Synaptophysin and insulin-like growth factor-1 immunostaining in the central nucleus of the inferior colliculus in adult ferrets following unilateral cochlear removal: A densitometric

Synaptophysin and Insulin-Like GrowthFactor-1 Immunostaining in the Central

Nucleus of the Inferior Colliculus in AdultFerrets Following Unilateral CochlearRemoval: A Densitometric Analysis

JUAN CARLOS ALVARADO,* VERONICA FUENTES-SANTAMARIA, SAMUEL R. FRANKLIN,JUDY K. BRUNSO-BECHTOLD, AND CRAIG K. HENKEL

Department of Neurobiology and Anatomy, Wake Forest University School of Medicine,Winston-Salem, North Carolina

KEY WORDS synapses; IGF-1; hearing loss; quantitative image analysis; plastic-ity; adulthood

ABSTRACT In the present study, unilateral cochlear ablations were performed inadult ferrets to evaluate possible time-dependent modifications of synaptophysin andinsulin-like growth factor-1 (IGF-1) in the central nucleus of the inferior colliculus(CNIC). Using densitometric analysis, synaptophysin and IGF-1 immunostaining wereassessed at 1 (PA1) and 90 (PA90) days after cochlear ablation. The results demon-strated that 1 day after the lesion there was an increase in the levels of synaptophysinimmunostaining bilaterally in the CNIC compared to control animals. That increasewas no longer present at 90 days after the ablation. Overall levels of IGF-1 immuno-staining at PA1 were increased significantly within neurons and neuropil. However, atPA90, only IGF-1 immunostaining contralateral to the lesion was elevated comparedto control animals, although elevation was less than that observed at PA1. Theseresults suggest that cochlear ablation appears to affect synaptophysin and IGF-1 pro-tein levels bilaterally in the CNIC. Synapse 61:288–302, 2007. VVC 2007 Wiley-Liss, Inc.

INTRODUCTION

Asymmetric hearing loss has been reported to resultin a broad range of plastic changes along the auditorypathway including trans-synaptic degeneration, pro-tein synthesis, synaptic rearrangements, axonal prun-ing due to fiber degeneration, and sprouting (Alvaradoet al., 2005; Hashisaki and Rubel, 1989; Hyson andRubel, 1989; Illing et al., 1997, 2000; Illing, 2001; Mor-est et al., 1997; Potashner et al., 1997; Syka, 2002;Tierney et al., 1997). The magnitude of these changesin the different auditory nuclei varies depending onthe age of the animal when hearing loss occurs (Changand Merzenich, 2003; Hardie et al., 1998; Illing andHorvath, 1995; Illing et al., 1997; Moore, 1990; Mooreand Kitzes, 1985; Moore and Kowalchuk, 1988; Mosta-fapour et al., 2000, 2002; Rubel and Fritzsch, 2002).Specifically, in adult animals, in the inferior colliculus(IC), the major processing site for nearly all monauraland binaural auditory brainstem pathways (Drugaand Syka, 1984; Majorossy and Kiss, 1994; Moore,1988; Nordeen et al., 1983; Oliver et al., 1997), plasticchanges following unilateral cochlear ablation have

been shown to take place to adapt to perturbations ofhearing that could affect binaural processing. Accord-ingly, loss of cochlear integrity induces in the contra-lateral central nucleus of the inferior colliculus (CNIC)in adult ferrets (McAlpine et al., 1997) and gerbils(Mossop et al., 2000), a rapid and significant increasein the number of neurons that are stimulated from theintact ear. Moreover, an upregulation in the calretininimmunostained plexus in the contralateral CNICappears at 24 h and remains at 90 days after the lesionin adult ferrets following unilateral cochlear ablation(Alvarado et al., 2005).

Growth factors are signaling proteins that may regu-late activity-related plasticity in adult animals by

Contract grant sponsor: National Institute of Health; Contract grant num-ber: DC00813.

*Correspondence to: Juan Carlos Alvarado, Department of Neurobiology andAnatomy, Wake Forest University School of Medicine, Winston-Salem, NorthCarolina. E-mail: [email protected]

J.C.A. and V.F.-S. contributed equally to this work.

Received 14 August 2006; Accepted 5 December 2006

DOI 10.1002/syn.20373

Published online in Wiley InterScience (www.interscience.wiley.com).

VVC 2007 WILEY-LISS, INC.

SYNAPSE 61:288–302 (2007)

translating modifications in neuronal activity intophysiological, biochemical, and morphological changes(Bramham and Messaoudi, 2005; Dore et al., 1997;Russo et al., 2005; for review). In particular, insulin-like growth factor-1 (IGF-1) is a polypeptide hormonewith pleiotropic systemic effects. In the nervous sys-tem, IGF-1 has been implicated in neuroprotection,promoting neuronal survival after injury (Torres-Aleman, 1999) as well as in synaptic plasticity (Aberget al., 2006; Russo et al., 2005; Torres-Aleman, 1999,2005), modulating processes of synaptic efficacy by reg-ulating neurotransmitter release, neuronal excitabil-ity, and synapse formation (Aberg et al., 2006; Niblocket al., 2000; O’Kusky et al., 2000; Russo et al., 2005;Shi et al., 2005; Torres-Aleman, 1999, 2005). Consider-ing the broad range of changes reported in the CNICafter unilateral hearing loss (Illing et al., 1997, 2000;Illing, 2001; Syka, 2002; for review), it might be ex-pected that changes in synaptic proteins may occur inadult animals and that those changes could be accom-panied by alterations in IGF-1. To test this hypothesis,in the present study, we used immunohistochemicalmethods and densitometric analysis to evaluate possi-ble modifications in synaptophysin and IGF-1 immuno-staining in the CNIC in adult ferrets after unilateralcochlear ablation.

EXPERIMENTAL PROCEDURESAnimal subjects

All animal protocols were approved by the AnimalCare and Use Committee of Wake Forest UniversitySchool of Medicine and conformed to the National Insti-tutes of Health standards. Data were obtained from 9adult, 6 experimental, and 3 unlesioned control ferrets.After the cochlear ablation, the experimental animalssurvived 1 (PA1, n ¼ 3) and 90 (PA90, n ¼ 3) days.

Unilateral cochlear ablation

Cochlear surgery was performed as described previ-ously (Fuentes-Santamaria et al., 2003). Briefly, experi-mental ferrets were anesthetized i.m. with a mixtureof ketamine (30 mg/kg) and xylazine (4 mg/kg) andredosed, as needed, during the procedure. A postauricu-lar incision was made under aseptic conditions. Aftersoft tissue dissection, the external auditory canal wasidentified and followed to the tympanic membrane. Thecochlea then was exposed through the right bulla andremoved with a forceps. Any remaining cochlear con-

tents were aspirated using a Pasteur pipette, and theincision was sutured. A heating pad was used to main-tain body temperature during the surgery and recoveryfrom anesthesia. Following surgery, the animalsregained consciousness under supervision at which timethey were returned to their cages and maintained withfree access to food and water for the appropriate sur-vival time. The extent of the cochlear ablation wasassessed by microscopic inspection of the dissected bullaafter the sacrifice and perfusion of the animal.

Immunohistochemistry forsynaptophysin and IGF-1

Control and experimental animals (after each postop-erative survival time), were anesthetized deeply with anoverdose of ketamine (50 mg/kg) and xylazine (5 mg/kg)delivered i.m. The thorax was opened and a fixative of4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 Mphosphate buffer (pH 7.4) was perfused through theheart using a peristaltic pump at a rate of 25 ml/min.The brains were removed and immersed in the same fix-ative for 3 h at room temperature followed by immersionin 30% sucrose at 48C overnight for cryoprotection. Coro-nal sections of 50 mm were cut frozen on a sliding micro-tome, placed in 0.1 M phosphate buffer (pH 7.4), andprocessed in three adjacent series. This section thick-ness was used based on pilot experiments demonstrat-ing that 50 mm sections were optimal in thicknessbecause they could be handled readily without tearingand careful analysis revealed complete antibody pene-tration through the thickness of the sections. The firstseries of sections was stained with cresyl violet. The twoother series of free-floating sections were incubatedovernight at 48C in primary antibody, either to synapto-physin (1:2000, mouse antisynaptophysin monoclonalantibody, MAB5258, Chemicon International, TemeculaCA) or to IGF-1 (1:800, rabbit anti-IGF-1 monoclonalantibody, PAB-Ca, GroPep, Adelaide, Australia) dilutedin a solution consisting of Tris-buffered saline (TBS)with 0.2% Tritron X-100 (TBS-Tx). The tissue then waswashed and incubated in a 1:200 dilution of biotinylatedanti-mouse or anti-rabbit secondary antibody, respec-tively (Vector Laboratories, Burlingame, CA) for 2 h atroom temperature. The Vector biotin-avidin procedure(Hsu et al., 1981) was used to link the antigen–antibodycomplex to HRP that then was visualized with diamino-benzidine histochemistry. Sections were washed thor-oughly, mounted on gelatin-coated slides, air-dried,dehydrated in ethanol, cleared in xylene, and cover-slipped with cytoseal (Stephens Scientific, Camden, NJ).

Immunohistochemical controlsof the antibodies specificity

Control experiments were carried out by incubatingsections in the absence of either primary antibodies orsecondary antibodies. No immunostaining was detected

Abbreviations

CNIC central nucleus of the inferior colliculusDCIC dorsal cortex of the inferior colliculusECIC external cortex of the inferior colliculusHRP horseradish peroxidaseIC inferior colliculusIGF-1 insulin-like growth factor-1PA1 one day survival time after cochlear ablationPA90 ninety days survival time after cochlear ablation

289SYNAPTOPHYSIN AND IGF-1 IN ADULT FERRET IC

Synapse DOI 10.1002/syn

under these conditions. The specificity of the synapto-physin antibody was established previously in our labusing Western blot analysis (Fuentes-Santamaria et al.,2005). To establish specificity of the IGF-1, the antibodywas preabsorbed at room temperature for 2 h with 5, 10,20, and 30 fold excess of LongTMR3IGF-1 (the immuno-gen used to produce the antibody) prior to application tothe sections. As shown in Figure 1, a 30-fold excess ofthe LongTMR3IGF-1 completely abolished the IGF-1 im-munostaining in the sections. In addition, the specificityof the antibodies used in the present study has been pre-viously described by other authors (Csaba et al., 2002;Degger et al., 2000; IGF-1: Hill et al., 1999; synaptophy-sin: Kawai and Senba, 2000; Kawasaki et al., 2003;Khan et al., 2003; Masliah et al., 2001).

Analysis of the immunostained sections

The IC anatomical subdivisions were defined accord-ing to previous studies in ferrets (Moore et al., 1983;

Moore, 1988). The immunostained sections were exam-

ined with brightfield illumination using a Nikon Opti-

phot research photomicroscope and the images were

captured with a Spot CCD color video camera, model RT

Slider (Diagnostic Instruments Inc.) attached to the

microscope. To avoid differences in light intensity of the

captured images, all images were digitized at the same

light intensity in the microscope (9 on the microscope

scale). A color image was digitized and the red channel

was used to obtain an image containing a grayscale of

pixel intensities from 0 (white) to 255 (black).The densitometric analysis could be affected by

slight variations present in the captured images due tothe section thickness, nonuniformities in the lightsource, and irregularities in the microscope optics(Caicedo et al., 1997; Lohrke and Friauf, 2002; Mize,1985; Riquelme et al., 2001; Russ, 1990). To correct forthese possible sources of errors, a normalization pro-cess was used. To accomplish unbiased normalization

Fig. 1. High magnification digital images showing the specificity of IGF-1 in the CNIC in controlferrets (see Experimental Procedures). The antibody was preabsorbed with 5 (A), 10 (B), 20 (C), and 30(D) fold excess of the competitive control (human LongTMR3IGF-1). Notice that the IGF-1 immunostain-ing in the sections was abolished completely after adding a 30-fold excess (D). Scale bar ¼ 100 lm.

290 J.C. ALVARADO ET AL.


of the images, an algorithm based on the signal-to-noise ratio principle (Herborn et al., 2002) was appliedusing the Scion Image software (Scion Corp, versionbeta 4.0.2). According to this principle, the ability toresolve individual structures in an image depends onthe relationship between the gray level intensity of thestructures (signals), and the mean overall intensity ofthe image (noise). Thus, structures with signals thatare clearly different compared to the image noise canbe resolved more readily than those structures withsignals similar to the intensity level of the image noise.The algorithm uses the signal intensity of the regionof interest (ROI), the noise level of the overall image,and the cross-sectional area of the ROI to correct grayvalues. Consideration of each of these features is cri-tical for the unbiased analysis of a given image (Brightet al., 1998). The algorithm normalizes each pixel(Mize, 1985), adjusting the grayscale range of the image(Alvarado et al., 2004). This procedure enhanced theimage gray level, allowing an adequate comparisonand better visualization of the labeling in the IC(Fuentes-Santamaria et al., 2003; Mize et al., 1988).Immunostained profiles were defined as profiles withmean gray level above the threshold for detection thatwas set as two standard deviations above the mean op-tical density of the whole image (Alvarado et al., 2004,2005; Fuentes-Santamaria et al., 2003, 2005). Ineach one of the 9 ferrets, the densitometric analysis foreach antibody was performed in three coronal sectionsevenly spaced along the rostrocaudal axis of the CNIC.For each side, data from all fields in each animal werepooled.

Synaptophysin was analyzed as described previously

(Alvarado et al., 2004; Fuentes-Santamaria et al.,

2003, 2005). The synaptophysin immunostained sec-

tions were digitized using a 40� objective. Three sam-

pling fields (6.39 � 104 mm2 each) were defined across

the tonotopic axis of the IC including the areas of low,

intermediate, and high frequencies (Fig. 2). Data from

all fields in each animal were pooled. Given that the

immunostaining can be used as a relative measure of

antigen concentration (Huang et al., 1996; Lin and Tal-

man, 2000; Yao and Godfrey, 1997), the mean gray

level of the immunopositive profiles within CNIC was

used as an indirect indicator of the amount of synapto-

physin. The immunostained area, used to estimate the

area occupied by synaptic endings, was calculated as

the sum of the areas of all immunopositive pixels in

the field (Alvarado et al., 2004; Benson et al., 1997;

Fuentes-Santamaria et al., 2003, 2005). To measure

the immunostained area in each field, only immuno-

positive profiles between 0.47 and 85 mm2 were

counted in this analysis, including both small puncta

and perisomatic profiles (Alvarado et al., 2004;

Fuentes-Santamaria et al., 2003, 2005). Thus, in syn-

aptophysin immunostained sections, two indices were

measured: (1) the mean gray level within the CNICand (2) the immunostained area.

For the densitometric analysis of IGF-1, the immu-nostained sections were digitized using a 20� objec-tive. Similar to synaptophysin, three sampling fields(25.57 � 104 mm2 each) were defined across the tono-topic axis of the IC (Fig. 2), and data from all fields ineach animal were pooled. The mean gray level of theCNIC was used as an indirect indicator of the amountof the IGF-1 and provides a general estimation regard-ing the effect of unilateral cochlear ablation on theIGF-1 immunostaining throughout the nucleus. Sinceany changes in the overall levels of immunostainingcould be due to either changes in immunostained neu-ropil or neurons, both the mean gray level within neu-rons and the mean gray level within neuropil weremeasured (Alvarado et al., 2004; Fuentes-Santamariaet al., 2005). Thus, in IGF-1 immunostained sections,three indices were measured: (1) the mean gray levelwithin the CNIC, (2) the mean gray level within neu-rons, and (3) the mean gray level within neuropil.

Image processing

Adobe Photoshop (version 5.5) and Canvas (version6.0) were used to adjust size and brightness in thepreparation of the figures. Other than the normaliza-tion procedure described earlier, no manipulations ofthe images were performed.

Statistical analysis

All the data were expressed as means 6 standarddeviations (SD). Data were analyzed statistically usinga two-way analysis of variance (ANOVA): group (con-

Fig. 2. Nissl-stained section showing the selected fields for thequantification of synaptophysin and IGF-1 immunostaining withinthe CNIC. Fields (1, dorsolateral; 2, central; and 3, ventromedial)are indicated by white squares. Scale bar ¼ 1 mm.



trol, PA1 and PA90) and side (left and right). Duncan’sposthoc analysis was used to evaluate the effect of theablation among groups. Statistical significance wasdetermined at a level of P < 0.05.

RESULTSSynaptophysin immunostaining in the

CNIC in control ferrets

In adult animals, as in young ferrets (Fuentes-Santamaria et al., 2003), the synaptophysin immuno-staining in the CNIC appeared predominantly as punc-tate deposits in the neuropil (arrowheads in Figs. 3Aand 3B) and also as profiles surrounding immunonega-tive somata (arrows in Figs. 3A and 3B). Cortical areasof the IC (dorsal cortex, DCIC, and external cortex,ECIC, in Fig. 2) are not considered here. Densitometricanalysis revealed that the mean gray level of synapto-physin immunostaining was 194.64 6 2.17 in the leftand 193.75 6 2.68 in the right CNIC (Fig. 4A), andthe immunostained area was 1976.98 6 29.85 and1989.32 6 16.73 mm2 in the left and right, respectively(Fig. 4B).

Synaptophysin immunostaining in theCNIC in ablated ferrets

Qualitatively, in adult animals with unilateral coch-lear ablation, synaptophysin immunostaining appearedsimilar to that in control animals. The immunostainedregion of the CNIC comprised mainly punctate depos-its in the neuropil (arrowheads in Figs. 3C and 3D forPA1, and in Figs. 3E and 3F for PA90) and perisomaticprofiles surrounding immunonegative somata (arrowsin Figs. 3C and 3D for PA1 and in Figs. 3E and 3F forPA90) at the two survival times.

Quantitatively, analysis of synaptophysin immuno-staining at PA1 revealed that the mean gray level was193.68 6 2.00 contralaterally (left side) and 194.60 62.57 ipsilaterally (right side) (Fig. 4A), and the immu-nostained area was 2326.08 6 19.97 and 2244.47 645.46 mm2 for the contralateral and ipsilateral sides,respectively (Fig. 4B). Densitometric analysis of synap-tophysin immunostaining at PA90 demonstrated thatthe mean gray level of the CNIC was 197.57 6 2.79contralaterally and 199.25 6 2.61 ipsilaterally (Fig.4A), and the immunostained area was 2040.52 6 30.64mm2 contralaterally and 2067.88 6 57.66 mm2 ipsilat-erally (Fig. 4B). When control and ablated ferrets werecompared, ANOVA analysis revealed no significanteffect of the cochlear ablation at any survival time onthe mean gray level of synaptophysin immunostainingin either left or right sides (Fig. 4A). In contrast, therewas a main effect of the cochlear ablation on the syn-aptophysin immunostained area, F(2,48) ¼ 23.24, P <0.0001. Further evaluation with Duncan’s posthocanalysis demonstrated that at 1-day of survival timethere was a small but significant increase in the immu-

nostained area on both the left (contralateral to thecochlear ablation) and right sides (ipsilateral to thecochlear ablation) compared to that in control (P <0.0001) and 90-day survival time ferrets (P < 0.001)(Fig. 4B). No significant differences were found be-tween control and PA90 animals in the synaptophysinimmunostained area in either left or right sides(Fig. 4B).

IGF-1 immunostaining in the CNICin control ferrets

Analysis of sections immunostained for IGF-1 in con-trol cases, revealed lightly immunostained neuropil aswell as neurons that in some cases were darkly immu-nostained throughout the IC (Figs. 5A– 5F). Similar tosynaptophysin immunostaining, regions of the IC out-side the CNIC (DCIC and ECIC in Fig. 5A) will not beconsidered here. In the CNIC, immunostained neuronswere distributed throughout the nucleus (Figs. 5C and5D). The immunostaining within these neurons waslocated mainly in the cytoplasm as well as in someproximal ends of the primary dendritic processes (Figs.5E and 5F). However, no IGF-1 immunostaining wasobserved within the nucleus (Figs. 5E and 5F). Densi-tometric analysis demonstrated that the mean graylevel within the CNIC was 84.23 6 2.36 on the left and86.71 6 1.78 on the right. The mean gray level withinneurons was 148.77 6 4.39 and 148.71 6 1.71 in theleft and in the right CNIC, respectively. In addition,the mean gray level in the immunostained neuropilwas 76.25 6 1.57 in the left and 77.35 6 4.01 in theright CNIC, respectively.

IGF-1 immunostaining in the CNICin ablated ferrets

In ablated animals, the general pattern of distribu-tion of IGF-1 immunostaining at PA1 (Fig. 6) andPA90 (Fig. 7) was similar to that in control ferrets(Fig. 5). In both experimental groups, most CNIC neu-rons were darkly immunostained and distributedthroughout of the nucleus. Conversely, the neuropilwas lightly immunostained (Figs. 6C and 6D for PA1,7C and 7D for PA90). In addition, as shown in Figures6E and 6F for PA1 and in Figures 7E and 7F for PA90,the IGF-1 immunostaining was darkest in the somata,but in some cases it also extended into a few proximaldendritic processes. However, 1 day after the cochlearablation the immunostained somata in the CNIC onboth sides appeared to be somewhat darker than incontrol and PA90 animals (compare Figs. 6C and 6Dwith Figs. 5C and 5D and Figs. 7C and 7D).

Densitometric analysis of the experimental animalsshowed that at PA1 the mean gray level within theCNIC was 100.73 6 4.93, on the left, contralateral tothe ablation and 99.45 6 2.11 on the right, ipsilateralto the ablation. The mean gray level within neurons



was 165.63 6 2.88 in the contralateral and 156.21 62.36 in the ipsilateral CNIC, and the mean gray levelin the neuropil was 94.43 6 4.21 and 95.11 6 3.40 in

the contralateral and ipsilateral CNIC, respectively(Fig. 8). At 90 days after the unilateral cochlear abla-tion, the mean gray level within the nucleus was 89.12

Fig. 3. High magnification digital images illustrating the pattern of distribution of synaptophysinimmunostaining in the CNIC in control (A,B) and 1 day (C,D) and 90 day (E,F) survival time ferrets.Punctate immunostaining was abundant in the neuropil (arrowheads) and present, but less common,around the cell bodies (arrows). Scale bar ¼ 25 lm.



6 2.36 contralaterally and 87.72 6 5.22 ipsilaterally.The mean gray level within neurons was 153.95 63.90 in the contralateral and 152.83 6 3.39 in the ipsi-lateral CNIC, and the mean gray level in the neuropilwas 83.92 6 2.11 in the contralateral and 88.89 6 3.38in the ipsilateral CNIC (Fig. 8).

ANOVA analysis demonstrated a small but signifi-cant effect of the survival time for each of the three de-pendent variables, F(2,48) ¼ 37.12, P < 0.00,001, forthe mean gray level within CNIC; F(2,48) ¼ 18.67, P <0.00,001, for the mean gray level within neurons; andF(2,48) ¼ 47.11, P < 0.00,001, for the mean gray levelin the neuropil. The Duncan’s posthoc analysis indi-cated that 1 day after cochlear ablation the mean graylevel within both CNIC were higher than that in theleft (P < 0.0001) and the right (P < 0.0001) sides incontrol animals and also higher than the contralateral(P < 0.001) and ipsilateral (P < 0.001) sides at PA90ferrets (Fig. 8A). The mean gray level within neuronsin the contralateral CNIC at PA1 was higher than that

in the ipsilateral CNIC (P < 0.05). In addition, themean gray level within neurons at PA1 was higherthan that in control ferrets in either left (P < 0.0001)and right (P < 0.05) sides and also higher than thatcontralaterally (P < 0.001) but not ipsilaterally inPA90 ferrets (Fig. 8B). The mean gray level in the neu-ropil at PA1 also was increased compared to that in ei-ther the left (P < 0.0001) and the right (P < 0.0001)sides in control ferrets or the contralateral (P < 0.001)and ipsilateral (P < 0.0001) sides at PA90 animals(Fig. 8C). Finally, after 90 days of survival time onlythe mean gray level in the neuropil contralateral tothe lesion was higher than that in the left side in con-trol ferrets (Fig. 8C) (P < 0.05).

DISCUSSION

In the present study, using densitometric analysis,the modifications in synaptophysin and IGF-1 immu-nostaining in the CNIC were evaluated in adult ferretsat 1 and 90 days after unilateral cochlear ablation. Thepresent results demonstrate significant changes inboth synaptophysin and IGF-1 immunostaining (sum-marized in Fig. 9) in the CNIC of experimental ani-mals. Specifically, 24 h after the lesion, there was asmall but significant upregulation in the synaptophy-sin immunostained area bilaterally in the CNIC thatwas no longer present at 90 days after the ablation. At1 day after the ablation, there was also a small but sig-nificant upregulation of IGF-1 immunostaining, seenas a bilateral increase in both the mean gray levelwithin neurons and the mean gray level in the neuro-pil. However, at 90 days after the ablation, only theupregulation in mean gray level in the neuropil contra-lateral to the lesion persisted although it was smallerthan that observed at 1 day after the ablation. Theseresults reveal a transitory alteration in synaptophysinand IGF-1 protein levels bilaterally in the CNIC thatoccurs within 24 h after the cochlear ablation.

Following unilateral hearing loss, a series of tran-synaptic modifications occur along the auditory path-way that include morphologic, metabolic, and physio-logic changes (Illing et al., 1997, 2000; Illing, 2001;Syka, 2002; for review). Although these changes areless pronounced when the hearing loss occurs in adultanimals compared to younger animals, they still occur,having important functional implications for struc-tures such as the IC, the major processing site for vir-tually all monaural and binaural auditory brainstempathways (Druga and Syka, 1984; Majorossy and Kiss,1994; Moore, 1988; Nordeen et al., 1983; Oliver et al.,1997). In the CNIC, unilateral cochlear ablation leadsto an increase in the number and/or terminal distribu-tion of cochlear nucleus neurons projecting ipsilater-ally to the IC on the intact side (Moore, 1994). In addi-tion, upregulation both in the number of calbindinimmunostained neurons (Forster and Illing, 2000) and

Fig. 4. Bar graphs showing the mean gray level of synaptophy-sin immunostaining (A) and the synaptophysin immunostained area(B) in the control group and in experimental animals. No significantdifferences were observed between sides in either group or betweengroups in the mean gray level (A), however, the immunostainedarea at 1 day after the unilateral cochlear ablation was larger thanthat in control and 90 day survival time ferrets (B). Error bars indi-cate standard deviation and asterisks (*) significant differences.



Fig. 5. Low (A,B) and high (C–F) magnification digital images illustrating the pattern and distri-bution of IGF-1 immunostaining in the left and right IC in a control ferret. In the CNIC, the immuno-staining was symmetric on both sides (A,B). A lightly immunostained neuropil (C,D) and neurons thatwere most lightly immunostained were distributed throughout the nucleus. Some darkly immuno-stained neurons (C–F) were also evident. Scale bar in A–B ¼ 500 lm; B–C ¼ 100 lm; D–E ¼ 25 lm.



in the calretinin immunostained area (Alvarado et al.,2005) have been reported to occur in the CNIC contra-lateral to the lesion. Interestingly, electrophysiologic

changes also have been reported in the CNIC. In par-ticular, unilateral cochlear ablation leads to a rapidand significant increase in the number of neurons in

Fig. 6. Low (A,B) and high (C–F) magnification digital imagesillustrating the pattern and distribution of IGF-1 immunostaining inthe contralateral and ipsilateral ICs 1 day after unilateral cochlearablation. Similar to that in control ferrets, there was a lightly immu-

nostained neuropil (C,D), and neurons that were mainly darkly im-munostained (C–F). Notice that the immunostained neuropil and neu-rons were apparently darker bilaterally than those in control animals.Scale bar in A–B ¼ 500 lm; B–C ¼ 100 lm; D–E ¼ 25 lm.



Fig. 7. Low (A,B) and high (C–F) magnification digital images illustrating the pattern and distri-bution of IGF-1 immunostaining in the contralateral and ipsilateral ICs 90 days after cochlear abla-tion. The immunostaining consisted of a lightly immunostained neuropil and neurons (C,D) that wereimmunostained darker apparently compared to those in control animals but lighter compared to thoseat 1 day survival time ferrets. Scale bar in A–B ¼ 500 lm; B–C ¼ 100 lm; D–E ¼ 25 lm.



the CNIC contralateral to the lesion that respond tostimulation of the intact ear (McAlpine et al., 1997;Mossop et al., 2000).

In the present study, synaptophysin, an integralmembrane presynaptic protein of the neurosecretoryvesicles, (Sokolowski and Cunningham, 1996), wasused as an indirect marker of synapses (Alvaradoet al., 2004; Benson et al., 1997; Davies et al., 1998;Fuentes-Santamaria et al., 2003, 2005; Kadish andVan Groen, 2002, 2003; Kadish et al., 2002; Masliahet al., 1990, 1991; Stroemer et al., 1998). The distribu-tion of synaptophysin immunostaining found here wassimilar to that reported previously in young ferrets(Fuentes-Santamaria et al., 2003). Compared to con-trol ferrets, no changes in the overall mean gray levelof synaptophysin immunostaining were present at ei-ther 24 h or 90 days following unilateral cochlear abla-tion. On the contrary, there was a bilateral increase inthe synaptophysin immunostained area in the CNICat 24 h after the ablation. However, this increase wasno longer present 90 days after the lesion. Such anincrease in the synaptophysin immunostained area inthe absence of a change in the overall mean gray levelmay be brought about by three possible mechanisms.First, it could be the result of an increase in the num-ber of terminals projecting to the CNIC after unilateralablation (Moore, 1994). However, in adult ferrets anincrease in the number and/or terminal distribution ofcochlear nucleus neurons projecting ipsilaterally to theIC on the unoperated side has not been shown to occurprior to 2 year after the lesion (Moore, 1994). Since theincrease in the immunostained area in the presentstudy occurs within the first 24 h, it is unlikely to bedue to an increase in the number of the synaptic termi-nals in the CNIC. Second, this increase could be theresult of an upregulation in synaptophysin withinvesicles. This increase in the concentration of synapto-physin within the vesicles, without changes in thenumber of vesicles, would lead to an increase in themean gray level and not to an increase in the immuno-stained area. However, the present data do not reportsignificant differences in the mean gray level of synap-tophysin immunostaining. Finally, the increase couldbe the result of an increase in the number of synapticvesicles within the existing terminals in the CNIC.Consistent with this latter possibility, it has been dem-onstrated in the MSO an increase of the number ofvesicles within terminals following unilateral cochlearablation (Russell and Moore, 2002). Therefore, ourfindings are consistent with an increase in the numberof synaptic vesicles rather than an increase in thenumber of terminals or an upregulation in synapto-physin within the vesicles.

Modifications in synaptophysin immunostaining inthe cochlear nuclei (Benson et al., 1997; Fuentes-Santamaria et al., 2005) and in the in the superior oli-vary complex (Alvarado et al., 2004) have been reported

Fig. 8. Bar graphs showing the nucleus mean gray level of IGF-1 immunostaining (A), the mean gray level within IGF-1 immuno-stained neurons (B), and the mean gray level in the IGF-1 immuno-stained neuropil (C) in the CNIC in control and experimentalgroups. The nucleus mean gray level of IGF-1 immunostaining (A)was significantly higher at 1 day after the ablation in the contralat-eral and ipsilateral sides compared to that in control and 90-daysurvival time ablated ferrets. The mean gray level within IGF-1 im-munostained neurons (B) was also significantly higher compared tothat in control ferrets and to the contralateral side at 90 days afterthe ablation. In addition, the mean gray level in the IGF-1 immuno-stained neuropil (C) was higher at 1 day after the ablation com-pared to that in control ferrets and 90-day survival time ferrets,while the contralateral side at 90 days after the ablation was higherthan the left side in control animals. Error bars indicate standarddeviation and asterisks (*) significant differences.



previously after unilateral cochlear ablation. Taken to-gether, these previous studies and the present resultssuggest that disruption of the auditory pathway bycochlear ablation induces modifications in the bio-chemical and/or morphological properties of the synap-ses that are reflected in changes in synaptophysin im-

munostaining. Since several studies have suggestedthat biochemical and/or morphological changes of thesynapses could modify synaptic efficacy (Buchs andMuller, 1996; Jones, 1999; Nikonenko et al., 2002; Shiet al., 2005; Toni et al., 1999, 2001), it is possible thatthe upregulation in the synaptophysin immunostained

Fig. 9. Schematic diagram summarizing the distribution of syn-aptophysin and IGF-1 immunostaining in the CNIC in control (A)and cochlear ablated ferrets (B,C). Twenty-four hours after cochlearablation an upregulation in the synaptophysin immunostained areawas found in the CNIC bilaterally (B). This upregulation was nolonger present at 90 days after the ablation (C). In addition, thesechanges were accompanied by an increase in IGF-1 immunostainingbilaterally in both the mean gray level within neurons and themean gray level in the neuropil at 1 day survival time ferrets (B).

Observe that only the increased mean gray level in the neuropilcontralateral to the lesion persisted 90 days after the deafferenta-tion (C) although it was smaller than that observed at 1 day sur-vival time animals (B). Circles indicate either synaptophysin immu-nostained profiles or IGF-1 immunostained neurons and differentgray colors represent different levels of synaptophysin and IGF-1immunostained neuropil or neurons in the CNIC. Upregulation inthe immunostaining in ablated ferrets compared to control animalsis indicated by darker gray colors.



area found in this study at 24 h after the cochlear abla-tion could lead to an increase in synaptic efficacywithin the CNIC. Consequently, this upregulationcould represent a rapid but transitory compensatorysynaptic response to the loss of auditory inputs follow-ing the unilateral cochlear ablation.

Although the molecular mechanisms mediating ac-tivity-related plasticity in adult animals have not beenelucidated fully, it has been proposed that growth fac-tors are involved in this phenomenon (Bramham andMessaoudi, 2005; Dore et al., 1997; Russo et al., 2005;for review). One of these growth factors, IGF-1, hasbeen studied widely as a possible treatment for severalneurological diseases as well as brain lesions (Doreet al., 1997; Russo et al., 2005; for review). In adultmammals, IGF-1 has been related to neurogenesis,including proliferation, differentiation, and neuronalmaturation in specific brain areas (Aberg et al., 2000,2006; Anderson et al., 2002; Bondy, 1991; Carson et al.,1993; Lichtenwalner et al., 2001; Russo et al., 2005;Torres-Aleman, 2005; Ye and D’Ercole, 2006). More-over, IGF-1 has been associated with synaptic plastic-ity (Aberg et al., 2006; Russo et al., 2005; Torres-Ale-man, 1999, 2005) by regulating neurotransmitterrelease, neuronal excitability, and synapse formation(Aberg et al., 2006; O’Kusky et al., 2000; Russo et al.,2005; Torres-Aleman, 1999, 2005).

The present results reveal a bilateral upregulationin IGF-1 immunostaining within neurons and neuropilin the CNIC in the first 24 h after the cochlear abla-tion, consistent with a role for this growth factor in theacute upregulation seen here in synaptophysin.Although the upregulation of IGF-1 declined after 90days postlesion, it was still present in the neuropil con-tralateral to the ablation. After deafferentation of thehippocampus, IGF-1 levels also have been reported toincrease rapidly within the first hours and days afterthe lesion before declining to normal levels. Thisincrease appears to be related to axonal sprouting thatmay be involved in the reorganization and restorationof neural connections (Guthrie et al., 1995; Kar et al.,1993; Woods et al., 1998). Thus, deafferentation-induced upregulation of IGF-1 may play an importantrole in the trophic support of synapses. However, thepersistence of elevated IGF-1 in the contralateralCNIC 90 days after the lesion, without similarincreases in synaptophysin, suggests that IGF-1 isinvolved not only in the initial synaptic response butalso in long-lasting changes within the CNIC.

The contralateral CNIC is particularly affected byunilateral cochlear ablation because it receives inputsmostly from nuclei that receive direct projections fromor are driven by the cochlea on the opposite side (Cantand Benson, 2003; Druga and Syka, 1984; Majorossyand Kiss, 1994; Moore, 1988; Oliver et al., 1997;Nordeen et al., 1983). The majority of the anatomical,biochemical, and functional changes reported in adult

animals in the CNIC contralateral to the ablationappear rapidly and are enduring (Alvarado et al.,2005; Forster and Illing, 2000; McAlpine et al., 1997;Moore, 1994; Mossop et al., 2000). Given the timecourse of the modification in the IGF-1 immunostain-ing found in the present study, it is possible that IGF-1may be involved in mediating some of these changes.Specifically, the fact that IGF-1 immunostainingwithin the CNIC contralateral to the ablation wasupregulated at 24 h and was still present in the neuro-pil 90 days after the lesion suggests that this growthfactor may be involved not only in short-term changesbut also in long-term changes that occur in the audi-tory midbrain in adult animals (Alvarado et al., 2005;Forster and Illing, 2000; McAlpine et al., 1997; Moore,1994; Mossop et al., 2000).

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Synaptophysin and insulin-like growth factor-1 immunostaining in the central nucleus of the inferior colliculus in adult ferrets following unilateral cochlear removal: A densitometric