inbasal regions of the cochlea while sense controlsshowed no such labeling. At P10, OHCs hybridizedstrongly for OM mRNA in basal regions whereasOHCs hybridized more weakly for OM in more apicalregions longitudinally along the cochlear spiral. ByP22, strong OM transcript label was found throughoutthe cochlear spiral with no significant basal-apicalgradient. In the adult, OM hybridization was muchweaker than observed at either P10 or P22. Takentogether with the relative decrease of OM expressionfound with RT-PCR, the decreased intensity of OMhybridization may be due to a down-regulation in OMmRNA expression in adult tissues.

Protein Quantification

Results from our RT-PCR and in situ hybridizationexperiments suggest that OM mRNA expressionpeaks around P10 and P22 and may be significantlylower in adults. To test whether OM protein levelsfollow mRNA expression levels, protein levels weredetermined (Table 1). As expected, rats at P2 showedno OM protein. From P5 to P10, protein levels in-creased significantly. At P10, P20, and adult ages,OM protein levels were not significantly differentalthough protein levels in the adult did show a slightdecrease. The ages that had the highest levels ofprotein, P10 and P20 with 2.0 and 2.4 ng OM/�g dryweight respectively, decreased to 1.77 ng/�g in theadult. For comparison, adult protein levels of OM in

Figure 2 Photomicrographs of in situ hybridization on organ of Corti sections show OM transcriptlabeling of cochlear sections from P2 through adult ages. The scale bar represents 20 �m in allimages. (A) There is no visible transcript labeling anywhere within the organ of Corti. (B) Transcriptlabeling is found only in OHCs. (C) Higher relative levels of transcript labeling are seen in OHCsat P10. (D) OHCs continue to demonstrate dark transcript labeling at P22. (E) In the adult, therelative intensity of OM transcript labeling decreases.

Table 1 Levels of Oncomodulin Protein Detected at P5, P10, P20, and Adult Ages

Age of Animal

P2** P5* P10 P20 Adult

Protein (ng OM/�g dry wt) -- 0.5 � 0.07 2.0 � 0.71 2.4 � 0.96 1.8 � 0.12Sample number 2 2 5 9 3

Protein levels are in ng OM/�g dry weight. Using a one-way ANOVA, P5* is significantly different at the p � 0.01 level (indicated byasterisk). No protein was detected at P2**.

Parvalbumins in the Rat Organ of Corti 483

guinea pigs were 4.57 � 1.04 ng/�g (n � 8). Previ-ously published results (Thalmann et al., 1998) foundadult levels of 3.44 ng/�g in guinea pigs and 1.45ng/�g in rats. Western blotting (data not shown) alsoconfirmed these quantitative 1D gel results, whichwere generally consistent with our mRNA experi-ments.

Immunocytochemistry

Based on our transcript expression studies, OM and�PV have different developmental expression pat-terns. To localize protein expression patterns at thecellular level, antisera against OM and �PV wereused on cochlear sections. Additionally, to identifyputative efferent terminals, we used antisera againstsynapsin. At P2, P5, and P10, �PV immunoreactivitywas abundant in IHCs, OHCs, and spiral ganglionfibers and cell bodies. Within the organ of Corti at P2,IHCs, OHCs and Deiters’ cells labeled for �PV im-munoreactivity [Fig. 3(A)]. In all such cases, �PV-immunoreactive labeling was distributed evenlythroughout the cell, and there was no obvious basal-apical gradient. Also at P2, synapsin immunoreactiv-ity was mostly below the IHCs and just beginningbelow OHCs. At P5, �PV immunoreactivity was ex-tensive [Fig. 3(B)]. IHCs labeled as intensely asOHCs, and afferent fibers were also labeled. Intensesynapsin immunoreactivity was seen below both IHCsand OHCs at P5 and P10, consistent with previousreports of efferent terminal synaptogenesis in the co-chlea. By P10, �PV immunoreactivity began to di-minish in OHCs, but remained intense in the IHCs[Fig. 3(C)]. In the adult organ of Corti, �PV immu-noreactivity was restricted to IHCs as reported previ-ously (Pack and Slepecky, 1995; Soto-Prior et al.,1995).

OM immunoreactivity was not detected anywherewithin the organ of Corti at P2 [Fig. 3(D)], was barelyvisible in OHCs by P5 [Fig. 3(E)], and was intensewithin OHCs by P10 [Fig. 3(F)]. Unlike �PV, OMimmunoreactivity was always restricted to the OHCsand was never seen in the IHCs or any other cells,although nonspecific secondary labeling was observedat P5 in supporting cells and at later ages in the striavascularis. At P5, OM immunoreactivity was re-stricted to basal portions of the cochlear spiral. AtP10, OM immunoreactivity was found in both ex-treme apical and basal portions of the cochlear spiral,but a clear spiral gradient of OM immunoreactivityexisted. Immunoreactivity was strongest in midbasalregions of the cochlea, weaker at extreme basal re-gions, and weakest towards the extreme apical region.This OM immunoreactivity pattern is consistent with

the general pattern of cochlear maturation. By adultages, there was no obvious apical-basal gradient forOM immunoreactivity.

OM and �PV Expression in Culture

To test whether PV expression is independent of themedial olivocochlear efferent neurons, the organ ofCorti was isolated at P0, P4, and P6 and placed inculture for a maximum of 6 DIV. Organs isolatedfrom P8 or younger animals and placed into culturefor 0 DIV were used as control preparations. Ages P0,P4, and P6 were chosen because they span the periodwhen efferent innervation is exclusively below IHCsto being below both IHCs and OHCs. As a furthercontrol, organs from different ages were processed forimmunoreactivity simultaneously. Table 2 summa-rizes the results for �PV and OM immunoreactivity inculture. As shown in Figure 4, high levels of �PVimmunoreactivity were seen across all ages and cul-ture periods. As with noncultured, fixed tissue, �PVimmunoreactivity labeled IHCs (red) strongly. In P06-DIV organs, not only was the �PV immunoreactiv-ity in IHCs high, but also the immunoreactivity ofOHCs stained more intensely than noncultured fixedorgans [Fig. 4(A)]. During this same culture period,there were few, if any, spiral ganglion fibers labeled.When compared with control cultures at 0 DIV [Fig.4(B,D)], organs maintained in culture for 4 and 6 DIVhad higher �PV immunostaining intensities in theOHCs [e.g., Fig. 4(B,C)].

In contrast to �PV immunoreactivity in culture, themajority of organ of Corti cultures demonstrated OMimmunostaining only if they were isolated after P4(Table 2 and Fig. 5). Organ of Corti cultures isolatedat P0 (nine out of nine experiments) were negative forOM immunoreactivity at least through 6 DIV. Simi-larly, most organ of Corti cultures isolated at P4 (19out of 21) were negative for OM immunoreactivity atleast through 4 DIV; only two out of 21 cultures hadany OM immunostaining, which was extremely weak.In P4 cultures, OM was detected occasionally inblood tissue cells that sometimes lined the basal andlateral areas of the organ. Organs isolated at P6 dem-onstrated OM immunoreactivity as did organs isolatedfrom control P8 organs at 0 DIV. Figure 5 shows thelabeling of OM in P0 6-DIV, P4 2-DIV, P4 4-DIV,and P8 0-DIV organs. There is a lack of OM immu-noreactivity in all cases except the control P8 organ[Fig. 5(D)]. These data suggest that the onset of OMexpression, but not the maintenance of OM expres-sion, is altered in culture.

Because OHC efferent innervation begins betweenP2 and P4 in rats (Bruce et al., 2000; Bergeron et al.,

484 Yang et al.

Figure 3 The postnatal expression of �PV and OM immunoreactivity at three different ages isshown in the organ of Corti in these confocal renderings generated from stacks of individual opticalslices. In all cases, only midbasal regions of the organ of Corti are represented. The scale barrepresents 20 �m in each panel. (A) �PV immunoreactivity (red) is found in IHCs, OHCs, andDeiters’ cells (DCs). Efferent labeling as indicated by synapsin immunoreactivity (green) is foundmostly below IHCs although some weakly labeled terminals are present below OHCs. (B) �PVlabeling remains fairly consistent in the IHCs and OHCs, but has significantly decreased in Deiters’cells. Synapsin immunoreactivity is more intense below the OHCs. (C) �PV immunoreactivityremains robust in IHCs but has decreased in OHCs. (D) OM immunoreactivity (red) is not found inthe organ of Corti. Synapsin immunoreactivity (green) is found mostly below IHCs as in (A). (E)OM immunoreactivity is just detectable above background levels in OHCs and is not present inIHCs. Synapsin immunoreactivity is similar to (B). (F) OM immunoreactivity is intensely expressedin OHCs. [Color scheme can be viewed in the online issue, which is available at http://www.interscience.wiley.com]

Parvalbumins in the Rat Organ of Corti 485

2003), the failure of immature OHCs to express OMimmunoreactivity in culture could be due to the ab-sence of efferent synapses as one of several possibil-ities. To test this idea in culture, the presence ofsynaptic terminals was investigated using antiseraagainst synapsin, which is a synaptic terminal marker,and against the high-affinity choline transporter,which is specific for cholinergic axons and terminals(Che et al., 2002; Lips et al., 2002; Bergeron et al.,2003). At the start of the culture period, we confirmedthat organs isolated prior to P2 do not have OHCefferent terminals present while organs isolated afterP4 do have OHC efferent terminals. At P0 0-DIV,terminals beneath the IHCs were synapsin immuno-reactive as seen in noncultured organs [Fig. 4(A)].However, spiraling synapsin-positive fibers were seenin P0 4-DIV and 6-DIV cultures. Because these spi-raling fibers contacted IHCs and OHCs and weresimilar to afferent fibers also labeled by �PV, weconcluded that synapsin immunoreactivity labeled af-ferent fibers in cultured organs and thus could not bea reliable indicator of efferent terminals. On the otherhand, choline transporter immunoreactivity was neveridentified in spiraling processes. By P4, choline trans-porter immunoreactivity is observed only below IHCs[Fig. 4(B)]. In P4 2-DIV organ cultures, choline trans-porter immunoreactivity was virtually absent belowIHCs and was not detected after 4 DIV. Spiralingsynapsin-positive fibers were observed under all threerows of OHCs in the P4 2- and 4-DIV organ cultures.By P8, the choline transporter immunoreactivity wasbelow all three OHC rows, although the first row wasstill labeled somewhat more strongly than the othertwo rows [Figs. 4(D) and 5(D)]. Thus, on the basis of

choline transporter labeling, cholinergic efferent ter-minals are not present in organs removed before P4,and when present at isolation, they cannot be detectedafter 2 DIV.

DISCUSSION

Expression and Regulation of OM and�PV

This study is the first investigation of the expressionof PV isoforms, OM and �PV, during cochlear de-velopment. Our results indicate that OM mRNA ex-pression begins around P4, peaks between P7 andP22, and is only found in OHCs. These results areconsistent with previous studies on OM (Sakaguchi etal., 1998; Thalmann et al., 1998; Harter et al., 1999),which found OM only in OHCs within the organ ofCorti. Past investigations of �PV in the inner ear havefocused on late postnatal and adult animals and havereported that �PV is expressed only in IHCs (Packand Slepecky, 1995; Soto-Prior et al., 1995). Thisstudy is the first to observe �PV expression in OHCsas early as P1. In such cases of early �PV immuno-reactivity, IHCs label generally more intensely thanOHCs. It appears that immature OHCs initially ex-press �PV but not OM, and as OHCs mature, OMexpression is up-regulated while �PV expression isdown-regulated. This differential regulation of OMand �PV in IHCs and OHCs is further demonstratedin culture experiments where OM expression is atten-uated but �PV expression continues. After at least 4days in culture, �PV is not only expressed in IHCs but

Table 2 Immunostaining Summary from Culture Experiments Presented for OM and �PV

Age Label

Days In Vitro (DIV)

0 DIV 2 DIV 4 DIV 6 DIV

Negative Positive Negative Positive Negative Positive Negative Positive

P0 OM 2 0 -- -- 2 0 5 0�PV 0 3 -- -- 0 2 0 2

P4 OM 3 0 10 1 9 1 -- --�PV 0 4 0 6 0 6 -- --

P6 OM 2 3 2 3 -- -- -- --�PV 0 1 0 5 -- -- -- --

P8 OM 0 7 -- -- -- -- -- --�PV 0 5 -- -- -- -- -- --

-- Not attempted.The goal of these culture experiments was to assess whether IHCs and OHCs expressed either �PV or OM immunoreactivity if isolated

prior to or after the period of efferent innervation (P2–P4). After P8, cultures were susceptible to hypoxic trauma. Because maximal expressionalso occurred around P8, the total age (i.e., postnatal days � days in vitro) of the organ cultures was limited to 8 days. In all 0-DIV cultures,the organs were isolated and placed in culture media under the same conditions as longer-term cultures. These 0-DIV cultures were fixed within6 h of being placed into media.

486 Yang et al.

appears to be up-regulated in OHCs. This potentialup- regulation in OHCs is different from the normalpostnatal �PV expression in that the OHCs are la-beled as intensely as IHCs. Because the developmen-tal onset of OM expression is coincident with thearrival of cholinergic olivocochlear terminals and de-pressed in the isolated organ of Corti preparation, wehypothesize that efferent innervation may play a rolein the regulation of the parvalbumin proteins.

OHC Unique Proteins

In addition to OM, there are several other proteinsexpressed exclusively by OHCs within the cochlea.The most recently discovered protein is prestin,whose role as the membranous motor protein involvedin rapid electromotility and enhancing cochlear sen-sitivity has recently been elucidated (Lewit-Bentleyand Rety, 2000; Zheng et al., 2000). Prestin-inducedrapid electromotility occurs on the order of tens ofmilliseconds. Recent studies of prestin expressionduring development in the rat found that it is ex-pressed in the membrane of OHCs as early as P0, withthe biggest increase in expression occurring betweenP6 and P9, and reaching adult levels by P9 in the baseand P12 in the apex. Similar to OM expression, pres-tin is expressed exclusively by OHCs during devel-opment. However, unlike OM expression, OHCs arecapable of expressing prestin, albeit at reduced levels,under culture conditions (Beurg et al., 2001).

Prestin aside, other proteins unique to OHCs in-clude the �9/�10 nAChRs and rSK2, the type of SKchannel found primarily in the rat organ of Corti. Inrats, it has recently been shown that the mRNA for �9and �10 is present in OHCs at birth, and reaches peakexpression in OHCs around P10 (Simmons and Mor-ley, 1998; Morley and Simmons, 2002). In the adultrat, �9 levels are substantially reduced in IHCs while�10 levels are below detection in IHCs. Moreover, �9and �10 both show an apical-basal gradient in OHCswith higher expression in basal portions than in api-cal. The one study that has focused on rSK2 expres-sion in the cochlea during development found thatrSK2 in the organ of Corti was restricted to OHCs andwas first detected at P2 and reached peak expressionat P21 (Dulon et al., 1998).

OM and �PV Development in Culture

The differences in OM and �PV expression in cultureare consistent with the idea that some exogenousfactor(s) related to efferent innervation may suppress�PV and up-regulate OM expression in OHCs in vivo.There are several concerns that can be raised regard-

ing the possible effects of culture conditions on theexpression of �PV and OM in vitro. However, anumber of studies have shown that hair cells in theembryonic and postnatal organ of Corti develop nor-mally under culture conditions (Sobkowicz et al.,1982, 1984, 1993; Flock and Strelioff, 1984; Kelley etal., 1995; He and Dallos, 1999; Beurg et al., 2001; Heet al., 2001). First, studies of the cultured organ ofCorti from rat, mice, and gerbils have shown that theultrastructure of hair cells appears normal, indicatingthat hair cells develop morphologically in culture(Sobkowicz et al., 1984; He, 1997). Second, measure-ments of receptor and ionic conductances of culturedhair cells from mice and gerbils show they developwithout significant delay in culture as they normallydo in developing animals (He and Dallos, 1997;Rusch et al., 1998; He et al., 2001). Third, studies ofOHC-induced motility and electromotility show theyalso develop in culture similar to in vivo, suggestingthat culturing itself has little effect on the functionaldevelopment of hair cells (He and Dallos, 1999; He etal., 2001). These studies do not suggest that there isno influence of culture conditions on organ of Cortidevelopment; however, these influences seem to belimited to some delays in structural maturation (e.g.,opening of the tunnel of Corti) as well as increasingdisruption of the structural organization of the organof Corti after about 2 weeks in culture (Sobkowicz etal., 1993). Especially for studies of how efferent in-nervation may influence hair cell development, thereare virtually no alternative approaches currently avail-able other than to use organ culture methods. In fact,much of our knowledge about the influence of cho-linergic innervation at the neuromuscular junctioncomes from experiments performed in culture (Sanesand Lichtman, 2001).

Possibility of Neuronal Induction

Interestingly during development, the onset of OM,�9/�10 nicotinic receptors, and SK2 all seem to cor-respond with the arrival of the medial olivocochlearefferents underneath the OHCs, which occurs be-tween P2 and P4 in rats (Bruce et al., 2000), whilestudies done in gerbils and rats suggest that the onsetof acetylcholine-induced responses in OHCs beginson or after P6 and becomes functionally mature byP12 (Dulon and Lenoir, 1996; He and Dallos, 1999).Are the efferent neurons somehow inducing the ex-pression or up-regulation of these proteins that lead toOHC functional maturity? To date, there has beenlittle evidence of a direct influence of neuronal induc-tion. Several studies in gerbils of organ of Cortidevelopment in culture have found no evidence for

Parvalbumins in the Rat Organ of Corti 487

Figure 4

Figure 5

488 Yang et al.

efferent innervation affecting the development ofelectromotility (He and Dallos, 1997), acetylcholine-induced currents (He and Dallos, 1999), or nAChRs(He et al., 2001). However, these studies are based onthe assumption that efferent terminals are not presentin P0 gerbils when these organs are placed into cul-ture. But developmentally, P0 gerbils are roughlyequivalent to P3 or P4 rats (Simmons, 2002). Thus,efferent terminals may have already been underneaththe OHCs and may have triggered downstreamevents.

Recently, Beurg et al. (2001) reported that rapidelectromotility does develop in OHCs isolated from2-week-old cultures of P0–P1 organs of Corti fromthe rat. They suggest that this early postnatal devel-opment excludes the participation of neural innerva-tion in the postnatal maturation of the OHCs’ motileproperties. Other studies have also concluded thatelectromotility is an OHC intrinsic property that doesnot depend on the presence of the efferent system (He,1997). However, Beurg et al. (2001) also reported thatcultured OHCs presented a lower density of motorproteins (one-third) compared to freshly isolatedOHCs, suggesting that the expression level of themotor protein may very well be under the regulationof an extrinsic factor such as the cholinergic efferentinnervation.

Role of PVs in the Cochlea

�-PV is expressed in various tissues other than theinner ear, notably fast-twitch muscle fibers andGABAergic neurons, where it is associated with mod-

ulating the amplitude and duration of Ca2� transientsin metabolically active cells (Caillard et al., 2000;Chen et al., 2001; Vreugdenhil et al., 2003). Loadingof rat dorsal root ganglion neurons with �PV hasshown that this protein does not significantly alter thebasal cytosolic Ca2� concentration, when comparedto untreated control neurons (Pauls et al., 1996). How-ever, �PV not only reduces the increase in Ca2�

concentration during depolarization, but also signifi-cantly affects depolarization-induced changes in thecytosolic Ca2� concentration, such as its rate of rise,maximal peak, and rate of decay from its peak value.Therefore, �PV is capable of not only efficientlybuffering the rise of intracellular Ca2� concentrationsbut also modifying the cytosolic Ca2� transients,which may be relevant for regulating or modulatingCa2�-dependent cellular signaling (Pauls et al., 1996).In muscle, the theory that �PV functions as a relax-ation factor, by facilitating Ca2� transport from myo-fibrils to the sarcoplasmic reticulum, has gainedstrong support (Pauls et al., 1996). Although �PV hashigh affinity for Ca2�, it is unique among calciumbinding proteins found in hair cells in that it alsodisplays equal affinity to both Ca2� and Mg2�, andconsequently, binds Ca2� relatively slowly because itmust displace Mg2� before binding to Ca2� (Pauls etal., 1996). In vertebrate hair cells, �PV has beenassociated with adapting hair cells, suggesting that itis more likely to be involved with adaptation kineticsby keeping external Ca2� ions in the vicinity of thetransduction channels through which they enter (Bairdet al., 1997; Steyger et al., 1997). Unlike other cal-cium binding proteins such as calbindin-D28k, �PV

Figure 4 Images of organ of Corti cultures staining for �PV immunoreactivity (red), taken fromsingle confocal slices. The scale bars represent 25 �m in each panel. (A) �PV immunoreactivity isfound in IHCs and OHCs. Synapsin (green) immunoreactivity is found in radial fibers contactingIHCs and spiraling fibers contacting OHCs. (B) �PV immunoreactivity is found intensely in IHCsand weakly in OHCs. Terminals demonstrating choline transporter (ChT1) immunoreactivity(green) are shown contacting only IHCS. (C) IHCs and OHCs demonstrate intense �PV immuno-reactivity. Synapsin immunoreactivity is found in spiraling fibers that contact both IHCs and OHCs.(D) Both IHCs and OHCs demonstrate �PV immunoreactivity, but it is more intense in the IHCs.Terminals showing ChT1 immunoreactivity are present below IHCs and OHCs.

Figure 5 All images show immunostaining results in organs of Corti for OM (red) and were takenfrom single confocal slices. The scale bars represent 25 �m in all cases. (A) At P0-6 DIV, hair cellsare labeled by phalloidin (blue), however, OM immunoreactivity is not detected in the organ. (B)At P4-2 DIV, hair cells are labeled by phalloidin. Neither OM nor choline transporter (ChT1)immunoreactivity (green) is present in or below the hair cells. However, a blood tissue-type cell(macrophage-like) is also labeled red (asterisk). (C) At P4-4 DIV, hair cells are labeled by phalloidinand OM immunoreactivity is absent in OHCs. Spiraling synapsin-immunoreactive fibers (green)contact OHCs. (D) In the P8-0 DIV organ, OM immunoreactivity is present in OHCs and absent inIHCs. Choline transporter (ChT1) immunoreactivity (green) is also present below IHCs and OHCs.

Parvalbumins in the Rat Organ of Corti 489

in the adult mammalian cochlea is uniquely associatedwith IHCs and not found in OHCs and has a tonotopicgradient, with IHCs at the apex (low frequencies)staining intensely and IHCs at the base (higher fre-quencies) staining weakly (Pack and Slepecky, 1995).

OM is expressed in only a few places outside of theinner ear and is typically associated with tumors orembryonic and placental tissues. In tumor cells, therole of OM is unclear, but it may play a role in cellcycling, which is greatly influenced by Ca2� (Pauls etal., 1996). Calmodulin is important for G2-to-M cellcycle progression, and OM levels have been shown toincrease—just as calmodulin levels do—at the G1-to-S boundary (Blum and Berchtold, 1994). In normaltissue, OM has been found in the embryo duringimplantation (Brewer et al., 1989) and in the cytotro-phoblasts of the fetal placenta (Brewer and MacMa-nus, 1987). Our results suggest that macrophages orother blood tissue cells may also be capable of OMimmunoreactivity at least under culture conditions.Although the physiologic function of OM is still un-known in these systems, it has been speculated thatOM could play a role in the Ca2�-dependent motilityneeded for the invasive action of the cytotrophoblasts(Pauls et al., 1996).

Although �PV and OM share roughly 49% se-quence identity, they display large differences inCa2� ion binding properties and affinities (Pauls et al.,1996; Henzl et al., 2003). Whereas the two Ca2�

binding sites in �PV are nearly indistinguishable,high affinity sites, the two sites in OM are distinctlynonequivalent: one is a typical high affinity site whilethe other exhibits a lower affinity, but greater Ca2�

selectivity (Hackney et al., 2003). Because OM hassome calmodulin-like activities, it may be part ofsignal transduction pathways similar to those foundfor calmodulin, or another possibility is that OMsomehow counteracts the actions of calmodulin in anantagonistic fashion (Pauls et al., 1996). In OHCs, itappears that OM is more likely to be related to theefferent system than to the electromotility system,because the former is Ca2� and calmodulin dependentand OM could presumably work as a specialized Ca2�

buffering system suited to the unique action of theOHCs. This suggestion is especially intriguing be-cause the nAChR-SK linkage is thought to involvecalmodulin (Kong et al., 2000). However, this doesnot preclude the efferent system somehow influencingthe organization or expression of electromotility. Itseems significant that �PV, the main Ca2� buffer inadult mammalian IHCs, is expressed early in OHCsbut is then replaced by its � isoform when OHCsbecome functionally active. Thus, perhaps OM func-tions in a manner more suited to the unique function

of the OHCs after efferent innervation. One uniqueaspect of OHCs is the fast and slow inhibitory re-sponses that are both modulated by Ca2� stores asso-ciated with the subsynaptic cisternae. It is possiblethat slower, high-affinity Ca2� buffering in immatureOHCs is done by �PV interacting with the subsynap-tic cisternae. In this way, immature OHCs may func-tion like muscle where �PV is highly concentrated inmuscle sarcoplasm and may interact with the sarco-plasmic reticulum to facilitate Ca2� transfer (Pauls etal., 1996). However, whatever the function of �PV isin immature OHCs, with the arrival of efferent axonsand activation of the nAChR-SK linkage, the up-regulation of OM then replaces �PV.

Although IHCs are initially contacted by efferentaxons (reviewed by Simmons, 2002), neither in cul-ture nor in vivo is OM ever expressed in IHCs, con-trary to a recent report of PV-3 localization in turtlehair cells and guinea pig IHCs (Hackney et al., 2003).The fact that �PV levels appear to increase in OHCsin organ culture compared to in vivo further indicatesthat the arrival of efferents might normally, via eitherdirect or indirect means, down-regulate �PV expres-sion. In cultured organs isolated prior to efferent ar-rival, it may be that �PV expression does not diminishand thus results in compromises to OHC function.The diminished responses of cultured OHCs isolatedat birth would be consistent with such an interpreta-tion (Beurg et al., 2001). Further experiments arenecessary to clarify the roles played by �PV and byOM.

The authors wish to thank Adam L. Bergeron for sug-gesting that we use the choline transporter antisera andSteve L. Gum for his help with RT-PCR and culture exper-iments. We are indebted to Dr. Elena Ignatova for herextraordinary assistance with in situ hybridization. We alsothank Dr. Michael Henzl for his helpful comments. Thiswork was supported with an HHMI undergraduate researchprogram grant (D.Y.).

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