Neuroscience Letters 395 (2006) 149–154

Changes of parvalbumin immunoreactive neurons and GFAPimmunoreactive astrocytes in the rat lateral geniculate

nucleus following monocular enucleation

Daniel Gonzaleza,c, Irawan Satriotomoa,d,∗, Takanori Mikia, Kyong-Youl Leea,Toshifumi Yokoyamaa, Tetsuo Tougeb, Yoshiki Matsumotoa, Hong-Peng Lia,

Shigeki Kuriyamac, Yoshiki Takeuchia

a Department of Anatomy and Neurobiology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kagawa 761-0793, Japanb Department of Health Sciences, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kagawa 761-0793, Japan

c Third Department of Internal Medicine, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kagawa 761-0793, Japand Department of Comparative Bioscience, School of Veterinary Medicine, University of Wisconsin, 2015 Linden Dr., Madison, WI 53706, USA

Received 27 July 2005; received in revised form 21 October 2005; accepted 26 October 2005

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The expression of calcium binding proteins (CaBPs) has been linked to protection of neurons. The present study investigated thonocular enucleation on the distribution of parvalbumin immunoreactive (PV-IR) neurons and glial fibrillary acidic protein immuno

GFAP-IR) astrocytes in both the dorsal (dLGN) and ventral (vLGN) regions of the lateral geniculate nucleus (LGN). Our results demn increase in PV-IR neuronal density on the contralateral vLGN at 1-week post-enucleation (PE), which was maintained withouthange for 12 weeks. By contrast, PV-IR neurons in dLGN decreased significantly at all time point examined. The number of GFAP-IRhowed an initial increase from 1 to 4 weeks PE and then gradually decreased until 48 weeks in both regions of the LGN with contraredominance. The present results suggest that monocular enucleation results in variable expression of PV-IR neurons and GFAP-IR

he LGN complex, which may play an important role in neuronal degeneration and neuroplasticity of the rat visual system.2005 Elsevier Ireland Ltd. All rights reserved.

eywords: Enucleation; Lateral geniculate nucleus; Parvalbumin; GFAP; Neuronal degeneration

he rodent visual system is a good model to study centralervous system (CNS) injury and regeneration induced byonocular enucleation. Following retinal deafferentation,

hanges can be observed at different levels in the visual path-ays. Moreover, the highly-crossed retinal projections (about5–97%) in this species offer a clear advantage to compare both

he contralateral and ipsilateral sides after enucleation[8,9]. Inhe visual system, the LGN has been investigated frequentlyecause of its attractive position as the primary relaying nucleiodulating retinal-cortical transmission of visual information

20,27]. We suggest that functional differences between adja-ent divisions, dLGN and vLGN could correspond to differentechanisms of adaptation following monocular enucleation.

∗ Corresponding author. Tel.: +1 608 263 5013; fax: +1 608 263 3926.E-mail address: satriotomo@wisc.edu (I. Satriotomo).

Several studies suggest monocular enucleation resuprofound changes in the LGN[5,11,12,21]. Parvalbumin (PV)a commonly used neuronal marker[2], is increased followinvisual pathway injury[18,26]. Changes have also been noin other cell types, such as neuroglia. For example Gexpression is increased in astrocytes shortly following neudamaged[1,7,13]. Although many studies have used thneuronal and glial markers to investigate the cellular respto acute CNS injury, little is known about their responselong-term deafferentation. Furthermore, most studiesconcentrated on the mechanisms of neuronal plasticideveloping brain following CNS injury[30,32]. Howeverrecent evidence suggests that the adult brain is also capaCNS plasticity following neuronal injury[14,22]. Therefore, thpurpose of the present study was to evaluate the short- andterm effects of monocular enucleation on neuronal-astrointeractions in the LGN complex in adult rats.

304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.neulet.2005.10.067

150 D. Gonzalez et al. / Neuroscience Letters 395 (2006) 149–154

Eight weeks-old male Wistar rats from SLC (Shizuoka,Japan) were housed in cages under controlled conditions with aconstant temperature (23± 1◦C), kept in a 12-h light/dark cycleand received water and food ad libitum for a minimum 1-weekacclimation period prior to the onset of the experiment. Theanimals were divided into two groups: the control group (n = 7),and the experimental group (n = 35). The experimental group,which received unilateral enucleation in the left eye at 9 weeksof age, were allowed to survive for 1, 4, 12, 24, and 48 weeksbefore sacrifice. Monocular eye enucleation was performed bydissection of periorbital tissue with Irish scissors, resecting theoptic nerves, removing the left eye and then suturing the eyelid toprevent bleeding. Surgery was conducted under deep anesthesiawith sodium pentobarbital (60 mg/kg, i.p.). The experimentalprocedures were conducted in accordance with the NIH Guidefor Care and Use of Laboratory Animals. The Kagawa Univer-sity Animal Committee approved the procedures and every effortwas made to minimize both the number of animals used and theirsuffering.

For immunohistochemistry procedures, the rats from the con-trol group (n = 7) and the experimental group (totaln = 35,n = 7per group at each time point) were deeply anesthetized withsodium pentobarbital (60 mg/kg i.p.), and perfused transcar-dially with phosphate buffered saline (0.02 M PBS, pH 7.4)followed by a fixative of 4% paraformaldehyde in PBS. Thebrains were removed and cryoprotected in 30% (w/v) sucrosea st uno-h onsw rumw inm ) ora O,G eI tinc or6 xidef arya lden

onsa GNr dt thev asn beinn eena ctioi ronsa . Foe V, ant unoh eacas digt hs oi rom

overlapping images. Only PV-IR neurons or GFAP-IR astro-cytes, which exhibit their cell bodies, were identified as singlecells for counting. The number of IR-cells within the countingframe was counted manually and transformed into the numeri-cal density by applying the counting frame area. Data representthe mean± standard error mean (S.E.M.). Data was analyzedusing two-way analysis of variance (ANOVA). Post hoc testswere carried out where appropriate using Tukey–Kramer’s test.All statistical analyses were performed using Minitab statisticalsoftware withp values being less than 0.05.

Significant differences in PV-IR were found between theregions of the LGN in control rats. The vLGN had more PV-IRneurons and stronger labeling in neuronal soma and processesthan the dLGN (Fig. 1I and J), which had presented very scarceand weak PV-IR in control animals (Fig. 1A and B). Labelledneurons localized more centrally in vLGN and were surroundedby bundles of PV-IR fibers oriented parallel to the optic nerve.At higher magnification, the neurons were frequently ovoidor polygonal (multipolar) in shape, and the PV-IR distributedthroughout the perikarya and the proximal parts of processes(framed area). At 1-week PE, the number of PV-IR neuronsincreased and showed stronger homogeneous labeling in con-tralateral vLGN. The cell bodies and their processes appearedto be stained intensely compared to the ipsilateral side (Fig. 1Cand D). At 12 weeks PE, contralateral vLGN had a slight but sta-tistically significant reduction in the PV-IR neurons comparedtR phic.P earedta undt hosea( bvi-o , inc ilat-e f theL sses,w rea).S GNc ctsi th neu-r thed fP weda ever,t sig-n aftere

P-IRa enic-u ns.T d tot s inc hort-a g in

t 4◦C until the brains sank. Serial 40�m thick frozen sectionhrough the LGN were cut coronally and processed for immistochemistry for PV and GFAP. Briefly, free-floating sectiere incubated in following solution: 10% normal goat seith 0.2% Triton X-100 in PBS for 60 min, anti-parvalbumouse monoclonal antibody (1:200 dilution, Sigma, USAnti-GFAP rabbit polyclonal antibody (1:2000 dilution, DAKlostrup, Denmark) overnight at 4◦C, biotinylated anti-mous

gG or biotinylated anti-rabbit IgG for 60 min, avidin–bioomplex (Elite ABC Kit, Vector Lab., Burlingame, USA) f0 min and 0.05% diaminobenzidine–0.03% hydrogen pero

or 3–5 min. Control sections omitting the primary or secondntibodies were processed identically and consistently yieo labeling.

For a morphometric study, the number of PV-IR neurnd GFAP-IR astrocytes were counted in the dLGN and vLegions. The counting frame (350�m× 240�m) was set arounhe lateral portion of the dLGN and around the center ofLGN. An initial section that contained any sign of the LGN wumbered as the first section, with all subsequent sectionsumbered sequentially. For each animal, a number betwnd 4 was selected randomly in order to determine the se

n which counting was to begin. Subsequently, PV-IR neund GFAP-IR astrocytes were counted every 4th sectionsxample, the 1st, 5th, 9th, etc. sections were selected for Phe 2nd, 6th, 10th, etc. sections were chosen for GFAP immistochemistry, respectively. Seven to eight sections fromnimal were used in this study. Photographs (×400) taken fromelected sections were captured by CCD color camera,ized and uploaded into a personal computer. Photograpndividual cells were used in order to avoid interference f

d

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o 1-week PE (426.88± 6.72 versus 390.80± 7.17, p < 0.05).etinal fibers of the optic nerve also appeared slightly atroV-IR neurons were also confined more centrally and app

o be segregated at 12 weeks in the contralateral vLGN (Fig. 1End F). At 48 weeks survival time, PV-IR neurons were fo

o be significantly decreased in the vLGN compared to tt 24 weeks PE (332.76± 6.36 versus 355.88± 6.94,p < 0.05)Fig. 1G and H). Atrophy of the optic nerve became more ous on the contralateral side (white arrowhead) of the LGNontrast to the thick and well-stained optic nerve in the ipsral side (black arrowhead). PV-IR neurons in both regions oGN demonstrated a weak labelling in cell bodies and procehich was most apparent on the ipsilateral side (framed atatistical analysis of numerical density of PV-IR in the Lomplex (dLGN and vLGN) showed significant main effen time, side and time× side interaction (Table 1A and B). Posoc analysis with Tukey–Kramer’s test revealed that PV-IRons were predominantly packed in the vLGN compared toLGN in control rats. As seen inFig. 1I and J, the number oV-IR neurons in the vLGN on the contralateral side shosignificant decrease between 12 and 48 weeks PE. How

he number of PV-IR neurons in the dLGN demonstrated noificant change on either side between 12 and 48 weeksnucleation.

The LGN presented scattered but well-stained GFAstrocytes especially on the pial surface and in the interglate leaflet (IGL) located between dLGN and vLGN regiohe dLGN exhibited fewer labelled astrocytes compare

hose in the vLGN in the control rats. Labelled astrocyteontrol section had small stellate-shaped cell bodies with snd thin-ramified processes and weak GFAP-IR labellin

D. Gonzalez et al. / Neuroscience Letters 395 (2006) 149–154 151

Fig. 1. Photomicrographs of PV-IR neurons in the LGN of control (A, B) andenucleated rats (C–H). The PV-positive neurons have intense labeling in thevLGN compared to those in the dLGN (A, B). Sections in C and D, E and F and Gand H represent LGN regions at 1, 12 and 48 weeks after monocular enucleation,respectively. The sub-field of the vLGN (framed area) is magnified to show thedetail of PV-IR neurons in each time point, respectively. Note that at 1-weekpost-enucleation, the PV-positive neurons on the contralateral vLGN appearedto be increased in number and enhanced their IR-intensity. Atrophic optic nerve(white arrowheads) showed more prominent in the contralateral side compareto the ipsilateral side (black arrowheads). Graphics of morphometric analysisshowed the PV-IR neuronal density in the dLGN (I) and vLGN (J) regionsat different times post-enucleation. The continuous and broken lines representthe contralateral side and the ipsilateral side, respectively. dLGN, dorsal lateralgeniculate nucleus; IGL, intergeniculate leaflet; vLGN, ventral lateral geniculatenucleus. Scale bars: 200�m; 50�m in framed area.

both perikarya and proximal processes (Fig. 2A and B). Theexperimental sections at 1-week PE presented a notable increaseof GFAP-IR in both regions of the LGN with contralateral sidepredominance. The densely-stained astrocytes were observedto be homogeneously distributed throughout the LGN. TheseGFAP-IR astrocytes appeared to be transformed to reactiveastrocytes with many appearing heavily branched with stronglylabelled and thickened processes (Fig. 2D). At 12 weeks PE,the number of GFAP-IR astrocytes in the contralateral LGN

Fig. 2. Photomicrographs of GFAP-IR astrocytes in LGN of control (A, B) andenucleated rats (C–H). The GFAP-IR astrocytes have intense labelling in theIGL. Sections in C and D, E and F and G, H represent LGN regions at 1, 12 and 48weeks after monocular-enucleation, respectively. The sub-field of vLGN (framedarea) is magnified to show the detail of GFAP-IR astrocytes in each time point,respectively. Note that at 1-week post-enucleation, the GFAP-IR astrocytes wereincreased in number on the contralateral side. Those labelled astrocytes werecharacterized by dense, thick and hypertrophy of cell body with long processes.Graphics of morphometric analysis showed the GFAP-IR astrocytes density inthe dLGN (I) and vLGN (J) regions at different times post-enucleation. Thecontinuous and broken lines represent the contralateral side and the ipsilateralside, respectively. dLGN, dorsal lateral geniculate nucleus; IGL, intergeniculateleaflet; vLGN, ventral lateral geniculate nucleus. Scale bars: 200�m; 50�m inframed area.

decreased relative to 1 and 4 weeks PE and were distributedsparsely, while on the ipsilateral side no significant changeswere observed. Moreover, the entire area of contralateral LGNoccupied by GFAP-IR astrocytes appeared to be shrunken andbounded by a slightly atrophic optic nerve (white arrowhead). Inaddition, by 12 weeks PE, astrocytes showed a weak expressionof GFAP-IR and were characterized by smaller cell bodiesand thin/short processes (Fig. 2E and F). At 48 weeks PE, ourimmunohistochemical analysis revealed that very few GFAP-IRastrocytes with short and thin processes that were dispersed onboth sides compared to earlier survival times. The optic nerveappeared to be more atrophic, and the regions of the contralat-

152 D. Gonzalez et al. / Neuroscience Letters 395 (2006) 149–154

Table 1

A. Data on the number of labeled-cells (cells/mm2) in the rat LGN following monocular enucleationa

Time Side PV-IR GFAP

dLGN vLGN dLGN vLGN

Control (n = 7) R 67.22± 3.56 360.08± 7.01 194.48± 7.23 245.58± 6.41L 68.05± 3.32 361.34± 7.62 197.42± 5.52 246.31± 5.16

1 week (n = 7) I 32.76± 2.16 338.64± 5.48 241.17± 7.49 298.82± 8.37C 46.21± 2.48 426.88± 6.72 444.70± 9.05 500.00± 9.57

4 weeks (n = 7) I 18.90± 1.94 329.82± 5.21 247.05± 7.83 310.18± 8.38C 32.76± 2.97 400.83± 6.94 493.32± 8.83 542.35± 9.85

12 weeks (n = 7) I 12.12± 1.70 326.09± 4.02 255.29± 8.64 324.70± 10.42C 21.31± 1.72 390.80± 7.17 397.64± 9.43 463.12± 10.21

24 weeks (n = 7) I 10.08± 1.67 318.90± 6.90 225.42± 5.56 285.76± 8.70C 15.96± 2.18 355.88± 6.94 306.25± 8.88 428.62± 7.60

48 weeks (n = 7) I 8.82± 1.67 308.82± 5.22 203.35± 4.75 246.63± 6.15C 13.85± 2.63 332.76± 6.36 250.00± 6.67 373.10± 4.97

B. Results of two-way ANOVA of the above data

dLGN vLGN

d.f. F d.f. F

(1) ParvalbuminSide 1, 324 34.25** 1, 324 158.06**

Time 5, 324 164.76** 5, 324 22.81**

Side× time 5, 324 2.29* 5, 324 12.36**

(2) GFAPSide 1, 324 665.74** 1, 324 883.66**

Time 5, 324 143.42** 5, 324 125.49**

Side× time 5, 324 68.21** 5, 324 47.00**

dLGN, dorsal lateral geniculate nucleus; vLGN, ventral lateral geniculate nucleus; ANOVA, analysis of variance; d.f., degrees of freedom;F, F-value.a The values are presented as mean± S.E.M.; R, right part of control groups; L, left part of control groups; I, ipsilateral part of enucleated groups; C, contralateral

part of enucleated groups;n, number of animals examined.* p < 0.05.

** p < 0.01.

eral side became shrunken and smaller compared to those on theipsilateral side (Fig. 2G and H). Some glial cells demonstratedmild IR-labeling and were distributed sparsely (framed area).Quantitative analysis confirmed the significant main effectsof time, side, and time× side interaction (Table 1A and B).As shown inFig. 2I and J, the number of GFAP-IR astrocytessignificantly increased in the contralateral LGN complex from1 to 4 weeks after enucleation and then gradually decreasedduring the last 12 weeks of survival time. In contrast, thenumber of GFAP-IR astrocytes in the ipsilateral LGN increasedgradually up to 12 weeks and then decreased afterward.

The present study demonstrates differences between dLGNand vLGN in the number of PV-IR neurons in control rats andrats following monocular enucleation (Table 1A). Specifically,we found that PV-IR neurons are more concentrated in the vLGNthan the dLGN in control rats. The high prevalence of PV-IRexpression in vLGN could be due to the particular activation pat-terns in this neuronal sub-population. Interestingly, the presentstudy demonstrates significant enhancement of PV-IR neurons inthe contralateral vLGN at early time points PE. These results areconsistent with previous studies showing significant increasesin PV-positive neurons in the rat subvisual system in response

to deafferentation[18,27]. However, some reports conflict withthese finding with some investigators showing no significantchange[12] or even a reduction[31] in PV-IR in the LGN follow-ing monocular deprivation. The discrepancies may be explained,in part, by differences in age and species (e.g., rats versusmonkeys), the experimental design, or time post-enucleation.

Since functional denervation increased intracellular calciumconcentrations[35] and injury is associated with a transitoryincrease in PV-IR[18,27,33], PV may be important in survivalof injured neurons. We suggest that certain populations of PV-positive neurons concentrated in the vLGN region might havecapacity to protect against neuronal death from degenerativeprocesses by increasing intracellular Ca2+ during the first weeksafter optic nerve denervation. Indeed, we demonstrate that thenumber of PV-IR neurons remains constant and higher than con-trol rats until at least 12 weeks of PE. After 12 weeks, however,the number of PV-IR neurons decreases steadily for at least 48weeks PE. These data indicate that the regulation of intracellu-lar calcium persist for several weeks after injury. Taken together,our results suggest that monocular enucleation affects functionalactivity of different subregions of LGN and causes intracellularredistribution of PV.

D. Gonzalez et al. / Neuroscience Letters 395 (2006) 149–154 153

In a previous study, we observed that neuronal death isassociated with down regulation of calbindin D28k-IR (CB-IR)in the LGN complex after optic nerve denervation[11]. Mize etal. also showed that monocular enucleation caused a reductionof CB-IR in deafferented lamina of dLGN in the monkey[21].This reduction in CB-IR neurons in the LGN complex couldbe related to the functional dependence of specific neuronalpopulations on retinal input. Interestingly, there are differencesin neurotransmitter content between CB- and PV-IR neurons.Parvalbumin has been discovered to be associated with specificsubpopulations GABAergic neurons in the cerebral cortex,LGN and hippocampus[6,17,29]. On the other hand, CB hasbeen observed in the GABAergic and non-GABAergic neuronsin the rat cerebral cortex, and the pulvinar and lateral posteriornuclei of cat thalamus[10,23]. Thus, differences in PV- andCB-IR expression in the vLGN and dLGN regions could bedue to the presence of a subpopulation of neurons distinguishedby their neurochemical content and their functional activity inresponse to monocular enucleation.

Impairment of intracellular calcium buffering is a commonlycited phenomenon in aged neurons and is considered tocontribute to age-associated neurodegeneration in the CNS.Although we found a reduction of PV and CB immunoreactivitybetween 32 and 56 weeks old rats, the number of IR-positiveneurons remains unchanged (unpublished data). These data aresupported by an in-situ hybridization study on the hamster brains RNAe reaa sionr

ort-t ationm onst ctiont ctiva up t4 alsos veraw r daa ncei df her terme brisf cyteg

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neuronal activity modulates the function of glial cells, whichthen regulate the intra- and extra-cellular microenvironment ofneurons. In fact, it has been reported that astrocytes respond toneuronal activity by elevating their intracellular calcium, whichtriggers the release of their own chemical transmitter, whichin turn causing feedback regulation of neuronal activity andmodulation of local synaptic circuits[3].

In summary, both short- and long-term changes were notedin the PV and GFAP immunoreactivity in the dLGN and vLGNfollowing monocular enucleation. The differences in neuronalvulnerability between the diverse populations of cells affectedby disconnection from ganglion retinal cells may be a possibleexplanation for this phenomenon. We speculate that effectivebuffering of intracellular free calcium play an important rolein the recovery of certain populations of nerve cells after enu-cleation in adult rats. This phenomenon may reflect a plasticresponse from the diverse population of neurons in the LGN,which is associated with a significant reaction of perineuronalglial cells to optic nerve deafferentation.

Acknowledgments

This work was supported by a grant-in-aid for ScientificResearch from the Ministry of Education, Culture, Sports, Sci-ence and Technology of Japan (B 15790627). We thank Dr. TracyBaker-Herman and Dr. Frank Golder for their constructive com-m izueF

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howing no significance age-related changes in CB mxpression between 4 and 13 months yet started to decfterward. However, PV- and calretinin-mRNAs expresemain unchanged at all ages examined[16,24].

The qualitative and quantitative differences among sherm, sub-chronic and chronic effects of monocular enucleay reflect the difference between a reversible glial resp

o changes in neuronal activity and a permanent glial reao neuronal degeneration. We found that the number of reastrocytes and the GFAP-IR levels dramatically increasedweeks PE in the contralateral side LGN. GFAP-IR levels

howed intense expression in the contralateral LGN for seeeks and then gradually decreased to normal levels. Oure consistent with previous reports that GFAP-IR is enha

n rat dLGN and visual cortex as early as 6 h[5], and maintaineor at least 24 weeks PE[1]. Therefore, we suggest that teactive astrocytes that appear in the LGN after short-nucleation may be involve in removing of degenerative de

rom the lesion site, once this process is complete, the astroradually return to a non-activated state.

The reactive astrocytes altering neurons’ activity may bmportant component of activity-dependent neural modificauring neuronal regeneration. From this standpoint, boitro [25,34] as well as in vivo studies[19] have proven thotential of glial involvement in synaptic reorganization. Othrevious studies have also demonstrated that reactive astr

acilitate new synapse formation by releasing trophic fachat promote cell survival and neurite outgrowth at the lesioide[4,15,28]. This interaction between glial cells and neuray be important in the modulation of neuronal activity

n the regulation of synaptic transmission. We hypothesize

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ents and suggestions, Mr. Wakashi Nagata and Mrs. Mukutomi for their technical assistance.

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