THE JOURNAL OF COMPARATIVE NEUROLOGY 312~561-572 (1991)

Organization of Callosal Connections in the Visual Cortex of the Rabbit Following Neonatal Enucleation, Dark Rearing, and Strobe Rearing ANTONY M. GRIGONIS AND E. HAZEL MURPHY Department of Anatomy, Hahnemann University, Philadelphia, Pennsylvania 19102-1192 (A.M.G.)and Department of Anatomy and Neurobiology, Medical College of PennsylvaniaiEPPI, Philadelphia, Pennsylvania 19129 (E.H.M.)

ABSTRACT The organization of visual callosal projections was studied in (1)normal adult rabbits; (2) adult rabbits which had undergone monocular enucleation (ME) or binocular enucleation (BE) at birth; and (3) adult rabbits which had been deprived of normal visual experience during development by dark rearing (DR) or strobe rearing (SR). Previously published observations (Murphy and Grigonis, Behav Brain Res 30:151, 1988) on callosal organization in adult rabbits in which retinal ganglion cell activity was eliminated during development by intraocular tetrodotoxin (TTX) injections, are also summarized for comparison with these data. The tangential extent of the callosal cell zone was significantly larger than normal in DR, TTX, and ME rabbits, was unchanged in BE rabbits, and was significantly reduced in SR rabbits. An analysis of the laminar distribution of the callosal cells revealed a significant increase in the percentage of callosal cells in lamina IV in ME, DR, and TTX animals. Measurements of density of callosal cells showed a significant increase in the density of the callosal projection in ME and SR rabbits and a decrease in density in BE rabbits compared with normal. The data suggest that the mechanisms involved in the development of the tangential and laminar organization of the callosal cell zone are different. In addition, the data suggest that the mechanisms involved in the maintenance of callosal projections are different from the mechanisms involved in the elimination of callosal projections during development. The effects of these developmental manipulations on callosal organization in other mammals are reviewed and compared with the effects in rabbits. The data suggest that species differences in the degree of maturity of the visual system at birth and in the extent of callosal development at the time of eye opening, may underlie species differences in the effects of these manipulations on the organization of visual callosal projections during development. Key words: corpus callosum, competition, neural activity,deprivation, visual system, development

In the mature rabbit, the callosal projections linking the primary visual cortex of each hemisphere are centered on the cortical area in which the vertical meridian is represented, i.e., the lateral border of area 17 (striate,VI) and the medial border of area 18 (occipital, VII) (Hughes and Wilson, '69; Hughes, '71; Choudhury and Gent, '73; Swadlow, '77; Towns et al., '77; Chow et al., '81a; Symonds and Rosenquist, '84a,b). Similar visual callosal projection patterns have been described for other mammals (Choudhury et al., '65; Heimer et al., '67; Rhoades and DellaCroce, '80; Cusick and Lund, '81; Van Essen et al., '82; Gould, '84). In the rabbit, as in most mammals studied, this pattern of callosal projections represents the final stage of a developO 1991 WILEY-LISS, INC.

mental process in which initially widespread callosal projections, which are present throughout area 17 in the neonate, are eliminated, and only the appropriate projections at the 17/18border are maintained (Innocenti et al., '77; Chow et al., '81a; O'Leary et al., '81; Innocenti, '84; Rhoades et al., '84; Stanfield, '84; Olavarria and Van Sluyters, '85; Frost and Innocenti, '86; Innocenti et al., '86). The primate appears unique in that, in area 17, callosal cells are sparse and no transient callosal projections are produced throughout development (Dehay et al., '86, '89; Dehay and Kennedy, '88; Chalupa et al., '89). Accepted July 26, 1991.

562 The ontogenetic process, observed in rodents, carnivores, and lagomorphs, of elimination of exuberant projections and maintenance of appropriate projections can be disrupted by various experimental manipulations of the visual pathways in the neonate (for reviews, see Elberger, '82; Rhoades et al., '84; Frost and Innocenti, '86). However, published reports indicate wide variability in the type and extent of callosal abnormality observed (for reviews, see Rhoades et al., '84; Frost and Innocenti, '86). The sources of this variability may include differences in methods of identifying and analyzing callosal projections or differences in the experimental paradigms used to manipulate developmental events. In the present study, we investigate the effects of several different experimental manipulations, using identical methodology, and quantify the tangential extent, density, and laminar organization of callosal projections. This permits detection of subtle differences between the effects of different manipulations. We compare the effects of a range of experimental manipulations involving elimination of pathways (enucleation), elimination of neural activity with tetrodotoxin (TTX), and elimination of patterned neural activity by deprivation [dark rearing (DR)] and by stimulus-induced synchrony of neural activity [strobe rearing (SR)]. Species differences may also be a source of some of the variability in reported effects of early manipulations on callosal development and may provide important insight into the mechanisms involved. It has been hypothesized that callosal development may be influenced by (1)binocular competition; ( 2 ) visual experience; and (3) retraction and sprouting of projections. Species differences in (1)the extent of binocular overlap in the visual projections, (2) the extent of callosal maturity a t the time of eye opening, and (3) the extent of retraction and sprouting of projections at birth and in response to enucleation could underlie species differences in the effects of enucleations and deprivation on callosal development. Most of the published studies have used cats or rodents as experimental subjects. In cats, monocular enucleation (ME) results in a callosal organization that is much more abnormal than that resulting from binocular enucleation (BE) (Innocenti and Frost, '79), but in rats the effects of ME and BE on callosal development are similar (Olavarria and Van Sluyters, '84a; Olavarria et al., '87). This difference between cats and rodents may be a function of the difference in the extent of visual cortex which is binocular and in which, therefore, binocular competition is disrupted by ME. In rodents, visual projections are predominantly contralateral and the visual fields predominantly monocular (Hughes, '71; Lund et al., '74). Thus, a much smaller proportion of visual cortex is influenced by binocular competition in rodents than in cats (see Fig. 1). Since the visual projections in rabbits are predominantly contralateral (Provis and Watson, '911, as they are in rodents, we hypothesized that the effects on callosal development of ME would be similar to the effects of BE in rabbits, as they are in rodents. In cats, the visual callosal projections are still immature at the time of eye opening, and abnormal visual experience can influence callosal development (Lund et al., '78; Lund and Mitchell, '79a,b; Innocenti and Caminiti, '80; Berman and Payne, '83; Innocenti et al., '85; Clarke and Innocenti, '86). In contrast, in rodents, visual callosal projections mature before eye opening (Olavarria and Van Sluyters, '85) and the evidence suggests that visual deprivation does not alter callosal development in rodents (Cusick and Lund,

A.M. GRIGONIS AND E.H. MURPHY '82). Since visual callosal projections in rabbits are still immature at the time of eye opening (Grigonis et al., 'go), as they are in the cat, we hypothesized that abnormal visual experience (DR, SR) in rabbits would alter callosal development. In terms of the degree of maturity of the visual system at the time of birth, the rabbit appears to be at a stage intermediate between the more mature visual system of the cat and the less mature visual system of the rat, mouse, and hamster. For example, the ipsilateral and contralateral retinogeniculate projections are already segregated at birth in the cat (Chalupa and Williams, '84), overlapping in the rodent (So et al., '84) and largely segregated in the rabbit (Grigonis et al., '86; but see Crabtree, '90). Similarly, the expansion of the ipsilateral retinocollicular zone in ME animals is much greater in the rodent (Lund et al., '73; Jen and Lund, '81) than in the rabbit (Chow et al., '73), and has not been reported in the cat. In maturity of the visual cortex, based on criteria of cortical depth, lamination, neuronal morphology, synaptogenesis, and functional development, the newborn rabbit (Hunt and Goldring, '51; Rose and Ellingson, '70; Mathers et al., '74, '78; Mathers, '79; Muller et al., '81; Murphy and Magness, '84; Grigonis et al., '88) is intermediate between the newborn cat (Marty and Scherrer, '64; Huttenlocher, '67; Rose and Lindsley, '68, Marin-Padilla, '71; Cragg, '72; Winfield, '83) and rodent (Rose and Ellingson, '70; Wolff, '76, '78; Lund and Mustari, '77; Miller, '81; Blue and Parnavelas, '83; Bahr and Wolff, '85). There is good evidence that neonatal lesions result in altered projections which may reflect both sprouting of intact axons and failure of retraction of "exuberant" immature projections (Campbell et al., '85; Grigonis et al., '86; Wree et al., '86). The extent of both sprouting and maintenance of exuberant projections is related to the degree of CNS immaturity at the time of a lesion (Goldberger, '86). For example, retinocollicular projections in ME rabbits are significantly expanded if the lesion is made prenatally, less expanded if the lesion is made on the day of birth, and not expanded if the lesion is made on the second postnatal day (Chow et al., '73, '81b). Therefore, if the effects of ME on callosal development are secondary to sprouting or maintenance of immature projections, the effects in the rabbit should be intermediate between those observed in the cat and the rodent.

MATERIALS AND METHODS Experimental subjects Dutch belted rabbits, which are pigmented, were used in all experiments. Table 1shows the number of animals used in each experiment. Rabbit cortex is mature by postnatal day 28 (for review, see Murphy, '841, and all rabbits used in these experiments were aged 35-60 days, and were bred in the laboratory.

Experimental conditions Dark rearing. Two pregnant rabbits were placed in a light-controlled room several days before giving birth, and the rabbit pups were reared in total darkness. Enucleation. Rabbit pups were operated upon within 24 hours after birth. Each animal was deeply anesthetized by inhalation of halothane and nitrous oxide. During anesthesia, one or both eyes were totally excised, using sterile precautions. The ophthalmic artery was cauterized

RABBIT VISUAL CALLOSUM FOLLOWING NEONATAL ENUCLEATION Cat

563

TABLE 1. Width of Callosal Zone'

Rodent

Tangential extent Adult

ME CZ

BE

Animal

Condition

R399 R455 R697 R763 R819

Normal Normal Normal Normal Normal Normal Normal

~ n n

R881 Mean A449 R450 R453

R697

Fig. 1. Diagrammatic representation of the spatial extent, in the cat and the rodent, of the regions receiving input from three major projections to area 17: (1)the retinogeniculocortical projection from the contralateral eye (C); (2) the retinogeniculocortical projection from the ipsilateral eye (I);and (3) the callosal zone (CZ). The regions which lose their normal input following monocular enucleation (ME) or binocular enucleation (BE) are shaded. Because the ipsilateral projection is so small (5%) in rodents, the total affected (shaded) area following BE (C + I) is only slightly greater than that affected following ME (C), and the binocular region of cortex, where binocular competitive interactions can occur, is very small. In contrast, in cats the ipsilateral projection is significant (40%). The total affected (shaded) area is significantly different following BE compared with ME, and binocular competitive interactions can occur throughout most of area 17. The ipsilateral projection is small in rabbits, as in rodents. Therefore, if the relative effects of BE and ME on callosal development are determined by the total area affected or by the total area in which binocular interactions occur, then callosal organization in the rabbit following ME and BE should be similar to that observed in the rodent. For ME animals, the cortex contralateral to the enucleated eye is shown. Throughout the figure, the lateral border of area 17 is to the right of the figure.

Mean R686 R807 R809 Mean R522 R564 R565 RBI7 Mean R394 R513 R514 R515 Mean R615 R616 R618 R619 R623 R633 R685 R693 Mean R684 R688 Mean R821

R822 R823

and the eyelids were opposed and sutured together. Animals were kept warm and returned to their litter box 1-2 hours later. Strobe rearing. Two pregnant rabbits were placed in a light-controlled room several days before giving birth. Within 24 hours after birth of the pups, strobe illumination was initiated. Pups were reared in conditions of total light deprivation except for strobe illumination (flash duration: 10 psec; frequency: 4 Hz; 12 hours per day).

Cortical injections Retrograde tracing methods using horseradish peroxidase (HRP) and histochemistry were used to identify the cells which project through the corpus callosum to the contralateral visual cortex (callosal cells). Rabbits were first anesthetized with ar, injection (i.m.) of 2% xylazine and 0.1%Ace promazine (0.35 mgikg) mixed with 1%ketamine (3.5 mgikg). The skull was then exposed, a small hole drilled with a dental burr, and rongeurs used to remove an area of the skull unilaterally from approximately 2 mm posterior to the Bregma suture to the posterior limit of the cortex, and from approximately 1mm lateral to the midline suture to approximately 8 mm lateral. This procedure exposed the entire striate and extrastriate cortical areas. After the dura was retracted, multiple injections of 0.5 p1 10% HRP (Boehringer), were made unilaterally using a Hamilton syringe held in a specially constructed stereotaxic attachment. Injections were spaced at approximately 1mm intervals throughout all of the striate and occipital cortex, and the total volume injected was approximately 8-12 p1.

R828 R829 R831 Mean

ME contra ME contra ME contra ME contra ME ipsi ME ipsi ME ipsi

BE BE BE BE Dark reared Dark reared Dark reared Dark reared

(%)

23 30 30 18 21 26 29 25 (.021 51 61 41

38 46 f.O4J*' 90 21 24 27 (.01j

22 28 26 22 24 LO21 33 32

36 35

SEM -01 .01

.u1 .01 .02 .01 .01

.01

02 01 .01 .01

.02 .01 .01 .01

.01 .01 .01

.01

.oi .01

34 ( 01)*'

TTX TTX TTX TTX

Trx Trx TTX TTX TTX control TTX contrul Strohe Strohe Strohe Strohe Strobe Strohe

37 39 31 28 32 36

46 41 36 (.03J'" 25 22 24 1.01) 16 16 21

I7 15 20

-01 .02 .01 .02 .01

.a2 .02 01 .01

.01 .01 .01

01 01 01 .03

18(.01)"

'Expressed as the percentage of the rnediolateral extent of the striate cortex, with standard errors, for all cuntrol and experimental animals. Data from each experimental group were compared with normal (Mann Whitney test: '*'P< 0 02) BE, binocular enucleation; ME, monocular enucleation; TTX,tetrodoxin.

The tip of the needle of the Hamilton syringe was lowered 1.5-2.0 mm below the cortical surface and left in place for 2-4 minutes following each injection, in order to ensure ample diffusion. In some cases spread to the underlying white matter did occur; however, the callosal projection patterns observed in this material did not differ from those in which injections were more restricted. Immediately following the injections, the cortex was covered with gelfoam soaked in saline, and the wound was closed. In most ME animals, HRP injections were made in the cortex ipsilateral to the enucleated eye so that the callosal zone could be studied in the cortex which was deprived of most of its input, k , the cortex contrdlaterd to the enucleated eye. These animals are referred to as ME contra in Figures 2-5 and Tables 1 and 2. In a few ME animals, for control data, HRP injections were made on the other side, so that the callosal zone could be studied in the cortex which was deprived only of the small ipsilateral input, i.e., the cortex ipsilateral to the enucleated eye. These animals are referred to as ME ipsi. Twenty four hours after the injections, animals were deeply anesthetized with nembutal(50 mgikg i.v.1,given an

A.M. GRIGONIS AND E.H. MURPHY

564 anticoagulant (heparin, 500 U.S.P units i.v.1, and perfused transcardially using 9% saline with 1% sodium nitrite followed by 1.25%gluteraldehyde with 1%paraformaldehyde, pH 7.4.

Histological procedures Following perfusion, the brain was removed, placed in a solution of 30% sucrose in 0.1 M phosphate buffer, and stored overnight at 4°C. Sections (40 bm) were made throughout the visual cortex. Every Fourth section was stained with thionin in order to visualize the cytoarchitecture of the striate and occipital cortices (Rose and Malis, '65). The remaining sections were processed with tetramethylbenzadine (TMB), modified with glucose oxidase (Mesulam, '78; Itoh et al., '79), and counterstained with 0.2%neutral red.

Data analysis The callosal projection comprises a callosal cell zone (containing callosal cells which project to the contralateral cortex) and the callosal terminal zone (the region which receives projections from callosal cells). Although both can be visualized with HRP, we have restricted our analysis to the callosal cell zone. A dense callosal cell zone obscures the terminal zone to some extent in HRP material and other studies indicate that, although the cell zone is often somewhat larger in tangential extent than the terminal zone, early experimental manipulations such as those we use in this study usually result in similar abnormalities in both the cell zone and the terminal zone. Light- and darkfield light microscopy were used to visualize the callosal cell projection. The border of the striate and occipital cortices were observed in the Nissl-stained material, using the criteria of Fleischhauer et al. ('80). The striate visual cortex and its lamination were then drawn for each Nissl-stained section using a drawing tube attached to the light microscope. Drawings of each section of laminated striate cortex were then used as a template upon which the distribution of callosal cells from HRP-stained sections adjacent to the Nissl-stained section were projected. At a magnification of 100x , the callosal cell distribution containing HRP-labelled cells was superimposed onto the drawing of the corresponding Nissl-stained section, and the callosal cell distribution was drawn. The density and distribution of callosal cells was estimated by averaging cell counts over ten sections for each animal. Density was always highest at the 17/18 border. In order to compare maximal density across groups, a 6 x lo4 Fm2 area was outlined over laminae I1 and 111, in each section, at the lateral edge of area 17, and the number of HRP-filled callosal cells counted (Fig. 6). To determine callosal cell density throughout the mediolateral extent of the callosal zone, a 2.25 x lo4 p,m2 square was moved medially in 200 pm steps from the 17/18 border to the medial extent of the callosal zone, and the number of HRP cells per area counted (Fig. 3). Again, mean density at each sample point was determined from counts over ten sections for each animal. To control for differences between groups in total cell density, we also determined the average number of cells in the 6.25 x lo4 pm2 area at the 17/18 border in Nissl-stained adjacent cortical sections. The level of significance was determined at P < 0.05 for the results of the Mann Whitney test (Table 2). The tangential extent of the callosal cell zone was defined as the mediolateral length of the callosal cell distribution beginning at the 17/18 border and extending to the medial

limit of the callosal zone. The medial limit was defined as the point at which callosal cell density counts fell below 10% of the mean of the preceding sampled area. For each drawn distribution of callosal cells, the tangential extent was measured using a Neumonics planimeter. In order to control for between-animal differences in shrinkage of tissue and also for differences in the mediolateral extent of striate cortex at different rostrocaudal levels, the tangential extent of the callosal cell distribution for each section was expressed as a ratio between the mediolateral extent of the callosal cell zone and the mediolateral extent of the entire striate cortex. This ratio was determined for each section and averaged for each animal. These data were used to compare the tangential extent of the callosal cell zone across experimental conditions, and differences between groups were analyzed using the Mann Whitney test. The laminar distribution of labelled callosal cells was determined by superimposing the HRP-labelled cell distribution of each section onto a drawing of the cortical lamination determined from an adjacent Nissl-stained section. Cells were identified as falling within laminae 11-111, lamina IV,or lamina V. The percentage of callosal cells in each lamina was determined for each section and averaged for each condition. Differences between groups were analyzed using a Chi-square test.

RESULTS Callosal projection in the normal adult rabbit In order to assess changes in the distribution of callosal cells following neonatal enucleation, dark-rearing (DR), and strobe rearing (SR), we examined the callosal cell zone in seven normally reared adult rabbits. These control data confirm results of previous studies using both Nauta and HRP methods to examine the callosal projection in the rabbit (Hughes and Wilson, '69; Choudhury and Gent, '73; Swadlow et al., '78). The visual callosal cell zone forms an elliptical pattern centered on the border of visual areas 17 and 18. Within striate cortex, the normal callosal cell zone is restricted to the lateral 25% of area 17 (Table 1 and Fig. 4). Figure 2 shows a photomicrograph of a coronal section through the middle of the rostral-caudal extent of the rabbit visual cortex. Most callosal cells are located in laminae I1 and 111, with relatively few cells in laminae IV and V (Fig. 5 ) . These data are in agreement with previous reports of the laminar distribution ofcallosal cells in the rabbit (Chow et al., '81a). Table 2 shows the density of callosal cells at the 17/18 border. The mean density of HRP-labelled callosal cells in normal rabbits at the 17/18 border was 44 cells per 6.25 x lo4 km2(Fig. 6 and Table 2 ) . Callosal cell density decreases with distance from the 17/18 border (Fig. 3).

Callosall projection following monocular enucleation In area 17 contralateral to the eye enucleated on the day of birth, the callosal cell zone in ME rabbits is expanded medially into cortical regions which are acallosal in the normal adult. The tangential extent of the callosal cell zone region is 48%larger than normal (Figs. 2-4 and Table 1). A photomicrograph of the abnormally wide callosal cell distribution through the center of the callosal cell region of an ME rabbit is shown in Figure 2. The laminar distribution of callosal projecting cells was abnormal, with a 12% increase in the proportion of labelled cells in lamina IV of

RABBIT VISUAL CALLOSUM FOLLOWING NEONATAL ENUCLEATION

Fig. 2. Representative photomicrographs of HRP-labelled callosal cells in sections taken from the middle of the anteroposterior extent of primary visual cortex in a normal, dark reared (DR), strobe reared (Strobe),binocularly enucleated (BE), and monocularly enucleated (ME contra: cortex contralateral to the enucleated eye; ME ipsi: cortex

565

ipsilateral to the enucleated eye) rabbit, and in rabbits given intraocular injections of tetrodotoxin (TTX)or control vehicle (TTXCont). Large arrows show the 17/18 border. Small arrows show the medial border of the callosal zone (see text for method of defining the medial border). Scale bar = 500 pm.

A.M. GRIGONIS AND E.H. MURPHY

566

Normal 40 30

y-=%bw 0 0.0

0.5

1.0

1.5

1

M E Contralateral

-o--q 2.0 0.0

Distance from 17/18 Border (mm)

0.5

1.0

1.5

2.0

Distance from 17/18 Border (rnm)

ME lpsilateral 20

1.5

l 0o b - - 0.0

0.5

2.0

1.0

0.0

Distance from 17/18 Border (mm) 40

30

1

1.0

1.5

2.0

Dark Reared

1 0.0

0.5

1.0

1.5

2.0

0.0

Distance from 17/18 Border (rnm)

40 30

0.5

Distance from 17/18 Border (rnm)

1

TTX

40

20 30

0.0

0.5

1.0

1.5

0.5

1.0

1.5

2.0

Distance from 17/18 Border (rnm)

20

Distance from 17/18 Border (rnrn)

l T X Control

1

1 0.0

0.5

1.0

1.5

20

Disiance from 17/18 Border (mrn)

Fig. 3. Average callosal cell density, at sample points from the 17/15 border to the medial hmit ot the callosal zone, for normal and experimental animals (for abbreviations see legend for Fig. 2). See text for methods of counting cell density.

ME rabbits compared with normal (Fig. 5). Counts of HRP-labelled callosal cells showed a significant increase in the density of the callosal projection. The mean number of callosal cells at the 17/18 border was 9416.25 x lo4 km2in ME compared with 44 in normal rabbits (Mann Whitney test, P < 0.011, and the density of the callosal cell distribution remained high through much of the medial extent of the callosal zone (Figs. 3, 6 and Table 2). This increased density of callosal cells does not reflect a change in overall cell density since there was no significant difference between normal and ME animals in cell density, as determined in Nissl sections (Table 2). The callosal cell zone in the hemisphere ipsilateral to the enucleated eye was similar to that observed in normal rabbit cortex (Figs. 2-4 and Table 1).Aphotomicrograph of a coronal section through the cortex ipsilateral t o the enucleated eye is shown in Figure 2. Neither the density nor the laminar distribution of callosal cells is significantly different from normal (Figs. 3 , 5 , and 6 and Table 1).

Callosal projection following binocular enucleation The callosal cell zone in a rabbit which had both eyes removed on the day of birth is shown in Figure 2. Unlike ME, BE does not result in a tangentially expanded callosal cell zone; its tangential extent does not differ from normal (Fig. 4 and Table 1).The laminar distribution of callosal cells also does not differ from that observed in the normal rabbit (Fig. 5 ) . However, there is a significant decrease in the density of callosal cells in these animals. The mean number of HRP-labelled cells at the 17/18 border per 6.25 x lo4 pnZwas 26 in BE animals compared with 44 in normals (Mann Whitney test, P < 0.05) and the density declined rapidly medial to the 17/18 border (Figs. 3, 6 and Table 2). This decreased density of callosal cells does not reflect a change in overall cell density since there was no significant difference between normal and BE animals in cell density, as determined in Nissl sections (Table 2).

RABBIT VISUAL CALLOSUM FOLLOW NG NEONATAL ENUCLEATION 60

567

100,

I

-

80

-

--.

80-

'k

40-

Q

0 Q E

2

20 Normal ME Contra

M E Ipd

BE

TTX

T T X Cont

DR

Strobe

Condition

Normal ME Contra ME Ipsl

Fig. 4. Average mediolateral width of callosal cell zone in the striate cortex, expressed as percent of total mediolateral width of area 17 for normal and experimental animals (for abbreviations see legend for Fig. 2). **, P < 0.01 (Mann Whitney test).

TABLE 2. Density of HRP-Labelled Callosal Cells and of Cortical Cells Counted in Nissl Sections, in Normal, ME, BE, TTX, Dark Reared, and Strobe Reared Animals'

Animal

Condition

R399 R455 R697 R763 R819 R871 R881 Mean R449 R450 R453 R697 Mean R686 R807 R809 Mean R522 R564 R565 R817 Mean R394 R513 R514 R515 Mean R615 R616 R618 R619 R623 R633 R685 R693 Mean R684 R688 Mean R821 R822 R823 R828 R829 R831 Mean

Normal Normal Normal Normal Normal Normal Normal ME contra ME contra ME contra ME contra ME ipsi ME ipsi ME ipsi

BE BE BE BE Dark reared Dark reared Dark reared Dark reared TTX TTX TTX TTX TTX TTX TTX TTX TTX control TTXcontrol Strobe Strobe Strobe Strobe Strobe Strobe

Callosal cell density/ 6.25 x 10' pmz (SEMI

Cortical cell density/

59.73 (9.38) 48.44 (7.10) 31.83 (6.74) 37.97 (8.89) 36.53 (3.871 47.16 (7.58) 45.74 (4.23) 44.11 (3.27) 73.69 (5.46) 118.72 (7.73) 97.65 (8.21) 84.78 (8.96) 93.71 (11.17)** 42.40 (4.73) 38.75 (3.67) 40.21 (6.74) 40.45 (1.30) 27.00 (1.22) 34.25 (8.48) 19.40 (4.01) 25.50 (9.57) 26.54 (3.52)* 29.00 (6.20) 42.51 (4.23) 44.25 (3.45) 37.50 (7.16) 38.32 (3.95) 40.80 (4.35) 21.75 (3.31) 22.00 (1.08) 18.80 (2.75) 31.50 (5.17) 21.51 (4.23) 23.75 (3.48) 23.25 (2.23) 25.42 (2.731** 26.75 (4.93) 31.75 (2.23) 29.25 (3.54)* 79.50 (5.47) 87.93 (8.52) 131.79 (6.43) 100.95 (3.47) 105.16 (4.62) 91.75 (7.23) 99.51 (8.181**'

159.79 (6.54) 129.25 (7.18) 136.50 (5.89) 181.51 (5.97) 153.25 (10.34) 167.52 (6.82) 159.75 (4.65) 155.37 (6.42) 154.25 (5.10) 159.25 (5.42) 178.75 (10.89) 182.50 (5.70) 168.69 (8.09) 164.50 (15.12) 157.75 (5.85) 122.95 (5.44) 148.33 (11.21) 131.01 (11.26) 118.00 (5.58) 167.25 (12.01) 150.50 (13.78) 141.69 (9.31) 150.75 (8.22) 156.75 (10.01) 163.25 (10.96) 164.75 (10.83) 158.88 (3.71) 117.00 (2.63) 141.25 (5.97) 133.25 (13.71) 142.75 (2.64) 149.00 (5.73) 133.75 (3.98) 132.50 (5.35) 128.67 (7.88) 134.77 (3.70)* 122.75 (4.88) 131.00 (8.71) 126.88 (5.83)* 190.00 (3.18) 197.00 (12.47) 187.50 (3.41) 181.00 (3.93) 197.25 (6.04) 175.25 (5.77) 188.00 (7.38)*

6.25 x

I Laminas 11-111

lo4 pm2

Callosal projection following dark rearing In DR rabbits, the tangential distribution of callosal cells is approximately 26% larger than in normally reared rab-

TTX

T T X Cont

OR

SR

RSY Lamina IV

0Lamina v

Fig. 5. Histogram of the percent of callosal cells in the striate cortex in laminae 11-111, IV and V in normal and experimental rabbits. In ME, DR, and TTX rabbits the percent of callosal cells in lamina IV was significantly higher than normal (for abbreviations see legend for Fig. 2). *, P < .05; **P < .02 (Chi-square test).

(SEMI

'See text for methods of assessing cell density. Data from each experimental group were compared with normal (Mann Wbitney test; *P < 0.05; **P < 0.02; ***P< 0.01). For abbreviations, see Table 1 footnote.

BE

Condition

100

...

..

1-

Normal

ME Contra

ME Ip8l

BE

TTX

TTX Cont

Strobe

Condition Fig. 6. Histogram of density of callosal cells at 17/18 border where callosal density is maximal, in normal and experimental rabbits. See text for methods of assessing density (for abbreviations see legend for Fig. 2 ) . *, P < 0.05; **, P < 0.02; ***, P < 0.01 (Mann Whitney test).

bits (Figs. 2-4 and Table 1).Figure 2 shows a photomicrograph of a coronal section through the middle of the rostrocaudal extent of the callosal projection, showing the expanded callosal cell distribution. The laminar distribution of callosal cells in DR rabbits (Fig. 5) shows an increase in the proportion of cells in lamina N compared with the distribution observed in normal animals (Chi-square test, P < 0.02), but the maximal density of callosal cells at the 17/18 border is not significantly different from normal (Figs. 3 , 6 and Table 2).

Callosal projection following strobe rearing The callosal cell zone in SR rabbits is 28% smaller than normal (Mann Whitney test, P < 0.02), (Figs. 2-4 and Table 1).Figure 5 and Table 2 show the laminar distribution and density of callosal cells in an SR rabbit respectively. The laminar distribution of callosal cells was not different from normal, but the density of cells at the 17/18 border was significantly higher than normal. The mean number of HRP-labelled cells per 6.25 x lo4 km2 a t the 17/18 border was 100 compared with 44 in normal rabbits (Mann Whitney test, P < 0.01). Although cell density measurements in Nissl sections also revealed a small but significant increase in cell density in SR rabbits, the density of callosal cells in SR rabbits was increased more than 100%

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pattern (Fig. 1)is similar to that seen in the cat (Innocenti and Frost, '80; Olavarria and Van Sluyters, '84b), but different from that seen in the rodent in which ME and BE ME BE TTX DR SR result in equally expanded callosal zones (Malach et al., '83; CC width + nc + + Olavarria et al., '83; Rhoades and Fish, '83; Lund et al., '84; Laminar N+ nc IV+ 1v+ nc + Densitv nc + Rhoades and DelllaCroce, '84). Since rabbits have predominantly contralateral retinofugal projections, like rodents, I + , increase; -, decrease;nc, nochange. CC, corpus callosum; DR, dark reared, SR, strobe reared. For other abbreviations see Table 1 footnote. we conclude that the relative effectiveness of ME and BE in modifying callosal projections is not dependent on the extent of binocular representation in the striate cortex. An alternative hypothesis which is supported by our data and cannot be accounted for by the small change in overall is that the relative effects of ME and BE vary with the state cell density. of maturity of the visual system at the time of enucleation. Callosal projection following intraocular The rodent visual system is much more immature at birth TTX injections than that of the cat and rabbit. Ipsilateral and contralateral Animals reared with retinal ganglion cell activity elimi- retinofugal projections to the lateral geniculate nucleus still nated in one eye by intraocular injections of TTX showed a overlap in the rodent at the time of birth, but are largely significant increase in the tangential extent of the callosal segregated in the lateral geniculate nucleus of the newborn cell zone in the visual cortex contralateral to the injected rabbit and cat. The degree of overlap or segregation of these eye (Mann Whitney test, P < 0.02) (Fig. 4 and Table 11, and projections at the time of enucleation may determine the a significant increase in the proportion of callosal cells in type and extent of enucleation-induced morphological lamina IV (Chi-square test, P < 0.05). A significant de- changes in the retinogeniculocortical projections, and these crease in callosal cell density was observed in TTX animals changes may subsequently influence callosal development. (Mann Whitney test, P < 0.02) (Figs. 2, 3, 6, and Table 2 ) . This hypothesis could be further tested by comparing the However, since a small but significant decrease in density effects of ME and BE in prenatal cats or in newborn ferrets. was also observed in TTX control animals and in Nissl Our hypothesis predicts that if enucleations were done sections, it is possible that these decreases reflect transneu- prior to segregation of the retinogeniculate projections in ronal effects of repeated eye injections. These studies have cats and ferrets, then the callosal zone would be equally been described in detail elsewhere (Murphy and Grigonis, expanded following ME and BE, as is the case for rodents. However, some role for binocular competition in shaping '88) and the data are included in Figures 3-6 to permit callosal projections in the rabbit cannot be discounted. comparison with data of the present study. Figure 4 shows that, across all experimental groups, the callosal zone width is most expanded in those conditions DISCUSSION which involve monocular manipulations (TTX and ME) The major findings of this study are that early manipula- compared with those which involve binocular manipulations of normal development result in abnormalities of tions (DR, BE,, SR). The mechanisms by which binocular visual callosal projections, and the pattern of abnormalities competitive effects could be operating when the visual differs for each manipulation. The tangential width of the projections are predominantly monocular are unclear. One callosal zone is increased in all animals except BE animals possibility is that, in neonates, not only are the retinotha(in which it is normal), and SR animals (in which it is lamic projections originating from the ipsilateral and consmaller than normal). Laminar organization is changed tralateral eyes exuberant and overlapping (Lund and Bunt, only in ME, DR, and TTX animals. Callosal cell density is '76; Linden et al., '81; Godement et al., '84; So et al., '84; greater than normal in ME and SR animals, and lower than Grigonis et al., '861, but the thalamocortical projections normal in BE animals. These findings are summarized, for may also be exuberant. If so, the area of cortex which receives binocular input, and which could therefore be clarity, in Table 3 . influenced by binocular competition, would be greater in the neonate than in the adult. We are currently investigatMethodological issues ing the postnatal development of these thalamocortical Differences between animals in density of HRP-labelled callosal cells could result from differences in the injection projections in the rabbit. If the effect of ME were simply to give a competitive site or from differences in the overall density of neurons advantage to callosal inputs, then lesions of the thalamus or within the region in which counts are made. However, data radiations should result in an expanded callosal zone from individual animals, presented in Tables 1 and 2, similar to that observed in ME animals. This has been indicate that measurements are consistent within experireported for rodent visual cortex (Cusick and Lund, '82; mental groups and cannot be attributed to variability in injections. With only four to six animals per experimental Fish et al., '85), but not for rodent parietal cortex (Wise and Jones, '78). The failure to observe expanded callosal projecgroup, the differences we observed are consistent and tions in parietal cortex following unilateral thalatomy is statistically significant. In addition, our counts of cell density in Nissl-stained sections (Table 2) indicate that, surprising, and suggests that visual and parietal cortex may except in TTX animals, the differences between groups in differ in other factors which influence callosal development, the density of callosal cells cannot be attributed to between- such as maturation or specific aspects of callosal organization. group differences in overall cell density. TABLE 3. Changes in Callosal Organization of ME, BE, TTX, DR, and SR Rabbits Cornoared With Normal

ME versus BE

TTX versus ME

In the rabbit, ME results in a callosal zone which is much more expanded than the callosal zone of BE animals. This

Comparison of TTX and ME rabbits indicates that absence of activity in retinal ganglion cells (RGC) has effects

RABBIT VISUAL CALLOSUM FOLLOWING NEONATAL ENUCLEATION on callosal development which differ from those of removal of the RGCs. The callosal zone is significantly bigger in ME animals than in TTX animals, both in tangential extent and in callosal cell density, suggesting that the two deprivations involve different mechanisms. Our previous observations that the critical period for modification of callosal organization by TTX injections occurs later than the critical period for ME also suggest that the mechanisms involved are different (Murphy and Grigonis, '88). Cell death and sprouting of intact pathways may be involved in the response to ME, whereas TTX injections may interfere with the stabilization of synaptic connections. Intraocular TTX injections in cats prevent maturational changes in formation of ocular dominance columns in the cortex and in synapse development in the lateral geniculate nucleus (Riccio and Mathews, '85; Kalil et al., '86; Stryker and Harris, '86). Neural activity may play a role both in stabilizing appropriate projections by correlated activity and in destabilizing inappropriate connections which do not have synchronous activity. Absence of activity then might result in a wide callosal zone but a low density of cells within this wide callosal projection.

BE versus DR BE and DR both involve binocular manipulations of retinal input, but their effects are different. The width of the callosal zone is expanded in DR but not BE animals, whereas callosal cell density is decreased below normal levels in BE but not in DR animals. A decrease in the number of callosal cells following BE has been reported in hamsters (Rhoades and Fish, '83) and cats (Innocenti and Frost, '80; Olavarria et al., '84b). These results, and the results from TTX animals (above) support the hypothesis that RGC activity plays a role both in stabilizing appropriate callosal projections at the 17/ 18border and in destabilizing inappropriate projections in medial area 17. In DR rabbits, although we observed some expansion of the width of the callosal zone, the effects of this deprivation were less severe than the effects of most other deprivations studied. In other mammals, minimal effects of DR have been reported. There is no change in the width of the callosal zone of DR hamsters (Rhoades et al., '84) or rats (Cusick and Lund, '82), and a slight but inconsistent narrowing of the callosal zone has been reported in DR cats (Lund and Mitchell, '79a; Frost and Dean, '86; Frost and Moy, '89). Thus, in the presence of RGC activity and the absence of competitive imbalance, callosal projections are minimally affected even in the absence of light stimulation.

Strobe rearing Of all deprivations studied, strobe rearing has unique effects. It is the only deprivation which resulted in a callosal zone narrower than normal, but it also resulted in the highest density of callosal cells within this callosal zone. The increased density of callosal cells suggests that strobe rearing stabilizes appropriate callosal projections at the 17/18 border more effectively than normal visual stimulation. Single-cell recording from the visual cortex of SR animals reveals receptive fields which are precise in orientation tuning, have normal ocular dominance characteristics, are normal or even smaller than normal in size, but which are deficient in direction selectivity-a y-aminobutyric acid (GABA)-mediated function (Cynader and Chernenko, '76; Sillito, '77; Pearson et al., '81, '83; Pearson, '83; Cremieux et al. '87). Thus, despite strobe-induced synchrony of

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neural activity, retinal stimulation in the absence of eye movements may provide a less noisy signal than retinal stimulation under normal conditions when eye movements may blur the signal. Both the observation of smaller than normal receptive fields and the observation of a narrowed callosal zone in SR animals support this interpretation. Such stimulation might provide optimal conditions for stabilization of synapses, resulting in increased callosal cell density. The effects of strobe rearing on callosal organization has been reported in only one other mammal-the hamster (Rhoades et al., '84). Unlike rabbits, the callosal zone was wider than normal in SR hamsters. The difference between rabbits and hamsters in the effects of strobe rearing on callosal development may be attributable to the fact that in rabbits, the eyes open before the callosal projections mature whereas in hamsters, the callosal zone reaches mature dimensions before the eyes open. Thus, hamsters do not experience patterned visual experience with stroboscopic stimulation until after the callosal projections are mature. The effects of diffuse stimulation experienced by SR hamsters before their eyes open may be similar to the effects of diffuse stimulation achieved by lens removal in SR goldfish. Retinotectal regenerating projections in these goldfish show a loss of precision similar to that resulting from dark rearing (Eisele and Schmidt, '88). In addition to the greatly increased density of callosal cells in SR rabbits, we also observed a smaller but significant overall increase in cell density in the supragranular layers of the striate cortex. We can only speculate on the possible significance of this observation. A Hebbian model (Hebb, '49) might predict that the synchronous neuronal activation caused by stroboscopic stimulation might consolidate synapses and this could reduce cell death. Developmental cell death has been documented in mouse neocortex, especially in granular and supragranular layers (Heumann and Rabinowicz, '82; Heumann et al., '78). However, the rabbit cortex is more mature at birth than that of the mouse and postnatal developmental cell death in rabbit visual cortex has not been documented. Alternatively, the increased cell density could reflect a developmental delay. Cell density is higher in younger than in older brains and the increased cell density may merely reflect a reduction of the neuropil. Heumann and Rabinowicz ('82) observed an increased neuronal density in mice 10 days after enucleation, with some recovery at 180 days. Since they observed no cell loss, these results suggest a delay in development of the neuropil. Further data are needed to confirm and interpret strobe-induced changes in cell density.

Mechanisms controlling callosal cell density There is evidence that the normal elimination of exuberant callosal cells involves not cell death but the retraction of a callosal axon collateral and the maintenance of an axon collateral projecting ipsilaterally to adjacent visual cortical areas (Innocenti, '81).It has not been established whether, when these callosal projections are maintained as a result of early experimental manipulations, they lose their ipsilatera1 projection, or maintain both collaterals. Double-label techniques would be needed to answer this question definitively. However, if the cells maintain an additional axon collateral, then soma size may be increased (Murphy et al., '90).

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Mechanisms controlling laminar distribution of callosal cells We observed some increase in the percentage of callosal cells in lamina IV in all experimental animals (Fig. 5), although this effect was significant only in DR and ME animals. Callosal cells in lamina IV are rarely seen in other mammals but their presence in rabbits has been confirmed by others (Swadlow et al., '781, and they may represent a unique population of callosal cells. Lamina N callosal neurons differ from callosal neurons in laminae 11, 111, and V in that lamina N neurons receive the major thalamic input. Thus, the effects of visual deprivation impact on lamina IV cells more directly than on most other cells in striate cortex, and this may enhance their susceptibility to such deprivation-induced modification of their callosal projections.

CONCLUSIONS While many factors clearly interact in shaping the development of area 1 7 callosal projections, our data, considered in the context of data from other studies using rodents and carnivores, suggest some conclusions about the mechanisms involved in callosal development. While these conclusions do not apply to area 1 7 of the primate, which appears to be unique in that callosal projections are sparse and never undergo a period of transient exuberance, they may prove applicable to primate area 18, which does have exuberant callosal projections during prenatal development. Further experiments will serve to test or confirm the conclusions below: 1. Visual deprivation (DR, SR, strabismus, and lid suture) modifies callosal development only in those mammals (rabbit, cat) in which callosal retraction is still ongoing at the time of eye opening. 2. The relative expansion of the tangential extent of the callosal zone following ME and BE depends on the maturity of the retinofugal projections and not on species differences in the degree of binocular interaction. 3. The tangential extent of the callosal zone is increased by manipulations which reduce or eliminate neural activity in the retinofugal pathways (BE, TTX), suggesting that neural activity plays a critical role in the elimination of exuberant projections. 4. The density of callosal cells is reduced by these same manipulations which reduce or eliminate neural activity in the retinofugal pathways (BE, TTX, and lid suture), suggesting that neural activity also plays a critical role in the stabilization of appropriate callosal projections. 5. The density of callosal cells is increased by two manipulations (ME, SR), which increase the synchrony of neural activity (SR) or the level of neural activity [in the intact retinofugal projections of ME animals (Clarke et al., '91)l.

ACKNOWLEDGMENTS We wish to thank Joyce Brown for her excellent histological technical assistance. The work was supported by NS26989.

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