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Neuroscience 154 (2008) 381–389

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APID REGULATION OF MICROTUBULE-ASSOCIATED PROTEIN 2 INENDRITES OF NUCLEUS LAMINARIS OF THE CHICK FOLLOWING

EPRIVATION OF AFFERENT ACTIVITY

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. WANG AND E. W. RUBEL*

irginia Merrill Bloedel Hearing Research Center, Department of Oto-aryngology–Head and Neck Surgery, University of Washingtonchool of Medicine, Box 357923, Seattle, WA 98195, USA

bstract—Differential innervation of segregated dendritic do-ains in the chick nucleus laminaris (NL), composed of third-rder auditory neurons, provides a unique model to studyynaptic regulation of dendritic structure. Altering the syn-ptic input to one dendritic domain affects the structure andength of the manipulated dendrites while leaving the otheret of unmanipulated dendrites largely unchanged. Little isnown about the effects of neuronal input on the cytoskeletaltructure of NL dendrites and whether changes in the cy-oskeleton are responsible for dendritic remodeling followinganipulations of synaptic input. In this study, we investigate

hanges in the immunoreactivity of high-molecular weighticrotubule associated protein 2 (MAP2) in NL dendrites

ollowing two different manipulations of their afferent input.nilateral cochlea removal eliminates excitatory synaptic in-ut to the ventral dendrites of the contralateral NL and theorsal dendrites of the ipsilateral NL. This manipulation pro-uced a dramatic decrease in MAP2 immunoreactivity in theeafferented dendrites. This decrease was detected as earlys 3 h following the surgery, well before any degeneration offferent axons. A similar decrease in MAP2 immunoreactivityn deafferented NL dendrites was detected following a mid-ine transection that silences the excitatory synaptic input tohe ventral dendrites on both sides of the brain. Thesehanges were most distinct in the caudal portion of the nu-leus where individual deafferented dendritic branches con-ained less immunoreactivity than intact dendrites. Our re-ults suggest that the cytoskeletal protein MAP2, which isistributed in dendrites, perikarya, and postsynaptic densi-ies, may play a role in deafferentation-induced dendriticemodeling. © 2008 IBRO. Published by Elsevier Ltd. Allights reserved.

ey words: deafferentation, dendritic plasticity, afferent reg-lation, cytoskeleton.

icrotubules are a major cytoskeletal component of neu-onal dendritic structure. It is well established that micro-ubule-associated protein 2 (MAP2) binding increases thetability of the microtubule network and dendritic structureSerrano et al., 1984; Kowalski and Williams, 1993; Yam-uchi et al., 1993; Sánchez et al., 2000; Harada et al.,

Corresponding author. Tel: �1-206-543-8360; fax: �1-206-616-1828.-mail address: rubel@u.washington.edu (E. W. Rubel).bbreviations: [Ca2�]i, intracellular calcium concentration; MAP2, micro-

ubule-associated protein 2; NL, nucleus laminaris; NM, nucleus magno-

bellularis; PBS, phosphate-buffered saline; XDCT, crossed dorsal co-hlear tract.

306-4522/08$32.00�0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reseroi:10.1016/j.neuroscience.2008.02.032

381

002). MAP2 has been proposed to play a fundamentalole as a regulator of dendritic morphology in the develop-ng and adult brain. This idea is supported by dynamicegulation of MAP2 expression and its involvement in dy-amic changes in neuronal morphology caused by excito-oxins (Siman and Noszek, 1988; Bigot et al., 1991; Felipot al., 1993; Arias et al., 1997; Faddis et al., 1997; Hoski-on and Shuttleworth, 2006; Hoskison et al., 2007), trau-atic brain injury (Taft et al., 1992; Folkerts et al., 1998), or

ocal ischemia (Pettigrew et al., 1996; Schwab et al.,998). In addition, manipulations of synaptic activity alterhe immunoreactivity for MAP2 in dendrites (Hendry andhandari, 1992; Steward and Halpain, 1999; Vaillant et al.,002), further suggesting that synaptic activity either di-ectly or indirectly regulates MAP2 expression.

The nucleus laminaris (NL) composed of third-orderuditory neurons in the avian brainstem, receives segre-ated binaural excitatory input from the two ears via theucleus magnocellularis (NM). The neurons of NL act asoincidence detectors and play an essential role in binau-al hearing (Carr and Konishi, 1988, 1990; Overholt et al.,992; Joseph and Hyson, 1993). This nucleus is thought toe analogous to the medial superior olivary nucleus ofammals (Burger and Rubel, 2008). NL provides a usefulodel for studying afferent regulation of dendritic structurend molecular mechanisms underlying this regulation (Dei-ch and Rubel, 1984; Sorensen and Rubel, 2006). Inhicks, differential innervation of the segregated dendriticomains of NL allows local manipulation of the excitatory

nputs to one dendritic domain, while leaving the otherendritic domain of the same cells intact to serve as aontrol. Specifically, neurons in NL contain a dorsal and aentral set of dendrites that are essentially symmetricalnd receive excitatory inputs almost exclusively from eitherhe ipsilateral or the contralateral cochlea via NM, respec-ively (Parks and Rubel, 1975; Rubel et al., 2004). Conse-uently, excitatory inputs to one set of NL dendrites canasily be silenced or altered by manipulating the ipsilateralr contralateral projection of NM neurons. Previous studiesave demonstrated a rapid retraction of the ventral NLendrites within hours after transection of the contralateralM projection, while the dorsal NL dendrites, receiving

ntact input, appear largely unaffected (Benes et al., 1977;eitch and Rubel, 1984; Sorensen and Rubel, 2006).

Little is known regarding the role of synaptic input on theytoskeletal structure of NL dendrites and whether changes

n the cytoskeleton are responsible for deafferentation-in-uced dendritic retraction. High-molecular weight microtu-

ule-associated proteins 2 (MAP2), including MAP2A and

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Y. Wang and E. W. Rubel / Neuroscience 154 (2008) 381–389382

AP2B, are of particular interest because of their direct as-ociation with microtubules in dendrites and perikarya, asell as colocalization with actin in dendritic spines andostsynaptic densities (see review, Sánchez et al., 2000). Toetermine whether dendritic MAP2 of NL neurons has aossible role in structural changes after alterations of synap-ic input, we examined changes in the immunoreactivity forigh-molecular weight MAP2 in NL dendrites following twoifferent manipulations of excitatory afferents from NM (Fig.). Our results demonstrate a rapid reduction of the immuno-eactivity for high-molecular weight MAP2 in the dendritesith reduced excitatory drive compared with dendrites that

eceived normal synaptic input.

EXPERIMENTAL PROCEDURES

hite Leghorn chicken hatchlings (Gallus domesticus) of 4–11ays of age were used. All procedures were carried out in accor-ance with the National Institutes of Health Guide for the Care andse of Laboratory Animals and approved by the University ofashington Institutional Animal Care and Use Committee. All

fforts were made to minimize pain or discomfort of the animalssed and to minimize the animal number.

ransection of the crossed dorsal cochlear tractXDCT)

nimals were anesthetized with a mixture of 40 mg/kg ketaminend 12 mg/kg xylazine. The animals were placed in a head holdernd surgery was conducted using the method of Deitch and Rubel1984). Briefly, the neck muscles were resected to expose theura covering the cerebellomedullary cistern. An ophthalmic knifeas inserted through the dura and the fourth ventricle and into therain stem so as to transect the XDCT at the midline. The woundas packed with Gelfoam and sealed with a tissue adhesive,iquiVet (Oasis Medical, Mettawa, IL, USA). The location andxtent of the transection were examined after further tissue pro-essing (see below). Only animals receiving a complete transec-ion of the XDCT were used for data analysis.

nilateral cochlea removal

he procedure described in Born and Rubel (1985) was used. Ani-als were anesthetized as described above. A small incision wasade to widen the external auditory meatus of the right ear. The

ig. 1. Differential innervation of NL dendrites and the arrangement ofllustrate the affected dendritic fields following unilateral cochlea remo

anipulation and deafferented axons and dendrites influenced by eac

ympanic membrane and columella were removed to expose the oval o

indow. The basilar papilla, including the lagena macula, was re-oved via the oval window using fine forceps, floated on water, andxamined with a surgical microscope to verify complete removal.nly animals with a complete removal of the basilar papilla, including

he lagena, were used for further tissue processing and data analy-is. The incision was sealed with LiquiVet adhesive.

mmunocytochemistry

fter survival times of 1, 3, 6, 14 h, or 1 week (for cochlea removalnly), the animals were transcardially perfused with 0.9% salineollowed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH.4). The brains were removed from the skull, postfixed overnight inhe same fixative, and then transferred to 30% sucrose in 0.1 Mhosphate-buffered saline (PBS; pH 7.4) until they sank. Complete-ess of the XDCT transection was verified under a microscope afteremoval of the cerebellum and incomplete cases were discarded.he brain was frozen, cut coronally at 30 �m on a freezing slidingicrotome, and each section was collected in PBS. Alternate serial

ections were stained for Nissl substance or immunocytochemicallyor MAP2 using peroxidase or fluorescent immunocytochemicalethods. Briefly, free-floating sections were incubated with primaryntibody solutions diluted 1:1000 in PBS with 0.3% Triton X-100vernight at 4 °C, followed by biotinylated anti-IgG antibodies (1:200;ector Laboratories, Burlingame, CA, USA) or AlexaFluor® second-ry antibodies (1:200; Molecular Probes, Eugene, OR, USA) for 1–2at room temperature. Monoclonal anti-MAP2 (cat. #MAB3418)ade in mouse was purchased from Chemicon International (Te-ecula, CA, USA). The immunogen is bovine brain microtubulerotein. The antibody binds specifically to MAP2a and MAP2b and isetected as a 300 kD band in Western blot analysis.

For peroxidase immunocytochemical staining, sections werehen incubated in avidin–biotin–peroxidase complex solutionABC Elite kit; Vector Laboratories) diluted 1:100 in PBS with 0.3%riton X-100 for 1 h at room temperature. Sections were incubated

or 3–5 min in 0.045% 3-3-diaminobenzidine (Sigma, St. Louis,O, USA) with 0.03% hydrogen peroxide, 125 mM sodium ace-

ate, 10 mM imidazole, and 100 mM nickel ammonium sulfate.ections were mounted on gelatin-coated slides and then dehy-rated, cleared, and coverslipped with DPX mounting mediumEMS, Hatfield, PA, USA). For fluorescent immunocytochemicaltaining, sections were mounted and coverslipped with Fluoro-ount-G® (SouthernBiotech, Birmingham, AL, USA).

ata analysis

elative changes between the deafferented and intact dendrites

eries. Schematic drawings of the chick brainstem in the coronal planer XDCT transection (B). Red color indicates the surgical site for eachulation. Abbreviations: NA, nucleus angularis.

the surgval (A) o

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Y. Wang and E. W. Rubel / Neuroscience 154 (2008) 381–389 383

uantified by comparing average optical densities of the immuno-taining between the dorsal and ventral NL dendritic domains.etween two and five cases of each survival time group were chosen

or this analysis. For each case, sections with fluorescent immuno-ytochemical staining were taken from the caudal and rostral portionsf NL at the levels comparable to those of Fig. 2A and 2C, respec-

ively. A photomicrograph of NL was taken using a 20� objective attotal magnification of 240� (see Fig. 3). Although the dorsal and

entral dendrites of each NL cell are normally of equivalent sizeSmith and Rubel, 1979; Smith, 1981), the width of the dendriticegions will vary between the dorsal and ventral regions in indi-idual sections due to slightly different slicing planes acrossases. A box covering the major dendritic MAP2 staining was

ig. 2. MAP2 immunoreactivity in the normal chick NL at the caudal (A, B),eroxidase and intensified with nickel ammonium sulfate. Scale bar�100 �m i

rawn first in the dendritic domain with a narrower width. Then thexact same size box was applied to the complementary domain.verage optical density of the box in the dorsal and ventral NL waseasured using Image J software (version 1.38X; National Insti-

utes of Health).We defined D and V as the average density of staining in the

orsal and ventral neuropil domains, respectively. The percentifference was calculated as the change in density of the deaffer-nted neuropil domain relative to the intact domain, which isV�D)/D�100 for the contralateral side and (D�V)/V�100 for thepsilateral side following cochlea removal. The percent differenceas calculated within the same photomicrograph from the sameection to avoid any possible inconsistencies caused by variations

middle (C, D), and rostral (E, F) levels. Sections were stained withn E (applies to A, C, E), 30 �m in F (applies to B, D, F).

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Y. Wang and E. W. Rubel / Neuroscience 154 (2008) 381–389384

ig. 3. MAP2 immunoreactivity in the caudal portion of NL before and 14 h after cochlea removal or XDCT transection. (A, B) In the normal NL, thetaining density of MAP2 immunoreactivity is similar between the dorsal and ventral dendrites. (C–F) MAP2 immunoreactivity in the ipsilateral (C, D)nd the contralateral (E, F) NL at 14 h following cochlea removal. Deafferented dorsal dendrites of the ipsilateral NL and ventral dendrites of theontralateral NL exhibited a distinct decrease in MAP2 immunoreactivity, as compared with the intact dendrites. (G, H) MAP2 immunoreactivity in NLt 14 h following XDCT transection. Deafferented ventral dendrites on both sides of the brain exhibited a distinct decrease in MAP2 immunoreactivity,s compared with the intact dorsal dendrites. (A, C, E, G) Confocal photomicrographs. (B, D, F, H) Spectrums resulting from the correspondinghotomicrographs using Image J software (version 1.38X; National Institutes of Health). These spectrum images use 16 colors to present different

evels of the intensity in a photomicrograph. They demonstrate that deafferented dendritic domains contain fewer dendritic branches with high intensity

f MAP2 immunoreactivity than the intact dendritic domain. Spectrum scale is presented on the lower right corner of the figure. Scale bar�100 �m.

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Y. Wang and E. W. Rubel / Neuroscience 154 (2008) 381–389 385

n immunostaining between cases and sections and variations inrightness across photos. The percent differences from all ani-als of the same group were averaged and plotted as a functionf survival time. All statistical comparisons used the Prism ver. 4oftware package (GraphPad Software, Inc., San Diego, CA,SA).

RESULTS

AP2 immunoreactivity in normal NL

n the chick, NL neurons are arranged in a single layer,xcept for the most caudolateral region of the nucleushere multiple layers of neurons are present. Dendrites ofipolar NL neurons are segregated into dorsal and ventralomains. Immunocytochemistry revealed a dense distribu-ion of MAP2 in the dendrites and neuronal cell bodieshereas the immunoreactivity was absent in the nucleus.ig. 2 illustrates a series of coronal sections across NL.AP2-immunoreactive dendrites are longer in the caudalnd lateral portion of the nucleus than those located moreostrally and medially, reflecting the gradient of dendriticength in NL (Smith and Rubel, 1979; Smith, 1981; Deitchnd Rubel, 1984). For sections with fluorescent immuno-ytochemical staining, the staining intensity is generallyigher in the caudolateral NL than the rostromedial NL.ections that were stained with peroxidase and intensifiedith nickel ammonium sulfate (see Experimental Proce-ures) exhibited a more uniform staining intensity acrosshe nucleus. However, at any particular location, the den-ity of MAP2 immunostaining in individual dendriticranches appeared similar between the dorsal and ventralendrites (Figs. 2 and 3A–B).

AP2 immunoreactivity in NL following eliminationf afferent action potential activity

wo methods were used to surgically eliminate the excita-ory action potentials to one set of NL dendrites: unilateralochlea removal and transection of the XDCT. The eightherve is the only excitatory input to NM neurons. Unilateralochlea removal silences all the excitatory inputs to the

ig. 4. High-magnification photomicrographs of MAP2 immunoreactivi

he ipsilateral NL. (C, D) From the contralateral NL. (B, D) Spectrum resultingide of the figure. Scale bar�20 �m.

psilateral NM and in turn to the dorsal dendrites of thepsilateral NL and the ventral dendrites of the contralateralL (Fig. 1A; Born et al., 1991). The XDCT contains theecussating NM axons that project to the ventral NL den-rites on both sides of the brain. A complete transection ofDCT at the midline eliminates all the excitatory inputs to

he ventral dendrites of NL neurons, while leaving thenputs to the dorsal dendrites intact (Fig. 1B). After cochleaemoval the cochlear ganglion cells and NM neurons begino die about 18–24 h after the surgery and by 48 h about0% NM neurons have degenerated (Rubel et al., 1990).fter XDCT transection, the afferent terminals from NM on

he ventral NL dendrites do not show any atrophic changesor at least the first 4 h, and then they begin to degenerateDeitch and Rubel, 1989). Fourteen animals received uni-ateral cochlea removal, 3 survived 1 h, 2 survived 3 h, 5urvived 6 h, 2 survived 14 h, and 2 survived 1 week. Eightnimals received a complete transection of XDCT, 3 sur-ived 1 h, 2 survived 3 h, 2 survived 6 h, and 1 survived4 h.

Changes in the pattern and intensity of MAP2 immu-ostaining following either manipulation were positivelyorrelated with post-surgery survival time and varied withhe location within the nucleus. At 1 h of cochlea removal,he pattern and average density of MAP2 staining showedo discernable differences between the dorsal and ventralendrites in any section of the nucleus, on both sides of therain. Notable differences between MAP2 staining in thewo dendritic domains were observed beginning at 3 host-surgery in the caudal portion of the nucleus and wereetectable up to 1 week following deafferentation, the long-st survival time examined. Low- and high-magnificationhotomicrographs in Figs. 3 and 4 illustrate the MAP2

mmunoreactivity in the caudal NL in a control and anxperimental case that survived 14 h following cochleaemoval. In contrast to the similar staining intensity be-ween the dorsal and ventral dendrites in the control (Fig.A–B), the deafferented dorsal dendrites of the ipsilateralL and the ventral dendrites of the contralateral NL exhib-

audal portion of the NL at 14 h following cochlea removal. (A, B) From

ty in the c from A and C, respectively. Spectrum scale is presented on the right

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Y. Wang and E. W. Rubel / Neuroscience 154 (2008) 381–389386

ted a lower density of staining than the complementaryendritic domain on the same side of the brain (Figs. 3C,E, 4A, and 4C). Corresponding spectrum images demon-trated that the deafferented dendritic domains containedewer dendritic branches with high levels of staining (Figs.D, 3F, 4B, and 4D). This observation was further con-rmed quantitatively by a dramatic decrease in the aver-ge optic density of MAP2 staining in deafferented den-rites (Fig. 5A and 5C). In the rostral portion of the nucleus,owever, differences in MAP2 immunostaining betweenhe deafferented and the intact dendrites were much lessronounced. Qualitatively, no difference was reliably de-ected in most cases across all survival times. Quantitativenalysis of the average optic density demonstrated a smallecrease of MAP2 staining in the deafferented dendrites inome cases with a survival times between 6 h and 1 weekFig. 5B and 5D).

Results of the percent difference calculations from thenimals that received unilateral cochlea removal were an-lyzed by one-way ANOVA, followed by appropriate postoc comparisons. Since there were no statistical differ-nces between the ipsilateral and contralateral sides in

erms of the amount of change in the deafferented vs.ondeafferented dendrite, data from the two sides of therain were combined to increase the power. However, thereere clear differences between the rostral (smaller) dendritesnd the caudal (larger) dendrites. Hence these two data-setsere subjected to separate analyses. ANOVA on the data

rom caudal dendrites yielded a significant main effect

ig. 5. Percent difference of the average optical density of MAP2 immhe survival time following cochlea removal in the ipsilateral (A, B) androm the rostral NL. The percent difference was calculated as the chan the ipsilateral side) neuropil domain relative to the intact (dorsal in thhe average percent difference from all animals in individual survival timata. The deafferented neuropil domains exhibited a notable decreaseA, C). This decrease was much less pronounced in the rostral NL (B

F(5,25)�19.75; P�0.001), and a highly significant trend to- m

ard increasing percent difference as a function of timeollowing cochlea removal (P�0.001; post test for linearrend). In addition, individual post hoc comparisons yieldedighly significant differences between the control groupnd each of the other groups beginning at 3 h after cochleaemoval (3 h, P�0.01; 6 h, 14 h, 1 week; P�0.001; Tukey’sultiple comparison test). As suggested by Fig. 5, thenalyses on the data from rostral NL dendrites did not yieldesults as clean as those from the caudal dendrites.

hether this was due to their smaller size and thereforereater percentage of error in the measurements, or dif-erences in their biology cannot be determined from ourata. ANOVA yielded a significant main effect (F(5,26)�.40; P�0.02) indicating that there was a significant reduc-ion of dendritic MAP2 immunoreactivity after cochlea re-oval on the deafferented dendrite. Post hoc linear trendnalysis also yielded a significant trend over time (P�0.01)ut only the 6 h group showed a reliable difference fromontrols (P�0.05; Tukey’s multiple comparison test).

XDCT transection resulted in decreases in MAP2 im-unoreactivity in affected dendrites following a time

ourse comparable to that in cases following cochlea re-oval (Fig. 6). Differences were first detected 3 h afterDCT transection and presented as long as 14 h later, the

ongest survival time examined. Fig. 3G–H shows an ex-mple at 14 h following XDCT transection. The ventralendrites on both sides of the brain contained less MAP2

mmunoreactivity than the dorsal dendrites. Similar to theases where one cochlea was ablated, this difference was

ing between the dorsal and ventral neuropil domains as a function oferal (C, D) NL. (A, C) Measured from the caudal NL. (B, D) Measurednsity of the deafferented (ventral in the contralateral side and dorsallateral side and ventral in the ipsilateral side) domain. Bar graphs are. Error bars are standard deviation. Each “�” indicates individual case

immunoreactivity compared with the intact domain in the caudal NL

unostaincontralatnge in dee contrae group

ost apparent in the caudal half of the nucleus. We did not

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Y. Wang and E. W. Rubel / Neuroscience 154 (2008) 381–389 387

erform statistical analyses on the data from animals withDCT transection because the n’s were too small. Thesexperiments were included only to determine if thehanges in MAP2 antibody reacted dendrites following thisanipulation are similar to our previous data using a vari-ty of other techniques for measuring dendritic changeBenes et al., 1977; Deitch and Rubel, 1984, 1989; So-ensen and Rubel, 2006). Clearly the results are similar.

DISCUSSION

ur results show that MAP2 immunoreactivity decreasesapidly and dramatically in deafferented dendrites of NLeurons, particularly in the caudal portion of the nucleus.e used two different methods to deprive one set of NL

endrites of its excitatory input: cochlea removal andDCT transection. Both manipulations cause an immedi-te cessation of action potentials of excitatory NM affer-nts to affected NL dendrites (Born et al., 1991) and bothppear to produce similar changes in MAP2 immunoreac-ivity in NL dendrites, as shown in the current study. Co-hlea removal does not directly damage the NM axons thatnnervate NL dendrites and deafferentation-induced celleath in NM does not occur for 2 days (Born and Rubel,985). Therefore, the changes in MAP2 immunoreactivity

n NL dendrites are likely to be a result of the elimination ofresynaptic action potentials rather than frank degenera-ive changes or the release of degeneration-related cyto-ines from the presynaptic endings. However, we cannotule out release of degradative products from silencedndings.

We failed to detect comparable decreases in MAP2

ig. 6. Percent difference of the average optical density of MAP2mmunostaining between the dorsal and ventral neuropil domains as aunction of the survival time following XDCT transection in the caudalL. The percent difference was calculated as the change in density of

he deafferented (ventral) neuropil domain relative to the intact (dorsal)omain. Data from the two sides of the brain were combined. Barraphs are the average percent difference from all animals in individ-al survival time group. Error bars are standard deviation. Each “�”

ndicates individual case data. The deafferented ventral neuropil do-ains exhibited a notable decrease in MAP2 immunoreactivity com-ared with the intact dorsal domain.

mmunoreactivity in rostral NL neurons. One possible ex- e

lanation is that neurons in rostral NL have thinner den-rites and/or contain less MAP2 than caudal NL neurons.e observed lighter staining density of MAP2 in the rostral

ortion than in the caudal portion of the nucleus. Relativehanges in MAP2 immunoreactivity may be obscured by

ow baseline levels of immunoreactivity. It is possible thathere is just a smaller change in rostral NL compared withaudal NL. Dendritic MAP2 of NL neurons may be regu-

ated to a varying degree, or by different mechanismslong the rostrocaudal axis. Further examination of MAP2xpression at an individual cell level may be necessary.

Decreases in the average density of MAP2 immunore-ctivity may be partially due to the retraction of deaffer-nted NL dendrites, which occurs as early as 1 h followingDCT transection (Deitch and Rubel, 1984). Whether co-hlea removal produces similar retraction of deafferentedL dendrites has not been studied within the survival timeeriod examined in the current report. A previous study onhe long-term regulation of deafferentation on dendritictructure reported a substantial preservation of the deaf-erented NL dendrites 4 weeks following cochlea removalRubel et al., 1981). Thus, it remains possible that thehanges to dendritic structure and cytoskeleton followingDCT transection and cochlea removal are regulated viaifferent mechanisms. On the other hand, this study alsoocumented sprouting of axonal arbors from the intact NMo the ‘deprived’ dendritic region at these long survivalimes. Regardless, the current study revealed that follow-ng either manipulation, many individual dendritic branchesn the deafferented dendritic domains appeared to containess immunoreactivity than those in the intact dendriticomain. These observations suggest that a major contri-ution to the total decrease of MAP2 immunoreactivityomes from the loss of MAP2 immunostaining in remainingeafferented dendritic branches. This loss prior to struc-ural changes of these dendrites suggests that changes inAP2 may be one of the first steps in deprivation-inducedendritic atrophy. Given that dendritic retraction takeslace as early as 1 h following XDCT transection, yetistinct changes in MAP2 immunoreactivity were found 3 hfter the surgery, MAP2 may play a more significant role inhe later retraction of dendritic branches, than the initialisruption of the balance of growth and retraction (So-ensen and Rubel, 2006).

The decrease observed in MAP2 immunostainingould result from either the degradation of MAP2 protein orhe alternation of its structure and/or binding properties, asreviously suggested in deafferentation-induced MAP2

oss in NM somata (Kelley et al., 1997). Light microscopysed in the current report does not allow us to determinehe nature of the changes in MAP2 immunoreactivity.mong all post-translational modifications of MAP2, phos-horylation is probably the most relevant to extracellularignals (for review, see Sánchez et al., 2000). MAP2 haseen found to be a substrate for many protein kinases andhosphatases, including c-Jun N-terminal kinase 1 (JNK1)nd stathmin, whose phosphorylations of MAPs are impor-ant in defining neuronal dendritic structure (Björkblom

t al., 2005; Ohkawa et al., 2007).

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Y. Wang and E. W. Rubel / Neuroscience 154 (2008) 381–389388

The decrease of MAP2 immunoreactivity, regardless ofhe cause, could be an indicator of changes in the normalunction of MAP2. In support of this idea, changes in MAP2mmunoreactivity appear to be associated with changes in

icrotubules in deafferented dendrites. A quantitativenalysis reported a 30–60% decrease in microtubulesensity in the initial portion of the ventral dendritic betweenand 48 h following XDCT transection (Deitch and Rubel,

989). This time course is comparable to the changes inAP2 immunoreactivity reported by the current study (seeig. 5), giving rise to the assumption that MAP2 loss orlternation may interrupt its function on microtubule as-embly and thus result in microtubule loss.

The rapid cellular events observed in NL dendritesollowing deprivation of afferent activity (overall dendriticetraction, interruption of the growth-retraction balance,ecrease in MAP2 immunoreactivity, and microtubule loss)ay be controlled by a single or separate mechanisms.mong a number of regulatory mechanisms controllingAP2 function (see review, Sánchez et al., 2000),

hanges in calcium signaling, especially rises in intracel-ular calcium concentration ([Ca2�]i), have been most ex-ensively studied following manipulations of synaptic in-uts. Excessive glutamate receptor stimulation induced byhysiological stimulation or exposure of glutamate or ago-ists of its receptors causes dendritic injury and/or MAP2

oss (Siman and Noszek, 1988; Bigot et al., 1991; Felipo etl., 1993; Arias et al., 1997; Faddis et al., 1997; Stewardnd Halpain, 1999; Swann et al., 2000; Vaillant et al.,002). Recent studies in hippocampal slices further dem-nstrated that these changes are calcium-dependentHoskison and Shuttleworth, 2006; Hoskison et al., 2007).ere we demonstrated that loss of excitatory synaptic

nput, which might be assumed to cause a decrease inCa2�]i, also produces MAP2 loss or alternation. This ob-ervation is consistent with a previous report in the visualortex following injections of tetrodotoxin into one eyeHendry and Bhandari, 1992). To our knowledge, a directink between decreases in [Ca2�]i and MAP2 loss or alter-ation has not been reported. Another possibility is that thehronic decrease in synaptic activity could lead to an in-rease in [Ca2�]i. This possibility is supported by the dra-atic increase of [Ca2�]i in deafferented NM neurons (Zir-el et al., 1995; Zirpel and Rubel, 1996). In NL dendrites,his switch may result from a slowdown of the calciumfflux induced by downregulation of plasma membranealcium ATPase isoform 2, one of the critical calcium effluxystem, following deafferentation (Y. Wang, unpublishedbservations). Investigation on changes in NL dendriticCa2�]i following deafferentation is required to confirm cal-ium signaling as one of the mechanisms by which deaf-erentation induces the observed change in NL dendriticAP2, and previously described changes to NL dendrite

tructure.

cknowledgments—The authors are proud to take part in thisribute to Kirsten Osen, a leader and model scientist in our field.he authors are grateful to Dr. MacKenzie A. Howard, Dr. Armin

. Seidl, Dr. Staci A. Sorensen, Ms. Kathryn M. Tabor, and Mr.

hristopher K. Thompson for criticisms of the manuscript. Theesearch was supported by NIDCD grants DC03829, DC04661nd DC00018.

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(Accepted 21 February 2008)(Available online 29 February 2008)