Visual Neuroscience (1992), 9, 471-482. Printed in the USA.Copyright © 1992 Cambridge University Press 0952-5238/92 $5.00 + .00

Monocular enucleation reduces immunoreactivity tothe calcium-binding protein calbindin 28 kD in theRhesus monkey lateral geniculate nucleus

R. RANNEY MIZE,1 QIAN LUO,1 AND MARGARETE TIGGES2

1 Department of Anatomy and Neurobiology, College of Medicine, University of Tennessee,The Health Science Center, Memphis

2 Yerkes Regional Primate Research Center and Departments of Anatomy and Cell Biologyand Ophthalmology, Emory University, Atlanta

(RECEIVED December 27, 1991; ACCEPTED April 4, 1992)

Abstract

The calcium-binding proteins calbindin (CaBP) and parvalbumin (PV) are important in regulatingintracellular calcium in brain cells. PV immunoreactivity is reduced by enucleation in the lateral geniculatenucleus (LGN) and by enucleation and visual deprivation in the striate cortex of adult monkeys. The effectsof enucleation and visual deprivation on CaBP immunoreactivity in the LGN are not known. We thereforehave studied cells and neuropil in the LGN that are labeled by antibodies to CaBP in normal and visuallydeprived Rhesus monkeys to determine if there is an effect on this calcium-binding protein. One group ofmonkeys had one eye removed 2 weeks to 4.3 years before sacrifice. A second group had one eye occludedwith opaque lenses from infancy without enucleation. A final group had one eye occluded long-term followedby short-term enucleation 2 weeks before sacrifice.

In normal monkeys, CaBP-immunoreactive neurons were found throughout the LGN. They were sparselydistributed within the six main laminae, and more densely distributed within layer S and the interlaminarzones (ILZ). The labeled ILZ neurons had a distinct morphology, with fusiform somata and elaboratedendritic trees that were confined primarily to the ILZ. Most CaBP-labeled neurons in the main layers haddendrites that radiated in all directions from the soma. ILZ and main layer cells labeled by CaBP thusprobably represent two different cell types.

Monocular enucleation with or without occlusion produced a significant reduction in antibody labeling inthe deafferented laminae. Field measures revealed an average 11.5% reduction in optical density in eachdeafferented lamina compared to its adjacent, nondeprived layer. The differences in field optical densitybetween deprived and nondeprived layers were statistically significant. CaBP neurons were still visible, butthe optical density of antibody labeling in these cells also was reduced. Occlusion without enucleation had noeffect. Thus, deafferentation, but not light deprivation, reduces concentrations of CaBP in monkey LGN.This effect is different than that seen in striate cortex of adult monkeys, where visual deprivation as well asenucleation alters CaBP immunoreactivity.

Keywords: Calcium, Immunocytochemistry, Visual pathways, Enucleation, Occlusion

Introduction

The calcium-binding protein calbindin 28 kD (CaBP) is con-tained in select populations of nerve cells where it is thought toplay a role in buffering intracellular calcium (Baimbridge &Miller, 1982; Baimbridge et al., 1982; Heizmann & Berchtold,1987; Heizmann & Hunziker, 1990) or in transporting calcium

Reprint requests to: R. Ranney Mize, Department of Anatomy,Louisiana State University Medical Center, 1901 Perdido Street, NewOrleans, LA 70112, USA.

Present address of Q. Luo: Department of Ophthalmology, LSUEye Center, Louisiana State University Medical Center, 2020 GravierStreet, New Orleans, LA 70112, USA.

across the cell membrane (Wasserman & Fullmer, 1982; Bron-ner et al., 1986). CaBP has been localized by immunocytochem-istry in both projection and interneurons in various brain nuclei(Jande et al., 1981; Baimbridge & Miller, 1982; Feldman &Christakos, 1983; Heizmann & Hunziker, 1990). In the visualsystem, CaBP has been found in a specific type of interneuronin monkey visual cortex that also contains the inhibitory neu-rotransmitters gamma-aminobutyric acid (GABA) (DeFelipeet al., 1989; Hendry et al., 1989) and within a few small neu-rons within the monkey lateral geniculate nucleus (LGN) (Jones& Hendry, 1989). The specific class(es) of LGN neuron that areimmunoreactive for CaBP antibody in the monkey have notbeen studied, but in the cat LGN CaBP neurons comprise sev-

471

472 R.R. Mize, Q. Luo, and M. Tigges

eral cell types, including some GABA-containing interneuronsand relay neurons thought to be X cells (Banfro & Mize, 1992;Demeulemeester et al., 1989, 1991).

Recently, CaBP and another calcium-binding protein, parv-albumin (PV), have been shown to be altered by visual depri-vation in adult monkeys. In Rhesus monkeys, two weeks ofmonocular enucleation are sufficient to reduce PV immunore-activity in the affected LGN laminae. This reduction appears tobe confined to retinal afferents and does not affect postsynap-tic LGN neurons (Tigges & Tigges, 1991). In adult cynomolgusmonkeys, Hendry and colleagues (Hendry & Carder, 1992) havedemonstrated that antibody labeling to CaBP and PV in visualarea 17 also is altered by monocular visual deprivation. PV im-munostaining is reduced in the ocular-dominance columns thatare functionally connected to an eye manipulated by intraocu-lar application of tetrodotoxin (TTX), a substance that blocksretinal ganglion cell activity. CaBP neurons are actually in-creased in number in the normal ocular-dominance columns af-ter this manipulation (Hendry & Carder, 1992). These findingsare significant because they show that the chemical propertiesof neurons can be altered by environmental modifications longafter the critical period of development in which dramaticchanges in cell structure and physiology take place (Sherman &Spear, 1982, for review).

In the present study, we have asked whether CaBP immu-noreactivity also is affected by various types of deprivation inthe adult and developing Rhesus monkey LGN-specificallywhether the effect can be produced both by denervation (enu-cleation of one eye) and by visual deprivation (monocular orbinocular manipulations) which leaves the anatomical connec-tions between the eye and the LGN intact.

Materials and methods

Tissue collection and perfusion

Sections from the LGNs of two normal Rhesus monkeys (Ma-caca mulatto) and 11 deprived monkeys (ten Macaca mulatto,one Macaca nemestrina) were studied. The monkeys experi-enced a variety of deprivation conditions (see Table 1 for theirrearing histories and ages at perfusion). Two of these 11 mon-keys were euthanized because of deteriorating health; monkeyR-001 was raised for 10 years with normal visual experience, buthad the right eye enucleated 2 weeks prior to perfusion; theother monkey (R-019) was a pigtail that had lost the right eyeat the age of 7 months. The remaining nine monkeys were partof ongoing studies of infantile aphakic amblyopia (Tigges et al.,1992) and myopia (Iuvone et al., 1991). These latter monkeyswere raised from birth under different types of monocular orbinocular deprivation conditions. Monocular manipulations in-cluded (1) continuous occlusion of one eye with opaque contactlenses (R-008, R-010, and R-012); monkey R-008 also receivedtopical application of apomorphine into the occluded eye; and(2) replacement of the natural lens of one eye with an intraocularlens (R-003). Binocular manipulations involved surgical removalof the natural lens of one eye and optical correction of the re-sulting aphakia with extended-wear contact lenses (EWCL)whereas the fellow eye was occluded with an opaque contactlens either part-time (PO) during the daylight hours (R-019,R-103, and R-929) or continuously (CO; R-933 and R-006). De-tailed information on contact lens design and fitting and rear-ing procedures have been published (Fernandes et al., 1988).

Table 1. Deprivation conditions

Monkeycode

R-001R-019R-010R-012R-008R-003R-103R-929R-006R-009R-933

Deprivationcondition

R

UMUMCOCO (Apom.)CO (Apom.)1OLANPANPANPANPAUC

L

UMUMUMUMUMUMPOPOCOPOCO

Eye enucleation(survival time)

R L

+ (14d)+ (4.3 y) -— _- -+ (14d)+ (15d) -+ (8 m)+ (14d)

+ (14 d)— —

Age atperfusion

10 y5y

19 m22 m20 m7 m

10 m31 m34 m35 m32 m

ANP: aphakia, optically corrected to a near point; Apom.: topicaladministration of apomorphine; AUC: aphakia, optically under-corrected; CO: continuous occlusion; d: days; IOL: natural lensreplaced by intraocular lens; L: left eye; m; months; PO: part-timeocclusion; R: right eye; UM: unmanipulated; and y: years.

In addition to these monocular and binocular deprivationconditions from birth, four of these monkeys had one eye enu-cleated 2 weeks and one monkey 8 months prior to perfusion(see Table 1). All of the monkeys with aphakic amblyopia ormyopia were sacrificed according to established procedures(Tigges et al., 1992). The NIH guidelines for the use of animalsin research were followed for all experimental procedures, in-cluding surgeries, enucleations, and perfusions. Surgeries wereperformed by an ophthalmologist under sterile conditions andgeneral anesthesia provided by the veterinary staff of the YerkesRegional Primate Research Center. The veterinarians also wereresponsible for postoperative care. Each animal was anesthetizedwith an overdose of sodium pentobarbital and perfused intra-cardially with normal saline followed by a 4% paraformalde-hyde-O.l'Vo glutaraldehyde fixative in a 0.05 M phosphatebuffer, pH 7.4 (with 4% sucrose added), followed by an addi-tional rinse of 5% sucrose in phosphate buffer. The brains wereblocked stereotaxically. Blocks containing the LGN were cryo-protected in 30% buffered sucrose and sections cut with a freez-ing microtome at 20 or 40 ^m. The tissue sections were thensent to Tennessee where they were treated for immunocyto-chemistry, mounted on glass slides, and analyzed.

Immunocytochemistry and data analysis

Two antibodies against calbindin were used. The first obtainedfrom Dr. Piers Emson was a polyclonal antibody raised insheep against purified CaBP from chicken gut. The second anti-body was a commercial monoclonal antibody raised in mouseagainst purified CaBP from chicken gut (Sigma, St. Louis,MO). The Sigma anti-calbindin-D is a monoclonal antibody(clone CL-300) specific to the 45 Ca2+ binding spot of 28 kDcalbindin-D (Celio et al., 1990).

For immunocytochemistry, sections of the LGN were rinsedin phosphate-buffered saline (PBS) and incubated in 1% so-dium borohydride (NaBH4) for 30 min. Following three addi-tional rinses of 10 min each in PBS, the sections were incubatedfor 30 min in 4% normal serum and then in the primary antisera,diluted 1:400 to 1:5000, for 16-18 h. Following three additional

Calbindin in the lateral geniculate nucleus 473

rinses of 10 min each in PBS, the sections were incubated for30 min in 4% normal serum and then for 30 min in the appro-priate secondary antibody with 1% normal serum added. Tri-ton X-100 (0.1-0.25%) was added to both the primary andsecondary antisera. After exposure to the secondary antiserum,sections were again rinsed three times for 10 min each in PBS,and incubated for 1 h in avidin-biotinylated horseradish perox-idase (Vector Labs, Burlingame, CA), followed by three morerinses in PBS for 10 min each and then reacted for 30 s to30 min in 0.05% 3,3'-diaminobenzidine tetrahydrochloride(DAB) in PBS with 0.003% hydrogen peroxide (H2O2). Allsections were then rinsed in PBS before mounting them on glassslides.

Data analysis included measurements of the intensity of im-munoreactivity in cells and neuropil of the deprived and non-deprived laminae and cell size in normal animals. The intensityof immunoreactivity (optical density, OD) of sections was mea-sured with a Joyce-Loebl Magiscan MD image analyzer usinga technique described in detail previously (Mize, 1989). Bothfield and neuron optical density were measured. The total fieldimmunoreactivity was estimated in four sections from monkeyR-001 by measuring the optical density of five fields positionedat approximately equal intervals across each main lamina of theLGN. The fields were positioned so as to avoid large blood ves-sels that would affect the measurements. Results were summedacross the four sections for each lamina in both the left andright LGNs. Percent reductions were calculated by comparingthe optical density value of an affected layer with its adjacentunaffected mate. Thus, on the side contralateral to the enucle-ated eye, affected layer 6 was compared to unaffected layer 5,layer 4 with layer 3, and layer I with layer 2. Similar compari-sons were made in the LGN ipsilateral to the enucleated eye.Differences were tested statistically, first using the nonparamet-ric Kruskal-Wallis one-way analysis of variance to test for over-all significant differences, and then the Mann-Whitney U testto compare differences between adjacent laminae. The opticaldensity of individual immunoreactive neurons was estimated byimaging each obviously labeled cell on the monitor and thentracing around the outer cytoplasmic membrane of the cell witha light pen attached to the image analyzer. The computer thencalculated both the size (area, minimum, and maximum diam-eter) and integrated optical density of the cell. Optical densityvalues were normalized for the size of the cell. Individual cellmeasurements were made from three monkeys as indicated inthe Results. Individual OD cell values were summed for eachlamina, first across sections, and then across animals. The datawere expressed as the percent difference between cells in the de-prived and nondeprived laminae. These differences were alsoanalyzed statistically using the Kruskal-Wallis and Mann-Whitney U tests.

The cross-sectional area of 205 calbindin-immunoreactive(CaBPi) neurons were measured in one normal Rhesus monkey.These measurements also were made with a Magiscan image an-alyzer using the technique described above.

Results

Normal monkeys: cell distribution and morphology

Calbindin-immunoreactive (CaBPi) neurons in the lateral genic-ulate nucleus (LGN) of the normal adult Rhesus monkey rep-resented a small subset of the total neuron population (Fig. 1).Many of the CaBPi neurons were located within the interlam-

inar zones (ILZ) between the main laminae (Figs. 1 and 2A).Others were scattered within each of the main laminae andmore densely within the S layer (Fig. 1). The immunoreactiveneurons in the ILZ were not uniformly distributed across themedial-lateral extent of the ILZ, but were often clumped intogroups of 3-4 neurons, separated by regions devoid of cells(Figs. 1 and 2A). CaBPi neurons within the main laminae weredistributed irregularly throughout each lamina and were foundboth within the center and near the upper or lower borders ofthe ILZ (Figs. 1 and 2).

Light immunoreactivity also was seen in the neuropil in eachof the main laminae. The neuropil immunoreactivity in the Slayer was more dense. This immunoreactivity was found in theproximal dendrites of CaBPi neurons and in smaller immuno-reactive profiles that could be dendrites or axons. However, nosmall densely immunoreactive puncta, typical of axon termi-nals, were seen within the neuropil.

Neurons in both the main laminae and the ILZ were mostlymedium-sized cells. Measurements of 205 CaBPi neurons showedthat the somata had a mean cross-sectional area of 264 /*m2

(Fig. 3). Only one CaBPi cell was larger than 500 jan2 in area,and very few neurons were below 200 ^m2.

Many CaBPi neurons in the ILZ had oblong, fusiform cellbodies with 2-4 proximal dendrites that rapidly branched intoa dense plexus of secondary and tertiary branches (Figs. 4A-4C). In some sections, the dendritic trees were well-labeled andcould be followed for 100-200 (im. In most of these cases, thedendritic trees were distributed parallel to the long axis of theILZ. A few dendrites dipped into the main laminae above andbelow the ILZ, but the bulk of the dendritic tree remainedwithin the boundaries of the ILZ. Although this distributionwas not an invariant feature of CaBPi cells in the ILZ, it wasthe most common pattern found. Only rarely did CaBPi neu-rons with somata in the ILZ have dendrites that extended deeplyinto the main laminae.

CaBPi neurons within the main laminae had more variablesomata and dendritic morphologies. Most neurons had oblongor stellate-shaped somata with dendrites that radiated in all di-rections from the cell (Fig. 5A). Other neurons had dendriteswhose principal orientation was perpendicular (Fig. 5B) to thelongitudinal axis of the layer. Although the dendrites of someCaBPi neurons could be followed for some distance, we foundno neurons in which dendrites could be followed into an adja-cent lamina. The dendrites of neurons whose cell bodies werenear the laminar border sometimes dipped into the ILZ andthen followed the long axis of the ILZ. However, the dendritesof most cells in the main laminae did not enter the ILZ. Basedupon the difference in dendrite arborization and distributionpatterns, we conclude that the neurons immunoreactive forCaBP in the main laminae represent a different neuron popu-lation than those in the ILZ.

Experimental monkeys

Two monkeys raised initially with normal visual experience, butwith one eye enucleated later, were used to determine the effectsof monocular enucleation on CaBP immunoreactivity in theLGN. We found that both short (R-001, 2 weeks) and long-term(R-019, 53 months) enucleation had pronounced effects uponCaBP immunoreactivity. The laminae connected to the enucle-ated eye showed obvious and consistent reductions in immuno-reactivity, as illustrated for monkey R-001 (Fig. 6). CaBPimmunoreactivity was reduced in laminae 1, 4, and 6 in the left

474 R.R. Mize, Q. Luo, and M. Tigges

• " • • • ' ; 'iKsSi! x: v '" : t f . I , • * •'"

" , - * * • • . : >

Fig. 1. Distribution of calbindin im-munoreactivity (CaBPi) in the lateralgeniculate nucleus of a normal adultRhesus monkey. CaBPi neurons weresparsely distributed within the mainlaminae and more densely within theS laminae and the interlaminar zones.Note the presence of light neuropil aswell as cell immunoreactivity. Scalebar = 500 /jm.

LGN contralateral to the enucleated right eye (Fig. 6A) and inlaminae 2, 3, and 5 in the right LGN ipsilateral to the enucle-ated eye (Fig. 6B).

This reduction in immunoreactivity in the deprived layers in-volved both neuropil and cell bodies. The reduction in neuropilimmunoreactivity is clearly illustrated in Fig. 6 where reducedlabeling is seen across the entire extent of the denervated lam-inae on both the left and right sides of the LGN. The effect isclearly a reduction in immunoreactivity in the deprived layersbecause the density of labeling is less in those layers than thedensity of labeling found in other thalamic nuclei and in the ba-sal ganglia. In addition, lighter immunoreactivity is not duesolely to a reduction in somata immunoreactivity becauseCaBPi neurons are only sparsely scattered through the mainlaminae. The reduction probably reflects decreased immunore-

activity in proximal and distal dendrites of these cells and pos-sibly a reduction in axon immunoreactivity as well.

To determine the combined reduction in both neuropil andcell immunoreactivity quantitatively, we measured the opticaldensity of five fields across the medial-lateral extent of eachlamina in four sections from animal R-001. We compared eachdeprived lamina with its nondeprived pair (6 with 5, 4 with 3,and 1 with 2) separately for the left and right LGNs in order toavoid variability in labeling caused by differences in overall la-beling intensity in sections reacted in different vials. Whensummed across four sections, the reduction in immunoreactiv-ity in the affected vs. unaffected laminae ranged from 9.05-12.6% (an average of 11.5%). Similar percentage reductionswere seen on both the left side (in which the contralateral lam-inae 1, 4, and 6 were reduced) and the right side (in which the

Calbindin in the lateral geniculate nucleus 475

Fig. 2. CaBP-immunoreactive neurons in the LGN of a normal adult Rhesus monkey. A: Labeled neurons in the ILZ betweenlaminae 1-2, 2-3, 3-4, and 4-5. B: Labeled neurons in main lamina 5. Broken lines indicate the approximate boundaries of theILZ. Scale bars in A = 100 fim; B = 50 ^m.

ipsilateral laminae 2, 3, and 5 were reduced). This consistent re-duction in immunoreactivity was seen despite the fact that sec-tions of the left and right LGNs had different overall intensitiesof immunoreactivity (Fig. 7). Although the percent reductionis modest, comparison of the standard errors of the mean in ad-

20

O

LU

oDCLUQ .

I

II

• • N = 205• • MEAN - 264.121,111'• • RANGE = 131.77-606.74MmJ

IlllulIlllllll... .150 300

AREA450 600

Fig. 3. Histogram of the cross-sectional area of 205 neurons measuredin the ILZ and main laminae of the LGN of a normal adult Rhesusmonkey.

jacent laminae illustrates the robustness of the effect (Fig. 7).Overall differences in immunoreactivity were significant at theP = 0.001 level using a nonparametric Kruskal-Wallis analysisof variance (left side: H = 42.3, df = 5; right side: H = 69.7,df= 5). Mann-Whitney t/test comparisons between adjacentlaminae showed significant differences in optical density valuesat least at the P = 0.0002 level. Similar reductions in field im-munoreactivity were observed qualitatively in the other two an-imals that had a unilateral enucleation but no occlusion.

Reduction in immunoreactivity also was observed in mon-keys that received both a short-term enucleation and were alsoraised under monocular deprivation conditions at or shortly af-ter birth. Monkey R-003 had the natural lens of the right eye re-placed with an intraocular lens (IOL); that eye was enucleated15 days prior to perfusion. Monkey R-008 had the right eye oc-cluded from birth; that eye was enucleated 14 days prior to per-fusion. Reduction in CaBP immunoreactivity in both neuropiland neurons was found in the denervated laminae of bothLGNs in these two animals. The same effect was observed inbinocularly deprived monkeys if these monkeys had one eyeenucleated prior to perfusion (Table 1). The binocularly de-prived monkeys had the natural lens removed surgically fromthe right eye and the resulting aphakia corrected optically withEWCLs. The fellow eye was occluded either CO or PO duringdaylight hours. The three monkeys with this treatment that alsohad one eye enucleated prior to perfusion showed a reduction

476 R.R. Mize, Q. Luo, and M. Tigges

Fig. 4. A-C: Morphology of CaBP-labeled neurons in the ILZ of a nor-mal adult Rhesus monkey. Note thatthe neurons have fusiform somataand dendrites which remain for themost part within the boundaries ofthe ILZ. Scale bar = 20 inn.

in CaBPi in both hemispheres in all layers connected to the enu-cleated eye. The reduction in immunoreactivity in laminae con-nected to an enucleated eye is thus a consistent and reproducibleeffect regardless of whether the opposite eye is deprived.

The effect of monocular enucleation on individual cell bodylabeling was as obvious as the reduction in neuropil labeling.An example of the reduction in cell labeling is shown in Fig. 8for monkey R-008. Cells in laminae 1, 4, and 6, which wereconnected to the unmanipulated left eye, were densely immu-noreactive (Figs. 8A-8C). By contrast, neurons in the deaffer-ented laminae 2, 3, and 5 also were immunoreactive, but clearlyless so than the neurons in layers 1, 4, and 6 (Figs. 8D-8F).These effects on cell immunoreactivity were also seen in other

animals. To determine the degree of the effect, we measured theoptical density of cells from the main layers of four of the mon-ocularly enucleated monkeys, two with short-term enucleation(R-001 and R-003), one with long-term occlusion and subse-quent short-term enucleation of the occluded eye (R-008), andone monkey of the binocularly deprived group with part-timeocclusion of the left and short-term enucleation of the rightaphakic eye (R-929). Reductions in CaBP immunoreactivityranging from 13.9 to 23.8% were found in cells in the deaffer-ented layers compared with cells in the adjacent nondeprivedlayers.

CaBP immunoreactivity in the LGN also was examined inmonkeys either monocularly or binocularly deprived from

Calbindin in the lateral geniculate nucleus 477

A I

Fig. S. Morphology of CaBP-labeledneurons in the main LGN laminae

, i of a normal adult Rhesus monkey.# i | A: CaBPi neuron with broad, pro-

fusely branching dendritic tree. B:CaBPi neurons with dendritic trees

*i oriented perpendicular to the longaxis of the lamina. Scale bar = 20 nm.

birth, but without enucleation of one eye. Long-term monoc-ular occlusion had no obvious effect on CaBP immunoreactiv-ity. Fig. 9 shows CaBP immunoreactivity in animal R-012 thathad the right eye occluded from birth until sacrifice (22 months,Table 1). Immunoreactivity in this animal was somewhat vari-able, probably due to uneven penetration of the antibody.Thus, for example, the lateral LGN was less densely labeledthan the central LGN. Despite this variability, considerable re-action product was found in both the neuropil and cell bodiesin all six main laminae and in lamina S. For the most part, thedensity of immunoreactivity in the affected laminae was as greatas in the laminae connected to the undeprived eye. Althoughthere was a slight reduction of immunolabeling in lamina 4 onthe left (contralateral) side and lamina 5 on the right (ipsilateral)

side, no differences in immunoreactivity were seen in the otherlaminae in this animal.

We also found no apparent differences in antibody labelingbetween laminae connected to the right and laminae connectedto the left eye under the condition of binocular deprivationfrom birth, where an optically corrected aphakic eye was com-bined with a part-time (R-009) or a continuously (R-933) oc-cluded fellow eye. The immunoreactivity patterns in these animalsdo not appear to be different from normal animals. Therefore,we conclude that the CaBP system in the LGN of Rhesus mon-keys is also resistant to binocular deprivation from birth.

Taken together, our results show that interference with thevisual inputs from birth by aphakia and/or occlusion, whichleaves the anatomical connections intact, has no apparent effect

478 R.R. Mize, Q. Luo, and M. Tigges

fj.' '• ' "• *v

Fig. 6. CaBP immunoreactivity in the LGN of a monocularly enucleated Rhesus monkey. A: Left LGN. B: Right LGN. Themonkey (R-001) had its right eye removed 2 weeks before sacrifice. Note the reduced immunoreactivity in the deprived lami-nae 1, 4, and 6 in the left LGN and 2, 3, and 5 in the right LGN. Scale bar = 1 mm.

on CaBP immunoreactivity of LGN neurons and neuropil inRhesus monkeys. In contrast, interruption of the anatomicalconnections between the eye and the LGN via enucleation re-sults in a pronounced reduction in CaBP immunoreactivity inthose LGN layers that were connected to the enucleated eye.

Discussion

CaBPi neurons in the LGN of normal monkey

Calbindin antibody labeled a small fraction of neurons in theLGN of Rhesus monkeys. CaBPi neurons were found mostcommonly in the ILZ, but were also scattered within each of themain laminae of the LGN and more densely within the S lam-inae. These CaBPi neurons probably represent two distinct celltypes.

The CaBPi neurons whose cell bodies lie within the ILZ wererelatively homogeneous in morphology. Most were medium-sized cells, many with fusiform cell bodies, and their dendriteswere largely confined to the region of the ILZ. These cells aresimilar to one variety of class C neuron described in a Golgistudy by Wilson and Hendrickson (1981). According to theseauthors, type C neurons were seen rarely, and had smooth, fu-siform cell bodies, and relatively short dendritic trees with fewspines. They were the only cell type described by Wilson andHendrickson (1981) whose cell body and dendrites lay almostexclusively within the ILZ. Wilson and Hendrickson (1981)suggest that these cells are projection cells, not interneurons.Horseradish peroxidase (HRP) retrograde transport and cortical

lesion studies show that ILZ cells project primarily to prestri-ate cortex (Benevento & Yoshida, 1981; Yoshida & Benevento,1981; Dineen et al., 1982). These neurons are likely to be thesame as those labeled by CaBPi in the ILZs.

The CaBPi neurons within the main laminae of LGN had adifferent morphology than those in the ILZ. Like ILZ cells,CaBPi laminar cells had mostly medium-sized cell bodies andcomplex dendritic fields that branched repeatedly. However, thelaminar cells differed from the ILZ cells in that they were scat-tered within the main laminae and their dendritic trees weremost often symmetrically distributed about the cell body or ori-ented perpendicular to the long axis of the lamina. In addition,the dendrites of the laminar cells rarely entered the ILZ andwere never seen to cross laminar borders. Thus, these cells prob-ably represent a different CaBPi cell type than that in the ILZ.The laminar CaBPi cells bear no clear relationship to any celldescribed in Golgi studies (Wilson & Hendrickson, 1981; Saini&Garey, 1981).

CaBPi neurons in the main laminae and the ILZ differ notonly in morphology, but also in their synaptic inputs. Althoughaxon terminals from the retina play a major role in the synap-tic circuitry of neurons in the main laminae, the ILZ receiveonly very few retinal terminals (Wilson & Hendrickson, 1981).Instead, projections from the superior colliculus, brain stem,and striate cortex predominate in the ILZ (Irvin et al., 1986).These cells must thus be influenced primarily by input otherthan that from the retina. There is also evidence for a functionaldifference. Neurons exhibit X- and Y-like properties in the par-vocellular and magnocellular laminae, whereas neurons in the

Calbindin in the lateral geniculate nucleus

12000 7

10000

RIGHT LGN

6000

Fig. 7. Histograms showing the reduction in mean optical density in thedenervated vs. nondenervated laminae of Rhesus monkey R-001, whoseright eye was enucleated 2 weeks prior to sacrifice. Error bars representthe standard error of the mean. A: Left LGN. B: Right LGN.

ILZ have been shown to be W-like in their response properties,at least in the Galago (Norton & Casagrande, 1982; Irvin et al.,1986). The cells in the main laminae and those in the ILZ arethus likely to differ in afferent input and physiology as well asin morphology.

It is unlikely that the CaBPi neurons are GABAergic inter-neurons. GABAergic neurons are far more numerous in theLGN of monkeys (Hendrickson et al., 1983; Hendry, 1991; ourown observations) than CaBPi neurons. In addition, they dif-fer in size and distribution, as most GABAergic neurons aresmaller, are concentrated within the center of the main laminae,and are rare within the ILZ. This conclusion is supported byJones and Hendry (1989) who failed to find CaBPi neurons thatco-localized GABA in double-labeling experiments in the LGNof M. fascicularis. If CaBPi neurons are GABAergic, they mustrepresent a very rare subtype of the total GABA-immunoreac-tive cell population in the LGN of monkeys.

The pattern of CaBP labeling in the monkey LGN differsdramatically from that seen in the LGN of the cat. In cat, be-tween 20 and 100% of LGN neurons are labeled by CaBP anti-body, depending upon the lamina examined (Demeulemeesteret al., 1989,1991; Banfro & Mize, 1992). In the A laminae,CaBPi neurons include small GABA-containing neurons andmedium-sized cells with cytoplasmic laminated bodies that arethought to be X cells (Demeulemeester et al., 1991; Banfro &

479

Mize, 1992). Virtually all neurons in the parvocellular C lami-nae also are labeled by CaBP antibody, indicating that W cellsalso are CaBP immunoreactive (Banfro & Mize, 1992). Directco-localization studies have shown that about 60% of CaBPicells are immunoreactive for GABA (Demeulemeester et al.,1991; Banfro & Mize, 1992).

The CaBPi neurons in monkey and cat LGN also differ inother features. In monkey, CaBP and parvalbumin, anothercalcium-binding protein, do not co-occur in the same neurons(Jones & Hendry, 1989). PV-immunoreactive neurons representa large percentage of all cells (Jones & Hendry, 1989; Tigges &Tigges, 1991), whereas CaBPi neurons are very rare in the mon-key LGN (Jones & Hendry, 1989; present study). In cat, bycontrast, CaBP and PV do co-occur in a significant number ofLGN neurons (Demeulemeester et al., 1989,1991) and both cal-cium-binding proteins are found in a large percentage of the to-tal cell population. The reasons for these species differences areunknown, but could relate in part to the differing functionalproperties of some cells in the LGNs of the two species, partic-ularly the presence of color opponent neurons in monkey.

Deprivation effects

Our results have shown that there is a significant reduction inCaBP immunoreactivity in laminae of the LGN that were de-nervated by eye enucleation two weeks to 53 months prior tosacrifice. By contrast, long-term monocular and binocular de-privation without enucleation had little or no effect on CaBPimmunoreactivity in the Rhesus monkey LGN.

Enucleation also affects another calcium-binding protein aswell as the neurotransmitter GABA in the visual system of adultanimals. In the LGN, PV immunoreactivity is reduced by 2weeks to 7 years of monocular enucleation. The reduced anti-body labeling appears to be confined to immunoreactive neu-ropil (Tigges & Tigges, 1991) and not cell bodies. Because PVantibody densely labels retinal axon terminals, it is likely thatthe effect is due to degeneration or loss of these axons (Tigges& Tigges, 1991).

GABA and its synthetic enzyme glutamic-acid decarboxyl-ase also are reduced by deprivation. Both short-term monocu-lar enucleation and tetrodotoxin (TTX) injection into one eyeof the adult monkey reduce immunoreactivity in the affectedlayers of the LGN (Hendry, 1991). However, the reduction inlabeled cell numbers does not occur until survival periods of3-4 weeks and may therefore be secondary to effects that oc-cur in the striate cortex (see below).

GABA also is reduced by deprivation in the adult cat LGN.Four weeks of monocular enucleation or TTX application re-duced the number of small, highly immunoreactive GABA neu-rons in affected LGN laminae (Luo et al., 1991). The numberof neurons darkly stained by cytochrome oxidase also was re-duced in the affected layers (Luo et al., 1991). Thus, resultsfrom several laboratories have shown that the chemical compo-sition of LGN neurons can be altered by brief periods of depri-vation in both adult monkeys and cats.

The nature of these activity-dependent alterations in adultanimals is uncertain. TTX, which blocks retinal ganglion cellaction potentials, is effective in reducing GABA immunoreac-tivity. Thus, reduced neural activity is sufficient to produce theeffect on this inhibitory neurotransmitter. On the other hand,we failed to find significant reductions in CaBP immunoreac-tivity after nearly 2 years of lack of visual experience using con-

480 R.R. Mize, Q. Luo, and M. Tigges

Fig. 8. CaBP immunoreactivity in neuronsfound in the denervated vs. nondenervatedlayers of Rhesus monkey R-008, whose righteye was enucleated 2 weeks prior to sacrifice.Note the reduced immunoreactivity in neuronsin the deprived layers 5, 3, and 2 (D,E,F) vs.the nondeprived layers 6, 4, and 1 (A,B,C).The enucleated eye also was occluded for 20months. Scale bar = 10 ^m.

tinuous opaque occluders, which indicates that prolonged visualdeprivation alone is not sufficient to produce an effect on thiscalcium-binding protein. Even 7 years of deafferentation ap-pears insufficient to alter neuronal PV immunoreactivity. Thus,the effects appear to vary with the chemical examined, with thelength of deprivation, and with the type of deprivation (dener-vation vs. reduced pattern stimulation and retinal activity).

The effects of monocular deprivation on adult monkey stri-ate cortex are more severe and more consistent. Unilateral enu-cleation, lid suture, and retinal ganglion cell blockade with TTXall reduce the number of GABA-immunoreactive neurons in theocular-dominance columns of area 17 of the adult cynomolgusmonkey. In cortex, the effects of enucleation and intraocularTTX application can be seen within 4-5 days (Hendry & Jones,1986; Hendry & Carder, 1992). Intraocular TTX applicationalso affects calcium-binding protein immunoreactivity in striatecortex. PV immunoreactive neurons in the affected ocular-dom-

inance columns are reduced in number, whereas the number ofCaBPi neurons is increased in the ocular-dominance columnsrelated to the normal eye (Hendry & Carder, 1992). The effectsin adult monkey striate cortex on both GABA and the calcium-binding proteins thus occur after shorter periods of deprivationand with blockade of retinal activity as well as enucleation.

The differences in effect on visual cortex and LGN suggestthat the mechanisms underlying the alterations are different inthese two structures. In the adult visual cortex, the effect on theGABA system is likely the result of a reduction in transmittersynthesis in the affected neurons because the reduction occursin neurons that are not directly denervated and occurs also af-ter lid suture, which eliminates pattern vision, and intraocularinjection of TTX, which eliminates neural activity, without de-stroying retinal ganglion cell axons (Hendry & Jones, 1986;Hendry & Carder, 1992). By contrast, the effect on CaBP im-munoreactivity in the LGN probably is caused by deafferenta-

Calbindin in the lateral geniculate nucleus 481

Fig. 9. CaBP immunoreactivity in the LGN of a monocularly occluded Rhesus monkey. A: Left LGN. B: Right LGN. The mon-key (R-012) had its right eye continuously occluded with an opaque contact lens 22 months before sacrifice. Immunoreactivityis variable but there is no consistent reduction in antibody labeling in the deprived layers 1, 4, and 6 on the left or deprived layers2, 3, and 5 on the right. Scale bar = 1 mm.

tion. Enucleation, which denervates LGN neurons, reducesCaBP reactivity, whereas monocular and binocular deprivationdoes not.

Acknowledgments

We are grateful to Dr. Robert Spencer of the Department of Anatomy,Medical College of Virginia, Richmond, and Dr. John Porter of the De-partment of Anatomy, University of Kentucky, Lexington, for the nor-mal tissue. Dr. Piers Emson of the Medical Research Council Group,AFRC, Institute of Animal Physiology and Genetics Research, Cam-bridge, England, provided us with his calbindin antibody. Grace Butlerhelped prepare the figures. She and C.J. Jeon assisted in the immuno-cytochemistry experiments. This work was supported by USPHS GrantsN1H EY-02973 (R.R.M.) and EY-05975 (Dr. Ron Boothe) from the Na-tional Eye Institute and by RR-OO165 from the National Center for Re-search Resources to the Yerkes Regional Primate Center.

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