Development of a crossed corticotectal pathway following cerebral hemispherectomy in cats: a quantitative study of the projecting neurons

ELSEWER

DevelopmentalBran Research 86 (1995) 81-93

Research report

Development of a crossed corticotectal pathway following cerebral hemispherectomy in cats: a quantitative stu&j of the projecting neurons P. David Adelson a, David A. Hovda a*b*c. *, Jaime R. Villahlartca b.c, Keith Tatsukawa b a Dic~isiotz of Neurosurgery,lJCL4 School of Me&cm-.LosAngel,?.% CA, USA

b Deportmentsof Psychiatryand tkbehauroral Scmce and of Arummy and Cell Lb&y, Mema/ Re~anfodonRLIcMh USA ’ Rrain Research instttute, iXL.4 School oJ.Me&cme, Los .4&es, CA W24, USA

Gnter,

Las&&s,

CA,

Accepted 4 January 1995

Ahstraet A hypothetical mechanism for the partial sparing of visual function in the contralateral visual 6eid following cerebral hemispherectomy early in life is the formation of a new wrticotectal pathway arismg from the remaining primaty visual w5ex (areas 17 and 18) that projects to the contralateral superior colliculus. To test this hypothesis, the left superior colliadus of intad adult and neonatal (S-15 days old) cats and of adult cats with a left cerebral hemispherectomy sustained neooatally (7-9 days old) or in adulthood, was injected with WGA-HRP and the brains were processed for combined TMB/DAB nktochemistry. The primary visual cortex was examined, labelled neurons were counted and the cross sectional area of theii somata was measured. The left primary visual cortex of intact adult animals exhibited a mean of 959.68 labelled cells f4065 (S.E.), with a mean soma size of 366.7 pm’ f 131.2. For the neonala ini& tipiS, there was a mean of 75.31 f 21.08 cells withinthe left primary visual cortex which exhibited a mean soma size of 249.56 pm* f 68.18. The peak cell size distribution for both intact groups was similar at 300 pm’. Viiually no labelled neurons were detected in the right primary visual cortex of intact animals (nc~nrtal or adult). For neonatal-hemispherectomized cats, the remaining right primary visual wrtex exhiiited a wart cell count of 351.09 f 126.3 cells, with a mean soma size of 436.1 pm* f 131.5, and a peak cell size distriition of 400 pm’. Finally, for adult-hemispherectomixed animals, the contralateral primaly visual cortex exhibited 68.27 f 20.13 neurons having a mean soma size of 486.6 pm25 143.2 with a peak cell size distribution of 500 km’. These results Mite that reor&zatiott of the cottiwtectal pathway occur i:: both adult- and neonatal-hemispherectomized cats but b uwtc pronounced in neonatal-iesii animals. In addition, the cells of origin of this reorganized pathway tended to be larger, perhaps in respoose to a greater axotml

Keywords: Sprouting; Nzuroplasticity;

Visual cortex; Superior colliculus

1. lntrodut!tion

functions both in man and in animals [8,9,17,2325,35,36,47]. Recently,we have reportedthat this spar-

Following cerebral hemispherectomyor unilateral neocorti~atresection early in life, many neurological functions exhibit a marked degree of sparing or enhanced recovery compared to the effects of the same

ing of function also includes visual field perception

lesion sustained in adulthood. This age-at-lesion effect haa been documented for sensorimotor and cognitive

. Corresponding author. Division of Neurosurgery, UCLA School of Medie, CHS 74-140, 10833 LeConte Ave., Los Angeles, CA 90024-6901, USA. Fax: (1) (310) 7Y4 “-. EMAIL: [email protected]~.~ 0165-XUl6/95/SO9.50 0 1995 Ekvier SSDI 01653806(95)00007-O

Science t. ‘. AU rights reserved

[20,21]. Specifically, we found that neonatalhemispherectomixed (NH) cats do not exhiiit the cornplete contralateral hemianopsia that is typical for adult-lesioned animals; but, show instead a partial sparing of the contralateral visual field out to 45”. A similar sparing of visual field function has also been repfiCd in patients who had undergone cerebral hemispherectomy as children [35,361. We [20,21] as well as others [37,%,40,41,521 have proposed that changed relationship3 and interactions between the visual cortex and the superior coUiius

82

ED. Ad&on et aL/Deuclopmmtal

(SC) following cortical resection may provide some insight into the mechanisms for the above sparing.

Others [40,52]have shown that visual orienting, which is lost in the contralateral visual field following a unilateral ablation of the occipitotemporal cortex can be immediately and completely restored by subsequently lesioning the contralateral SC or by sectioning the collicular commissure. According to Sprague (1966) [40]these. results suggested that each SC receives selective facilitatory input from the ipsilateral visual cortex (VC] and inhibitory input from the contralateral SC. Consequently, removal of one VC would ~uoduce a pnmounced inhiiition within the ipsilateral SC as a result of the loss of facilitatoty input. The subsequent removal of the inhibitory input from the contralateral SC would alleviate the inhibition, thus restoring visual orienting. Termed the ‘Sprague effect,’ this phenomenon has been the subject of more recent studies and it has become clear that control of the visuomotor capacity does not lie completely within the SC as the nigrotectal pathway also plays an impurtant role [52,53]. In an effort to determine whether the ‘Sprague effect’could explain the age-at-lesion sparing of visual field perception following neonatal hemispherectomy, metabolic studies of the SC and visual cortex of intact and cerebral hemisphere&on&d cats were conducted [1921] utilixing both cytochrome oxidase histochemistry and [“Cl 2deoxy-glucose autoradiography. The results showed that adult-hemispherectomixed (AH) animals had markedly depressed oxidative and glucose metabolism within the SC @lateral to the lesion compared to NH animals which exhiiited sparing of the contralateral visual fieid. The metabolic differences in the SC in this model could be explained as resulting from relative sparing from neuronal degeneration in the NH cats and/or as a consequence of reinnervation of the ipsilateral SC from the contralateral primary VC. Although, we recently reported that there was less neuronal 1~ within the SC in the neonatal than in the adult-lesioned animals [22,501the differences were not large and would be insufficient to explain the marked metabolic diierences. This sustained metabolic function suggests that the SC m the neonatal-lesioned animal is less inhiiited than in the adult-lesioned cats. Therefore we began to focus our attention on the possible reinnervation of the SC since a reorganized corticotectal pathway would alknv for the remaining visual cortex to provide facilitatory input to the contralateral SC and could explain the functional sparing in our neonatal-lesioned animals. The possibility of a lesion-induced reorganized corticotectal pathway would appear to be likely given our

Brain ?&search 86 (1995) 81-93

previous work, in which hemispherectomixed cats showed a reorganization of terminals of pathways descending from the remaining motor cortex and resulting in the reinnervation of partially deafferented structures such as the red nucleus, the thalamus, the dorsal colunm nuclei, and the spinal cord [15,18,48,49,51]. The present study specifically addresses this hypothesis as well as attempts to determine whether any reorganixed pathway in neonatal-lesioned animals is the result of sustained connection present early in development or whether it represents a new pathway that forms in response to the lesion. Finally, since it has been reported [4,16,17] that changes in soma size of the cells of origin typically occurs following axonal sprouting and that these changes correlate with increased functional activity, an examination of the soma size of putative sprouting neurons in the remaining VC was undertaken to further investigate, the source of the reorganization. Preliminary results of this study have previously been published [22]. 2. M8terlals 8nd

methods

2.1. Subjects Our study group consisted of 9 adult cats and 3 neonatal kittens. Three of the adults (2 males, 1 female) served as adult intact (AI) controls. The remaining 6 adults sustained a left cerebral hemispherectomy as neonates (7, 7, and 9 days of age, (2 males, 1 female)] or as yoag adults (3 males]. The 3 neonatal kittens (all males; 9, 11, and 1: .!ays of age] were studied as neonatal intacts (NI] in order to assess if the projections of the corticotectal pathway are bilateral at that age. The mean survival time was 634 days and 782 days for NH and AH respectively.

2.2. Surgery and

maintenance

procedure has been described previously in detail 1471.Briefly, surgery on the neonatal cats was performed under chlorpromaxine (5 mg/kg, i.p.1 and pentobarbital (lo-15 :ng/kg, 1.p.) anesthesia while only a standard dose of pentobarbital(35 mg/kg.l was used in the adult subjects. Hypothermia (30-35” C, rectal) was used in all cases. Using blunt dissection with a suction pipette, the left cerebral hemisphere was separated from the underlying thalamus at the level of the internal capsule and the caudate nucleus. Thereafter, the middle cerebral artery was ligated and settioned and the frontal pole dissected from the olfactory The

surgical

pig. 1. ‘ho C& fmm the tnimuy visual cortex retmgradely tilkd with the reaction $mduct following injectim of WGA-HRP in the left supxior edlictd~r. A: hum the kft bcmia~~bere of an intact adult cat. B: from the riaht hemiqbere of an adult cat hemispbmctomked nconstaUy.

P.D. A&km et OL/Dew~tol

Bram Research 86 (1995) 81-93

83

84

P.D. Ad&cm

et al /Lkwlqmmtal

bulb. The entire hemispherecould then be removed en f&xc.The cranialdefect was covered with the temporal muscle, which was reattached to the midline (in the adults) or with the bone flap in the neonates. After surgerythe animalswere graduallywarmed and placed in a recoverycage (adults)or rwemightin a thermostaticatly controlled incubator (neonates) prior to beiig returnedto their home cage. AU the animalswere closely monitoredfor the first few days of post-surgicalrecovery and, in addition to vkundassessments,all cats receivedperiodicneurological testing using selected tests from the battery descriid previously[8,47j.There were no complications and neurologicalevaluation indicated no remarkable differenceor deviationsfrom the previousreports.

Bmin

Research

86 (1995)

81-93

of the lesion and then coronal (SOam) serial, froxen sections were sequentiallycut throughout the brain and stained with thioninor processed for cytochrome oxidasc histochcmistryto assist in cytoarchitectonically defining the primaryvisual cortex (Areas 17 and 18). Adjacent to the histologicalsections, 100 pm coronal sections were taken every 400 pm in the adult and every 200 Nm in the kittens. These sections were cross-reacted with tetramethylbenxidine (TMB) foilowed with 3,3’diaminobenxidme(DAB) [321.All sections were then mounted and coverslippedfor examination under light microscopy. 2.5. Data c&e&on and analysis injection site

23. Horwadish

pemhhse

pmcedum

After completion of neurological and behavioral mating,each of the cats was anesthetixedusing either the adult or the neonatal protocol (see above). The cm&tom& were reopened in the previouslyoperated cats or initiatedin the intact controls in order to gain access to the tectai plate. A 5% WGA-HRP (wheat germ agglutinincombinedwith horseradiihpcroxidase) aohrtionwas made up and a 1 mm deep unilateral mjecthm[O.l ~1 (neonate) or 0.3-0.5 ~1 (adult)]of the rcfi SC was performed,under visual guidance,in order to fill it with the tracer. The injections were made through a sterilized IO-PI Hamilton syringe with a 27-gaugeneedle. The syringewas mountedon a stereotaxic carrierand the nead!e was intro&cd pcrpendicular to the horizontalplane.After allowingfor a IO-ruin pre-iqjuzkm equiliition period, the HRP was injected at a rate of 0.1 p1/4 min. After a post-injection equilibrationperiod of 10 min., the needle was withdrawn and the wound was sutured closed. 24. Tllpmpamtion ‘No days after the injection,the cats were administered a lethal dose of pentobarbital(150 mg/kgt i.p.) whereupontheywere intracardiallyperfusedwith 0.9% saline, glutaraldehyde(2%)/paraformaldehydc (OS 2%). fobwed

by sucrose

U_W).

AU solutions

were

suspended in 0.1 M phosphate buffer (pH = 7.4). The brain was removed and stored for at least 48 hours in two sequential increases of sucrose solutions (10 and 20%). Notes were taken regarding the gross anatomy

To study and compare the injectionsites, each of the sections throughthe SC was inspectedon an Olyntpus SZH microscopeunder low power (10 x ) and the areas of the reactionproductwere quantifiedby means of commerciallyavailablesoftware (JAVA; Jandel Scientific)integratedwith an AST/286 computer.Thereafter, calculationof injectionvolumewas based on the formula[311: V(mm3) =d(a,/2+a,+

...+an_1+a,/2)

where V is volume in cubic millimeters; a is the surface area of the indiidual section in mm2; 1,2,... n is the number of sections; and d is the distance between sectionsin millimeters(0.4 mm in the adultsand 0.2 mm in the NJ cats). 2.6. Countingof lab&d

neumns in the primary

visual

cort6x

Each of the HRP-processed sections through the entire’primaryVC (areas 17 and 18) of each brain was examinedunder light microscopy(OlympusBH-2) for retrogradelystained neurons. Consequently,the number of sections studied for each group of adult animals differed sliitly but not systematicaliyran& from 43 to 55 sections. For the neonatal intact animals3%f 5) sections were examined reflecting the smaller overall brain sixe. With the aid of a camera lucida drawing tube, the soma of each of the retrogradelylabeled neuronsconfuted to the primary visual cortex was drawn under 15OtJ X . For all available sections, these cellular profiles were compiledwith the criteriathat the cell could

Fii 2. An uampk of cells located within layer V of theprimuyvissslcortextAresa17 and 18) retmgradely filkd with HltPf&wiss A: medium power view (375 x ) of the rctrgnde lab&d neurona seen in the ICJIprimaty visual cortex of an tutactadult animat.B: similar view witbin tbe &hr primaryvisual cortex in a neonatakshed animat.Note the relattvespars& d rerogadely filkd cetk in this animal wqwed to the viw.4 cortexof the oppmite side in an intact adult wt. However,no labeled neurons were found within tbsri&visualcoltexofin~dukcats.

was-ructionwithW/DAB.

P.D. Ad&on ci .l./Lkvelopmental

be clearly identified by the accumulation of reaction product within the cytoplasm in the plain of section and that the boundaries of the cell along with processes appeared distinct (see Fig. 2). Given the HRP reaction technique used [28,34] along with the thickness of the section (100 am), the cell bodies of labeled neurons wcrc completely filled resulting in a dark, black profile allowing for the clear identification of several processes. Although this prohibited the identification of intracellular organelles (e.g. the nucleolus), the detailed profile allowed for a very high level of confidence in determining if the cell was in fact neuronal. In the rare cases where only a few grains were present in the cytoplasm, or when the profile, with pnxcsses, were unclear, the cells were not included in the study. Each drawing was then digitized on a graphics tablet (Jandel Scientific) utilizing commercially available software (SCAN, Jandel Scientific~ integrated with an AST Premium,%6C computer where the cells were counted

Brain l&samh

84 (EXL5)81-93

and the cross-sectionai area (am*) was determined for each of the neurons. The observed cell number for each animal was subjected to an Abercrombie adjustment to control for overestimation [l]. The mean soma size and the cell size distributions per group were then determined. The average cell sire was calculated for each animal and then collectively for each group. These values were then compared between groups (MamtWhitney VI. For cell sire distributions, the neurons were assigned to oxrsecutive 100 pm* categories, and the percentage of the total within each size category was calculated. In this way, comparative analyses could be made. 3. Results 3.1. Gtuss anatomy

Intact Adult

Adult Hexni

Intact Neonate

Neonatal Hemi

Fii 3. S&c&d cmmal cations of the brahIm at the kwl of the wpcrior CoUiculua kom each animal mup illustrating the injection sites within the left rupcrior mlliculw. Adult Hemi, adult cats bmispbe~ in adultho@ Nematal Hemi, adult cats bemispberectomized nunlaw.

P.D. A&&n et a: /DeuelopmenIal Bnun Reszcrch 86 (IW.5) 81-93

bemispherectomy was qualitatively similar in both ageat-lesion proups and included all of the dorsolateral and most of the midline neocortex. RJstral to the callosum there were midline cortical remnants in all cases. In AI-I animals, generally most cortical midline tissue under the sulcus (s.) genualis (rostrally) and s. cruciatus (caudally) was intact, including the gyrus (9.) rectus and parts of g. cingulus. In the neonatal-lesioned cats, one half or more of the cortex under these sulci was removed. Ventrally, the olfactory bulb and tract, the ttibercuhnn olfactorium and cortex prepyrifo&a were intact in both lesion groups. Dorsal to the

the ventral 3/4 of the rostra1 portion of the

caudate nucleus (less then half) was but was markedly atrophic. AIso in the the amygdala nuclei sustained little damage and in some cases portions of the putamen and claushum were spared. The thalamus was not surgically damaged in any of the cats. None of the brains sustained direct surgical damage to the right hemisphere or lower brainstem. 3.2. Injection site and LW&UPX?

I3 700

650 i

600

NE i 9

i 550 1

I

500

k? ,_

450

= 8

400

B 2

ii

350 300

250

___ 7””

I”I.CL He.a”alo

I;,‘&l

N.g

yd”,

Fii. 4. Surrrmuiy of (Al the mean numbcr of cclls ( f standard crmr

S.E.)

of the aam, S.E.) and (B) neuronal cett cross sxtional area (f for each groupwithin the primaryvisual cortex(side ipsilatcral to the superior a~lliculus in intact cats contralatercl cortex in lesioned cuimats~.in (A). cotc the substantial number of cells found within the contralateralvisual cortex of the neonatal-hemisphercetocdzcd cats (’ =P
Upon inspection of the injection sites it was evident that all injections were centered in the upper 2 layers of the left SC filling its entire dorsal-ventral extent (see Fig. 3). In one animal per group, the injection site extended beyond the SC and into tbe pcriaqueductaI grey, pretectal region and tbe inferior ~~Uicub~ Medially, there was no evidence of the injection inva&mg the contralateral colliculus in the hemispherectomized animals and laterally the injection did not extend past the border of the colliculus. In the intact animals there was a suggestion of the reaction product crossing the midline, however this spread bad no effect on the final outcome of ceU counts within the contralateral VC (see below) The average injection volume [in mm3, SE.1 for each of the individual groups was: AI, 106.89& 20.07; NI, IO.73f 3.41; NH, 113.% f 27.79 (but note that these cats were adults at the time of the study); and AH 200.86 It 47.58. 3.3. Cell counts In the intact animals the uptake of the tracer was seen almost exclusively in the visual cortex ipsilateral to the injection site (left) and only i;; !aye: V (SX Fig.

2). There was a total of 3 labeled neurons in tbe primary VC (Areas 17 and 18) on the side contralateral

al

to the injectionsite in the NI cats (2.0, 1.0 and 0.0 in the three animalsrespectively)and no cells at all were found there in the AI. In contrast,there was a substantial number of neurons with tracer in the contralateral (right or remain&l hemisphere in the lesioned animaIs. For the three AI animals,a total of 3.806 ceUs wxxe analyzedin the left hemisphere.WhiIe,the three NI animaIsa total of 286 cells were. anaIyzedin that hemisphere.It should be. noted that since the procedure for injectionof the HRP into the left SC required the retraction of the lefi occipital pole of the Intact animals,the correspondiog visual cortex suffered signiticautdamage. This damage was more robust in the NI and most IiieIy contributedto both the variability andtheIowerceIIcountintheNIanimaIs.AppIying Abercrombie’sadjustmentto these raw vaIues resulted in a mean &I count (f&E.) per AI cat of 959.68 f 406.5 in the left hemisphere,while in the NI cats, the mean ceU count was 75.31 f 21.08 and 0.756 f 0.43 for the left and right hemisphererespectively. For the lesioned animals,a total of 281 ceUs were analyzed in the remaining right hemisphere of the three AH animals,whereas, 1424 ceUs were anaIyzed in the righthemisphereof the three NH cats. Applying Abercrombie’sadjustmentto these values resultedin a mean ccII count of labeled neurons for AH cats of 68.27 f 20.13, while in the NH subjects,the mean ceU count was 351.09 f 126.3. ConsequentIy,both lesioned groups exhibited a significantincrease in the number of back-f&o &Is within the remainingvisual cortex (Mann-Whitney U, P c 0.05). Again, aU of the neurona! c&l bodies were :estricted Co layer V within the primaryVC in aU groups (See Fig. 4A). It shouldbe noted that the majorityof the variabiity between animalswas present within the ipsilateral,left hemisphereof the intact aqimaIs.This is most UkeIy due to the variable damage done to this hemisphere during the surgicaIretraction which was required in order to visualizethe SC (see Methods and Discussion se&ions).The variabilityin the lesioned groups represents factors inherent in the HRP technique and the numberof animalsstudied per group (n = 3). 3.4. Cmss-sectionalarea of neumnul soma For the intact animals,the mean soma size of the neuronsin the VC ipsilateralto the HRP injectionwas 366.7pm* (S.E. f 131.2)for the adultsand 9.6 f %.4 pm* for the neonates. The contralatera? VC of the IeJioncdanimais,NH and AH, had a mean soma size of 436.1 pm2 (S.E.f 131.5) and 486.6 cm2 (SE.* 143.2) respectively,which ten&d to be larger than in intact cats albeit not statisticaUysignifimt (See Fig. 4B). However, when the resulti W,KC‘Jlokcndawn in cell size categories,the groupsexhiiited distinctdifferences. The groupswere compared using the size range

distriiution and calculatingthe percentageof neurons within each range. In AI animals,most of the neurons ranged in size from 24Nlto 700 pm2, with a peak diim%utionat 300 pm2. This was similar for the NI cats. In the NH, though,this distributionwas shifted to the right extendmgbetween 200-800 pm2 and in the adult-lesionedanimak?,it was shifted even further to between 300 and 800 pm2. The respective peak percentage of distributionof the lesioned animalswas 400 pm2 for the NH and 500 pm2 for the AH. These results show that the dism%utionfor the lesioned animaIs was broader and shied to the right indicating that a greater proportionof neuronswere larger.Also, notable were the lower peaks and broader areas under the curve for the lesioned animals(See Fig. 5). 4. Dtseusston The results of this study documentthree main findings in regard to the changes in the corticotectalpathway following neonatal and adult cerebral hemispherectomy.First, both neonatal- analadult-lesioned cats exhibiteda crossed corticotectalpathway that was not present in intact age-matchedadult controls or in neonatal intactkittens.This suggests that this pathway is the result of axonal sprouting or reinnervationin response to the lesion rather than a lesion-induced sparing of a preexisting corticotectalprojection.Setond, this crossed pathwaywas much larger in the NH cats which had previouslydemonstratedless metabolic depression within the ipsilateralSC as weU as visual

sparing in the contralateral field of vision [22]. Third, the cells of origin of this reorganized pathway tended to be larger when compared to intact controls. We believe that this may have been in response to the expansion of their terminal fields resulting from reinnervation of the contralateral SC. Since we wanted to ascertain the strength of our findings, we examined every section reacted for HRP throughout the entire extent of the primaly visual cortex for each animal. As described in the methods section, this resulted in some variability in the total number of sections examined for each cat. In addition, given the HRP reaction procedure used, the nucleolus of the cells could not be identified and employed as the plane to count and measure neurons. Consequently cells could have been counted or drawn with a portion of their profile falling outside the plane of section. However, we attempted to adjust for this kind of sampling bias by correcting for split cell error using the Abercrombie formula [l]. The above methodological issues would be of a major concern if the results were of only marginal significance. However, two aspects of the results makes it extremely ud’&c$ that a sampling error could explain the current findings. First, the majority of the cells identified had multiple urocesses which were clearly identified (see Fig. 1). Consequently, in order to see a significant number of these processes the plane of section would most likely have to include a large portion of the cell soma. Second, the major finding of this study is derived from measurements within the right cerebral hemisphere, i.e., the side contralateral to the HRP injected SC with comparisons made between age-at-lesion groups and intact controls. At a minimum, this comparisons represent a five fold difference between NH and AH animals; and a difference between zero (0) cells in intact cats compared to 351.09

cells observed in the neonatal-hemispherectomized cats. It would be extremely difficult to argue that such a magnitude oi difference cnuid he explained in terms of sampling error. As mentioned above, the novel crossed ccrticotectal pathway occurred in both lesicned groups and could have resulted from either the sustaining of crossed projections present at birth and/or as a result of collateral axonal sprouting. The first possibility is suggested by the work of Edwards et al. (1979) [13] and of Chalupa et al. (1980) Ill]. Edwards et al. (1979) 1131 injected [3H] leucine unilaterally into the visual cortex in neonatal cats, and found tracer in both superior colliculi, although most of the label was detected within the SC ipsilateral to the injection. Unfortunately, in this study the injection was widespread and involved not only the visual striate cortex but the extra-striate cortex which is known to contribute to a normally present crossed corticotectal pathway I3.71. Another

study, reportrd in abstract form only (111, also used i3H] leucine injections into the VC and suggested that a crossed pathway existed in the neonatal cat, and therefore a similar pathway in neonatal-lesioned animals must represent a lesion-induced ded projection. This study has never been published in errenro and; therefore, it remains to be fully documented. The second possibility, i.e., that the crossed pathway was the result of coilateral sprouting from axons descending from the contralateral VC is supported by the present findings and by other reCcnt work [30]. In the present study, the HRP injectiin was fairly liited to the left SC and the cell drawings were obtained only from the neurons retrogradeiy filled in tbe primary VC (Areas 17 and 18). A crossed corticoteztai pathway could not be documented in intact adults and was only minimally present in the neonatal intact animals. In a recently pubhshed work by Lord Hummer and Behan (1992) [Ml, they characterized the topography and development of the cmticotectal pathway in neonatal cats and compared these to adult cats using topographically restricted injections of J%se&s u&7rir leukoagglutinin into the striate cortex. Even by ncizg t!ds pres*umabiy more sensitive measure, they did not observe a crossed pathway in either the intact neonatal or adult cats (personal commumcatmq, Lord ?!umm._ __ han, .93), and this contirms an earlier report by Tsumctc et al. (1983) [45] using HRP. Therefore, we believe that we have demonstrated a novel cmssed corticotectal projection and that this could only be generated via collateral axonal sprouting. This finding of axonal sprouting in the visuomotor system of adult cats reaffirms our previous results in the motor system in which a significant remodellmg of ccrticorubral terminals also occurred in both neonataland, tJt to a lesser extent, in adult-hemispherectomized animals as demonstrated by injections of i3H] leucine-proline into the motor cortex (49,511. At least two factors may contriiute to the remodelling in adult cats. First, it has been reported WI that in cats C-layer neurons of the do& lateral geniculate nucieus can exhibit lesion-induced axonal terminal sprouting until P126, srlggesting retention of capacity for reinnervation until relatively late in postnatal Me. Second, the extent of a unilateral cortical lesion may be important for the capacity to express terminal sprouting in adult cats. Consequently, a crossed corticorubral innervation is not seen in adult cats following a restricted unilateral sensorimotcr cortex ablation [2&B], and yet it is present after cerebral hemisphere&my 115,491. The age of two of the three intact kittens that we used (P9, 11 and 13) did not exactly match the age-atlesion of our NI animals @7,7 and 9) such that it could be argued that an exuberant bilateral corticoteztal projection might have been present in our kittens at W (and then retained as a consequence of hemispherec-

. .

PIand&_

P.D. Adelnmd aL/De~ial

tomy) but not at P9 or later on. This is interpretation is highly unliiely. Fit, as mentioned above, there is no evidence from the literature that a crossed projection exists at that age in kittens. Second, in all cases of exuberant projections within the kitten visual system reported in the IiteraNm [L?,7,3@43-451, the period of ‘exubcrtice’ fully encompassed the W-13 of our hittens and, furthermore, it extended considerably beyond this narrow range (from biih to over 8 weeks, depending on the projection being considered). Third, in none of the many studies that we have performed on reorganization of projections from the remaining neocortex after neonatal hemispherectomy, have we found anatomical (or any other) differences in the results for kittens between P5 and P15. In the M cats, there was a much lower level of ipsilateral retrograde fiUmg of visual cortex neurons compared to the AI animals. This was probably due to cortiad changes sustained by surgical retraction from performing the injection. The retraction that was netessary for visuakation of the tectal plate in the intact neonates caused significant damage to the left hemisphere which was less pronounced in the fully myelinated, harder brain of the adult i&cted animals. An alternative explanation would be that some of the young neurons failed to achieve satisfactory uptake and/or transport of the tracer. This is not unlikely since injections of tracers tend to diffuse more in kittens than in adult cats, and often the neurons are therefor less intensively labeied &Sj. Though conclusions ahout absohrte numbers for the ipailateral side could not be made using the HFtP method, the finding of minimal to no crossed fibers in the intact groups arising from the right primary VC was conclusive since this area was not disturbed intraoperatively. It should be noted that by using the HRP methodology, as in this study, the absolute number of cells which project across the midline in our experimental groups also can not as yet be ascertained with precisiin. It is likely that the use of this method did not label all the ceils of the ipsiiateral or crossed pathways from the injection site in either the control or operated animais. Consequently. this method may not have been sensitive enough to detect sparse crossed connections that may be significant. Previous studies of HRP transport noted that this method can only provide scaled estimates of the relative numbers of labelled cells in cases with approximately comparable injeckns [17j, to which we agree. Our injections in the SC were large and this raises the possibiity that some of the neurons labeled in the VC may have their axon rermiuals outside the SC. It is known that the striate visual cortex sends efferents from layen IV and V to tire lateral geniculate body, the subantkal visual relay centers such as the SC and pretectal region, as well as to certain thalamic nuclei,

Bmin R.?xmrh86 (199s) 81-93

though the corticotectal pathway arises almost solely from layer V. In this study, the regions involved with the largest injections were the periaqueductal gray, inferior colliculus and pretectal region. These regions are not known to receive projections from the contraiateral primary visual cortex [a,101and thus would not be expected to contribute to the iinding of a crossed projection. Furthermore, the SC injection in the AH group was the largest and yet these animals showed less crossed connections than NH cats. It is therefore unliiely that the injection spread to such a small extent contributed to any change in the interrelationship between groups. The anatomical findiigs reported in the current study may at least partially explain the differences in visual function behveeu neonatal- and adult-lesioned animals. As descriid previously [20,211, there is a partial sparing or reccnceryof visual field perception that occurs following cerebral hemispherectomy in the NH cat but not in adult-lesioned animals. The AEI exhibit a complete contralateral hemianopsia that is stereotypical and permanent. The proposed relationship is that there exists between the VC and the SC an interaction that aUows for visual field perception. Following cortical resection, there is an interruption of the normal inputs and thus loss of function. Sparing of fun&m may occur as a result of a functionally effective -cd pathways which only existed in our MI cats. Although both age-at-lesion groups exhiiited a reorganixed corticotectal pathway, it occurred to a much greater extent in the young animals. This suggests, that it is not simply the development of a projection from the remaining VC to the opposite SC which may account for the sparing of visual field perception in the NH animals. The later may occur because the new pathway attained a critical fiber ratio necessary for excitatory input to the SC in these animals. In contrast, this might not have occurred in the AH cat due to the low level of reinnervation. This ‘normal’function might be expressed because an appropriate percentage of connections/ synapses or a minimal threshold complement was achieved [4,14,2fl. However to directly test this hypothesis, it would be necessary to allow for recovery of visual field perception following neonatal hemispherectomy and then selectively lesion the newly formed crossed corticotectal pathway to verily if indeed the animals lost function. Within this context, one wonders whether a pharmawlogic regimen that wuld enhance axonal sprouting could have allowed the adult animals to also attain a critical anatomic remodelling thereby aehieving a functional recovery as well. A differential neuronai loss in visuai nuclei may also wntriiute to the better visual performance in NH * versus AH cats. We have previously reported that neural degeneration and nuclei atrophy in the SC is decreased when comparing MI to AH cats [22,39,50].

91

The differential neuronal sparing in these structures could explain the metabolic differences that were seen in the SC [21,22] and may be important in the sparing of visual field perception. Other structures that may play a role include the thalamus since a similar sparing from degeneration also occurs in the dorsal lateral geniculate nucleus (LGd) of the NH cat [39]. Since the central retino-geniculate connections are bilateral [12], decreased degeneratiozn cf the LGd and possible translaminar sprouting may contribute to the maintenance of the central retinal projections and thus preseme further visual perception. This age-at-lesion effect on neuronal loss, therefore, may be important in the developmental-dependent differences in visl?&ehavioral recovery. The present results indicate that in the lesioned animals, the cell size distributions were shifted toward the larger size ranges comparGd to the intact groups. This suggests that the cell bodies which form a crossed corticotectal projection in the lesioned animals rended to he larger whtn compared to the neurons contributing to Ihe unilateral projection in intact cats. This putative increase in neuronal size suggests that these neurons may hypertrophy in response to developing a greater axonal arborization in order to innervate both SC. Increased neuron size after lesioning has been reported previousiy in our hemispherecromized cats [39,46] as well as in other animal models [17,42], and has been attributed to the formation of additional synaptic arhorizations requiring more metabolic machinery to support the new connections. In addition, it has been proposed that a lesion-induced cellular hypertrophy represents the expression of new byproducts (e.g. proteins), in the establishment of functional contacts. However, as has been reported in the goldfish, thii type of hypertrophy is transient since with the

re-establishment of normal functional contacts, retinal ganglion cells regained their normal appearance [33]. It is unlikely that such a proposed transient hypertrophy could explain the results in the current study since the animals were maintained for over 6 months ery was stable [20,50]. In this chronic anstomical reorganization was most likely and therefore does not represent an

trophy in the inferior olive following unilateral decortication of the cerebellum in the cat. Their data suggested that since there exists a bilateral cerebelloolivary tract, the recurrent circuits that remained intact after the cercbellar ablation elicited the hypertrophic reaction within the inferior olive. Therefore, they hy-

pothesized that the lesion-induced olivary neuronal hypertrophy was an expression of a functional differenwithin

the

inferior-olivary

complex

Underscoring this observation is the work by Lord Plummer and Behan (1992) [30] who recently reported that the corticotectal pathway is in a state of active growth and remodelimg well until eight weeks postnatally. Therefore, our neonatal age-at-lesion group was well within this period of active change for this pathway. In this and our previous studies in :he sellso-

rimotor system, we have demonstrated that the brain of our neonatal-lesioned cats was able to anatomica@ remodel in several wars (i.e. reinnervation, differential neuronal loss and cerebral metabolism) and that this reorganization most likely plays an important role in their functional recovery. The anatomical plasticity reported in the current paper further testifies to the correlation between anatomical reorganization and behavioral recovery, and provides insight into the basis of the corticotectal interactions.

and recovmodel, the Frmanent acute phe-

nomenon. Boesten and Voogd (1985) [S] reported on a chronic, stable model in which they described neuronal hyper-

tiation

olivo-cerebellar relationships. Sii, according to our data, bilateral w~ections from the VC to the SC did not exist in the intact animals prior to decortication, a recurrent path could not have elicited the hypertrophic response at the time of injury. It remains possible that a change in the tecto-tectal interaction or lack of input to the ipsilateral SC signalled this response. A metabolic study of these altered neurons would elucidate if indeed the differential function occurred as a result of lcsioning. The cellular hypertrophy within the VC of the current study may have occurred, once the crossed pathway existed in both the lesioned groups, as a cellular expression of increased functional differentiation. The combination of fin&mgs within this study in the VC and in previous work on the red nucleus [15,49,51] highlights that even after reaching what is believed to hc end difierentiati~n, both lesioned groups retamed the ability for axonal sprouting. It is clear that this is an zn*romir3! reorganization that does not result in a functional recovery in the adult-lesioned cats. The young kitten though still retains not only a great degree of anatomical plasticity but functional plasticity as well.

based

on

Supported by Grants USPHS ROl NS25780 (J.R. Villablanca) and PO1 HD-OS9S8 Willablanca’s Project), HD-04612 (UCLA Ment. Retard. Res. Ctr.) and NS30308 (D.A. Hovda).

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-__.. ..__~.._r.

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Res

Evidenoc

,