Postnatal development of the visual corpus callosum: The influence of activity of the retinofugal projections

151

Behavioural Brain Research, 30 (1988) 151-163 Elsevier BBR 00720

Postnatal development of the visual corpus callosum: the influence of activity of the retinofugal projections a E. H a z e l M u r p h y a n d A n t o n y M. G r i g o n i s * The Medical College of Pennsylvania, Department of Anatomy, Philadelphia, PA 19129 (U.S.A.) (Received 9 December 1986) (Revised version received 24 April 1987) (Accepted 28 April 1987)

Key words: Tetrodotoxin; Enucleation; Callosal cell zone; Visual Experience; Development

Visual callosal projections were studied in normal adult rabbits, and in adult rabbits in which normal development was manipulated by monocular enucleation on the first or seventh postnatal day, or by abolition of retinal physiological activity by repeated application oftetrodotoxin ( T r x ) beginning on postnatal day 7. Animals given control vehicle injections, and animals enucleated on postnatal day 7 did not differ from normal in the tangential extent of their callosal zone which is limited to the lateral one-third of area 17. In contrast, animals enucleated on the day of birth and animals given T r x vitreous injections beginning on postnatal day 6-7 are similar in that the tangential extent of their callosal cell zone extends approximately through the lateral two-thirds of area 17. The results suggest that different mechanisms underly the effects of removal of the eye, and abolition of retinal activity, and that the critical period for the effective manipulation of these two mechanisms is different.

INTRODUCTION

An important principle in neural ontogeny is that projections that are widespread prenatally or at birth - exuberant projections - become restricted as maturity is approached 1°,2°,34.5t,52. This process, by which selected projections are maintained and inappropriate ones are eliminated, appears to play an important role in the selection of appropriate targets during development. The mechanisms underlying this selective elimination are not well understood but activitydependent influences in the competition of available space are considered i m p o r t a n t 21,3°,47,64, 80,87,88,96

The visual callosal projections offer an ideal

model system to investigate the mechanisms underlying this aspect of development, because they undergo extensive postnatal development, and the physiological and anatomical organization of their afferents and efferents are well studied. In adult mammals, visual callosal projections are largely restricted to the representation of the vertical meridian, which is located at the border of cortical areas 17 and 18, suggesting that they may play a role in the integration of information across the midline and in s t e r e o p s i s 19,31,32,50,68,71,72,74, 75,89,90,91,98-101. In most neonatal mammals, in contrast, the caUosal cell zone (the region containing cells with axons which project into the corpus callosum) is tangentially expanded in medial area 17. In most neonatal mammals, the callosal

" Some of the data reviewed here has been previously presented in abstract form24~6 and was also presented at the IBBS workshop on Hemispheric specialization and interhemispheric communication, held in Rotterdam on 20-21 March 1986. * Present address: Hahnemann University, Department of Anatomy, Philadelphia, PA 19102-1192, U.S.A. Correspondence: E.H. Murphy, Department of Anatomy, Medical College of Pennsylvania, 3300 Henry Ave., Philadelphia, PA 19129, U.S.A. 0166-4328/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

152 terminal zone (the region in which callosal axons terminate) is also tangentially expanded into medial area 17, although these exuberant axons in all mammals studied except rabbits are largely restricted to the white matter. Thus, the postnatal developmental sequence involves a tangential restriction of the callosal cell and terminal zones. Evidence suggests that most of this restriction involves a selective elimination of axons, rather than cell d e a t h 9'33-44'53'61'62"69'70"83'94 In the present study, we used the visual callosal projections of the rabbit to study some of the mechanisms underlying this postnatal elimination of initially exuberant callosal projections. In this paper, we will present only data on experimentally induced modifications in the tangential organization of the callosal cell zone in primary visual cortex. Investigations of changes in the tangential organization of the terminal zone, and changes in the laminar organization of both cell and terminal zones, are still ongoing. In the studies summarized below we have concentrated our attention on the influence that activity of the central visual projections, the retino-geniculo-cortical (RGC) projections, have on the postnatal selective narrowing of the initially exuberant callosal cell zone. The rabbit visual system is predominantly monocular and 90 % of the visual pathway is crossed 23. Thus, as shown in Fig. 1, the region of area 17 that is normally callosally linked in the adult is largely restricted to the small binocular region of primary visual cortex32,99,101. The remaining primary visual cortex is monocular, and is normally not callosally linked except by transient exuberant projections in the neonate 5. Monocular and binocular cortical areas therefore differ both in their RGC input (monocular or binocular) and in their callosal cell input (transient or stable). In rodents which, like rabbits, have a predominantly monocular visual system, monocular enucleation on the day of birth results in a tangentially expanded callosal cell zone which extends throughout area 17 in the cortex contralateral to the enucleated eye, suggesting that, in the absence of normally functioning RGC afferents, the exuberant callosal projections fail to undergo their normal postnatal elimination 13"2L46.s3.sT'63,7°'

D

binocular zone

]

callosal cell zone

I

ADULT t t t i

........

t t t i

o,

~ i

NEONATE i

fl

o e • • d ~ l l

..... "~I

ME (1)

Fig. 1. Diagrammatic representation of the regions of area 17 which are monocular or binocular, and the callosal projections zones in the normal adult, the normal neonate, and the adult monocularly enucleated on the day of birth [ME(l)]. The cortex contralateral to the enucleated eye is illustrated. Arrows indicate input from the dorsal lateral geniculate nucleus. Double arrows indicate the input originates from both eyes. Dotted arrows indicate the elimination or modification of this input.

81,86. Our first experiment was aimed at determining whether monocular enucleation at birth would modify the normal development of visual callosal projections in rabbits. EXPERIMENT 1

Monocular enucleation at birth

All studies used Dutch Belted rabbits, which have pigmented eyes, and which were bred in our animal facility. The animals used in each study are listed in Table I. In one series of animals (n = 3), monocular enucleation was performed under halothane anesthesia on the day of birth (for details of surgical procedure see ref. 29). Animals were then returned to the mother and reared normally until the age of 4-8 weeks (i.e. at an age when, in rabbits, the maturation of visual cortex is largely complete 66'67, and at least 2 weeks after the age when callosal projections have normally reached their mature organization 5. As shown in Table I, the average age at sacrifice for all groups was between 40 and 50 days. The largest range of age at sacrifice is in

153 TABLE I Number and age of animals in each group Treatment

n

Normal ME(l) ME(7) TTX TTX control

2 3 3 7 5

Age at initiation of treatment (days)

Age at sacrifice (days)

1 7 6,7 6,7

45, 56 48,49,52 44,45,67 26,32,33,37,42,60 32,45,4.6,50

the tetrodotoxin (TTX) group, and no differences were observed between the youngest and oldest animal in this group. At this time, animals were again anesthetized, and a Hamilton syringe was used to make multiple injections of 0.1 ~tl of 10 ~o horseradish peroxidase (HRP) in H:O at l-ram intervals throughout the primary visual cortex ipsilateral to the enucleated eye. The injection site extended from the bregma suture to the posterior limit of the cortex, and from the midline to at least 10 mm lateral to the splenial sulcus. This ensured that the injection site included the entire primary visual cortex (area 17), and also extended several mm lateral, medial and rostral to area 17. Following a survival of 24 h, animals were again anesthetized and perfused pericardially with 0.9~o saline followed by a mixture of 1 ~o paraformaldehyde and 1.259/o gluteraldehyde in 0.1 M phosphate buffer. The cortex was sectioned frozen at 50/~m, and treated with tetramethylbenzidine (TMB) modified by the use of cobalt-intensified o x i d a s e 1"45"59. Sections were faintly counterstained with Neutral red, and every third section was stained with Cresyl violet. These sections were used to identify the 17/18 border. Light- and darkfield optics were used to visualize the HRPfilled cells. The zone in which HRP-filled cells were locMized within area 17 was drawn and measured. The entire antero-posterior extent of area 17 was used for these measurements, and the width of the caUosal zone was expressed in each animal as a percentage of the total mediolateral extent of area 17 in order to control for possible differences in shrinkage. These data were compared with data obtained from normal animals (n = 2).

Fig. 2A shows photomicrographs (with higher magnification on the right) of HRP-fllled cells in area 17 of a normal rabbit (Fig. 2A). The cells are restricted to the region around the 17/18 border and are strictly limited in their tangential extent. Most of medial area 17 is free of callosal cells. In contrast, in Fig. 2B, are photomicrographs of the callosal cell zone of a rabbit in which the contralateral eye was enucleated on the first postnatal day [monocular enucleated, ME(I)]. Although the callosal cells are still most densely clustered around the 17/18 border, they extend much further than normal into medial area 17. Since Chow et al.5 reported that a dense callosal cell zone is present throughout area 17 in the normal 7-dayold rabbit, these data indicate that enucleation on day 1 results in an incomplete elimination of exuberant callosal cells. As illustrated in Fig. 1, exuberant projections in the most medial onethird of area 17 are eliminated in ME(l) animals, but many of the cells in the middle one-third of area 17 maintain their exuberant callosal projections into adulthood. Inspection of the material suggests that, even in the normal callosal zone at the 17/18 border, the density of callosal cells may also be greater than normal. The modification of callosal projections in ME(I) rabbits could result from imbalance in the functional input from the 2 eyes or from the morphological consequences of the enucleation, including degeneration of optic terminals, transneuronal degeneration of geniculate or cortical neurons, or compensatory alterations in the remaining intact pathways, such as sprouting, or failure of retraction of exuberant projections. The critical period for morphological consequences of enucleation, which could underlie abnormal callosal projections, may occur very early in development, since the sprouting and failure of retraction which may result from early lesions often occur only during a very limited critical period 7,8,12,48. For example, expanded retinocollicular projections are observed in ME rabbits only if they are enucleated on or before the day of birth 7,8,27, and the normal retraction of exuberant retino-geniculate projections in the rabbit is completed within the first postnatal w e e k 28,73. However, the functional consequences of such

154

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C ~

:i . . . . .

i:¸ :.~

Fig. 2. Photomicrographs o f HRP-filled callosal cells in area 17 of mature rabbits. For enucleates, the cortex contralaterat to the enucleated eye is illustrated. Low magnifications are shown on left (bar = 500/~m) and higher magnifications at right (bar = 250 #m). O n high magnification photomicrographs, the right arrow indicates the location between areas 17 and 18. The left arrow indicates the medial border of area 17. A: reared normally. B: monocularly enucleated on the first postnatal day [ME(l)]; C: monocularly enucleated on the seventh postnatal day [ME(7)].

155 morphological changes must occur later. In rabbits, functional activation of the central visual pathway is minimal prior to the second postnatal week. Although the retinal ganglion cells and the subcortical nuclei to which they project have some spontaneous activity before that time, the spontaneous activity is extremely low 58'79"93. Light-mediated activity cannot influence the retinofugal pathway of the rabbit until postnatal days 6-714'15'm2. Therefore, if the consequences of ME reflect exclusively an altered balance of functional activity from the two eyes, then ME at the time of onset of such functional activity should have the same effect as on day 1. However, if the consequences of ME reflect the altered function of intact pathways which have undergone sprouting, then ME after the fn'st postnatal week may not have the same effects on callosal development as ME on the day of birth. The next experiment explored this question.

callosal projections, (which are still exuberant and extend throughout area 17 on day 7). These findings suggested that, in the rabbit, as in the rodent ~3, visual experience may not have a significant influence on visual callosal development. However, it is now well estabfished that in the rabbit, as in the cat 92, visual experience can modify the functional organization of visual cortical neurons, and that the critical period for such plasticity extends into the second postnatal month. There is also evidence, in the cat 4'55's6, that visual experience can modify callosal projections even after significant retraction of exuberant callosal projections has occurred. Finally, data from both cats and rodents suggest that removal of the eye, and deprivation of visual experience may have quite different effects on callosal development 13"22"4°'54'7:.We therefore initiated studies in which we investigated the effects of modifying the activity of the RGC projections, beginning at the time of onset of light-mediated functional activation of these projections.

EXPERIMENT 2 EXPERIMENT 3

Monocular enucleation on postnatal day 7 In a second series of animals (n = 3), monocular enucleation was carried out on the 7th postnatal day, animals were allowed to survive for 4-8 weeks, and the callosal cell zone was studied following injection of HRP into the cortex ipsilateral to the enucleated eye, using the same methods described in Expt. 1. In this case, as shown in Fig. 2C, the cell zone appears normal. It is not expanded medially, and its density at the 17/18 border appears indistinguishable from normal. Thus, the critical period for the modification of callosal projections by ME is over by postnatal day 7. These results suggest that the effects of ME on the day of birth may involve the influence of pathways altered by reactive sprouting or by failure of retraction, rather than solely the influence of imbalance in activity from the 2 eyes. Thus, it appears that the critical period for such lesioninduced reorganization of pathways is over (1)before the onset of light-activated activity (which occurs around day 7 ~4"15a°2) and (2)before the occurrence of significant retraction of

The elimination of retinal ganglion cell activity with TTX TTX abolishes neural activity by blocking sodium channels. Therefore, repeated injections into the vitreous have the effect of eliminating the transmission of activity from the retina to the thalamus, without the associated changes resulting from removal of axons 3'95"96. Lyophilized TTX in citrate buffer was diluted with saline to produce a solution of 5 x 10- 3 M TTX. Vitreal injections were made 3 times/ weekly, under anesthesia [0.1~o Ace Promazine (0.35 mg/kg) and 1 ~o Ketamine (3.5 mg/kg) with 2 ~ xylazine (5 mg/kg)], to provide a continuous blockade of retinal activity (n--7). Injections were started on postnatal day 6 or 7 with an initial dose of 1/~1, the dose injected was increased with increasing weight of the animal, and injections were continued for at least 3 weeks. Identical procedures were carried out for control animals (n = 5), except that a 0.0035 M citrate buffer vehicle solution, pH 4.8, was used for control injections. The volume of vehicle solution injected

156

TTX

A

B

corltrol

TTX

+ +.

+

Fig. 3. Photomicrographs ofHRP-filled callosal cells in area 17 contralateral to the injected eye in mature rabbits. Magnification and calibration bars as for Fig. 2. A: following vehicle vitreous injections given 3 times weekly as controls for TTX injections. B: following TTX vitreous injections given 3 times weekly from days 6-7. Arrows indicate the area 17/18 border.

in these control animals was matched to the volume of TTX solution injected in experimental animals. Animals were inspected daily for signs of eye infection and they were weighed daily. Weight gain of litter mates given TTX or vehicle injections was similar, and the TTX appeared to cause no systemic effects. The dosage selected for TTX animals was similar to that used and found effective in blocking retinal activity in rabbits by Masland (personal communication) and in cats by Stryker ~nd H a r r i s 96. Animals were checked daily for direct and consensual light reflexes (which can first be detected around postnatal day 9) to monitor the efficacy of the treatment. Consensual reflexes are

not easily obtained in normal rabbits but were never obtained in TTX animals. Absence of the direct response in TTX animals could reflect blockade of either the pupillomotor system or of the retinal ganglion cells. However, Stryker and Harris observed that following cessation of TTX injections in kittens, blockade of retinal ganglion cells continued for 110 h after a TTX injection, and they also noted that retinal ganglion cell blockade remained complete for 24 h after complete cessation of the TTX-induced pupillomotor blockade. In our rabbits the efficacy of the TTX blockade can therefore be reasonably assumed, since the inter-injection time interval for our rabbits never exceeded 72 h, and since the direct

157 light reflex was always blocked. Following at least 3 weeks of this treatment, multiple injections of 10~ H R P were made throughout area 17 in the cortex ipsilateral to the injected eye, as described above for Expt. 1, in order to assess the callosal cell zone. Fig. 3 shows H R P labelled callosal cells in area 17 of animals given control vehicle injections (Fig. 3A) and animals given TTX injections (Fig. 3B). Control animals do not differ from normal (See Fig. 2A). In contrast, the callosal cell zone of TTX animals is expanded and fills the lateral two-thirds of the primary visual cortex. Its tangential extent is similar to that observed in ME(I) animals, but the density of labelled cells is clearly less than that observed in ME(l) animals. The results demonstrate a failure of retraction of medially located exuberant callosal cells following blockade of retinal activity beginning at day 7. The paradox presented by these data, which is discussed below, is that TTX injections initiated on day 7 can modify callosal development, whereas enucleation on day 7 is ineffective. The results of the series of experiments described above are summarized in Fig. 4, in which the tangential extent of the callosal cell zones in all groups of animals is shown. Animals given control vehicle vitreous injections, and animals enucleated on postnatal day 7 to not differ from normal, and their callosal cell zone is limited to the lateral one-third of area 17. In contrast, animals enucleated on the day of birth, and animals given TTX vitreous injections from days 6-7 are similar in that the tangential extent of their callosal cell zone extends approximately through the lateral two-thirds of area 17. DISCUSSION

The effects of monocular enucleation Our data indicate that monocular enucleation on the day of birth prevents the normal retraction of exuberant callosal projections, but enucleation on postnatal day 7 has no effect on this process of retraction. Expanded callosal projections have been reported in many other mammals following neonatal enucleation. In M E rats and hamsters, the callosal zones in the contralateral cortex fill

60

p

5o

e r

40

G e

30

n 2O

t

o

NORM

CONT

ME(7) TTX(7}

ME(l)

Fig. 4. Histogram of the average tangential extent of the callosal cell zone in normal (NORM) rabbits, and in rabbits enucleated on day 1 [ME(I)] or day 7 [ME(7)], given TTX or control vehicle (CONT) vitreous injections. Values shown are mean mediolateral extent of the callosal zone in area 17 expressed as a percentage of the total mediolateral extent of area 17.

most of area 1713"53'81'86. In M E cats, the callosal cell zone is wider than normal but does not extend throughout area 1739. In rabbit, the effect is intermediate between these two extremes, with the callosal cell zone f'tlling two-thirds of the mediolateral extent of area 17. Species differences in the degree of abnormality of the callosal zone following ME may reflect the relative balance of competitive inputs to cortex. The cat's visual system is predominantly binocular, so that much of the exuberant callosal zone receives input from both eyes. Monocular enucleation in the cat, therefore, would result in a much less severe reduction of the R G C input to this region of cortex, compared with the reduction in monocular systems, such as those of the rabbit and rodent (see Fig. 1). Our data partially support this interpretation, since the callosal zone abnormality in M E rabbits is much more extensive than that observed in ME cats.

158 However, it is less extensive than that seen in rodents. An alternative hypothesis to account for the differences in the extent of callosal zone in ME cats compared with ME rodents, is that the rodent visual system is much more immature at birth and therefore more capable of re-organization in response to early lesions. Our data in rabbit support the hypothesis that the effectiveness of monocular enucleation may be related to the immaturity of the system at the time of birth (for review see ref. 66). The rabbit visual system is intermediate between the rodent and cat in terms of its maturity at the time of birth, and its callosal abnormalities in response to enucleation on day 1 are also intermediate between cats and rodents. In addition, our data showing that enucleation on day 7 in rabbit does not affect callosal development further support the influence of the level of maturity of the system at the time of the enucleation. However, the specific mechanisms involved in the response to very early lesions are not clear. In rodents, the segregation of ipsilateral and contralateral inputs to LGd occurs postnatally and coincidentally with the onset of retraction or elimination ofcallosal axons. In addition, ME on day 1 in rodents results in an expanded ipsilateral retinogeniculate projection 12. It has been hypothesized that both these events may be causally related to the abnormal callosal projections of ME rodents 53'8~'83. Evidence in the rabbit does not support these hypotheses. In the rabbit, segregation of ipsilateral and contralateral retinogeniculate projections, although it occurs postnatally, precedes the onset ofcallosal retraction, and there is no expansion of the ipsilateral retinogeniculate projections in ME rabbits 5'27"73. Our data, therefore, support the hypothesis that the effects of ME on callosal development are related to the maturity of the visual system at the time of the enucleation and suggest that they depend on modification of remaining pathways. However, the more specific hypothesis, that the effects are related causally to failure of segregation of the retinogeniculate inputs or to expansion of the ipsilateral retinogeniculate pathway is not supported by our data. The specific modifications which are

causally related to callosal abnormalities, whether in the form of reactive sprouting of intact paths or of transneuronal degeneration, remain to be established.

The role of activity There is evidence that manipulation of afferent activity by deprivation, which interferes with normal cortical development 6,25"65,66,77can also interfere with normal callosal development, but the effects of deprivation appear to differ from the effects of enucleation. It has been hypothesized that such differences arise from the availability of cues derived from correlated activity of neighboring retinal ganglion cells in deprived animals, and the elimination of such cues by enucleation 2. Below, we review the evidence that the consequences of enucleation and deprivation are different and evaluate the hypothesis that correlated retinal ganglion cell activity can account for such differences. Most comparisons of the effect of deprivation and enucleation are confounded by the fact that enucleation has usually been performed on the day of birth, whereas deprivation cannot be effective until either the light-mediated retinogeniculate responses are functional (before which dark rearing, DR, cannot be effective) or the eyes are normally open (before which monocular deprivation, MD, and binocular deprivation, BD, cannot be effective). For example, in rodents, the onset of visual function occurs after the end of the critical period for the effects of enucleation on callosal development, so comparisons of the effectiveness of DR, binocular enucleation (BE) and ME in modifying callosal projections are not valid ~3,69.82. In cats 4, a significant difference in callosal abnormalities was reported following deprivation (BD) compared with enucleation (BE), but again these differences might be attributed to the temporal interval between the enucleation (performed on postnatal days 1-4) and the onset of effective deprivation (at least several days later). In fact, our studies, in which we compare the effects of enucleation on day 7 with the effects of TTX application beginning on day 7, provide the only data comparing callosal development following enucleation or manipulation of activity, in

159 which both treatments were initiated at the same time. The data demonstrate that the presence of a silent retina does have an influence on callosal development. However, our data do not support the hypothesis that the retina influences central visual pathways through cues based solely on correlated activity of neighboring retinal ganglion cells, since the TTX injections eliminated such cues. The mechanism by which the silenced retina modifies callosal development must be different from the mechanism by which enucleation modifies callosal development, since the critical period for the effects of enucleation is over before the TTX treatment is started. Thus, our data suggest that two different mechanisms underly the effects of enucleation and TTX injections, and that the critical periods for manipulation of these mechanisms is also different. As reviewed above, we suggest that the critical period for enucleation is over by day 7, and the mechanism involves some sprouting or failure of retraction of remaining pathways. The end of the critical period for the effects of TTX injections on callosal development is not yet established but is later than that for ME. The underlying mechanism for the effect of TTX is also not yet established, and at this point we can only offer some hypotheses. Since we have shown that silencing of retinal ganglion cells has an effect which differs from that of removing them, we postulate that transported trophic factors in the TTX-silenced neurons influence their postsynaptic target. The influence of trophic substances on their postsynaptic target, during development, is well documented (for review see ref. 11), and if these factors are proteins, it is known that their transport by TTX-silenced cells is unaffected, but their release and their uptake by the postsynaptic target of the TTX cells is reduced ~6,17. During the first week of life, physiological activity of the rabbit retinofugal pathways is minimal 58'79'93, and the synthetic machinery of the neurons is presumably directed predominantly towards growth and the synthesis of trophic substances which influence survival and differentiation of these neurons. At the end of the first week, functional activity develops, and the predominant mode of the neurons' synthetic ma-

chinery changes to one directed to neuronal transmission. If the onset of functional activity is prevented by TTX at this point, then not only are electrophysiological signals eliminated, but the shift from the growth mode of the cells' metabolism to the transmission mode may also be prevented. Therefore abnormal growth of retinofugal and thalamocortical projections may result 18'49'6°'84-86'88' 95,97. For example, TTX-silenced retinal ganglion cells may show sprouting or failure of retraction of their terminals 97, and a similar phenomenon has also been reported when the neuromuscular junction is rendered inactive by toxins or by curare TM. Reported differences in the size and activity of LGd neurons of kittens reared with TI'X injections also result95,47. Therefore, it is possible that axonal overgrowth (or failure of retraction) of TTX-silenced neurons results in excesses of trophic substances throughout the retino-geniculo-cortical pathway, and these excesses secondarily influence axonal growth of callosally projecting cells in the cortex. In conclusion, our data indicate that monocular enucleation and elimination of retinal ganglion cell activity have separable critical periods for their effects on callosal development, and therefore must involve different mechanisms. Other studies of development of the corpus callosum, suggest that two developmental mechanisms are involved: the elimination of exuberant axons, and the stabilization of remaining projections. We hypothesize that early enucleation disturbs predominantly the first of these mechanisms, and TTX injections disturbs the second. Evidence4° that the critical period for deprivation-induced failure of stabilization of callosal projections occurs later in development supports this hypothesis. Our results indicate that the mechanisms by which TTX-induced elimination of electrophysiological activity alters the organization of developing and regenerating systems (for review see ref. 87), do not reflect only the absence of activity, but must reflect the abnormal influence of trophic substances released by the TTX-silenced cells on the activity of their postsynaptic target.

160 ACKNOWLEDGEMENTS

This research was supported by Grants EY02488 and EY05420. We thank Dr. Tim Cunningham for valuable suggestions and criticisms given during the writing of this manuscript, and we thank Dara Tashayyod and Rosalinda DiRienza for expert assistance in many aspects of these studies. REFERENCES 1 Adams, J.C., Technical considerations in the use of horseradish peroxidase as a neuronal marker, Neuroscience, 2 (1977) 141. 2 Arnett, D. and Spraker, T.E., Cross-correlation analysis of the maintained discharge of rabbit retinal ganglion cells, ,1. Physiol. (London), 317 (1981) 29-47. 3 Archer, S.M., Dubin, M.W. and Stark, L.A., Abnormal development of kitten retino-geniculate connectivity in the absence of action potentials, Science, 217 (1982) 743-745. 4 Berman, N. and Payne, B.R., Alterations in connections of the carpus callosum following convergent and divergent strabismus, Brain Res., 274 (1983) 201-212. 5 Chow, K.L., Baumbach, H.D. and Lawson, R. Callosal projections of the striate cortex in the neonatal rabbit, Exp. Brain Res., 42 (1981) 122-126. 6 Chow, K.L. and Spear, P.D., Morphological and functional effects of visual deprivation in the rabbit visual system, Exp. NeuroL, 42 (1974) 429. 7 Chow, K.L., Mathers, L.H. and Spear, P.D., Spreading of uncrossed retinal projections in superior colliculus of neonatally enucleated rabbits, J. Comp. Neurol., 151 (1973) 307. 8 Chow, K.L., Ostrach, L.H., Crabtree, J.W., Bernegger, O., Baumbach, H.D. and Lawson, R., Anomalous uncrossed retinal projections fail to activate superior colliculus neurons in rabbits unilaterally enucleated by fetal surgery, J. Comp. Neurol., 196 (1981) 189. 9 Clarke, S. and Innocenti, G.M., Organization of immature intrahemispheric connections, J. Comp. Neurol., 251 (1986) 1-22. 10 Crespo, D., O'Leary, D.D.M., Fawcett, J.W. and Cowan, W.M., Minimal effect of intraocular tetrodotoxin on the postnatal reduction in the number of optic nerve axons in the albino rat, Neurosci. Abstr., 1985. 11 Cunningham, T.J., Naturally occurring neuron death and its regulation by developing neural pathways. In G.F. Bourne and J.F. Danielli (Eds.), Internal Review of Cytology, Vol. 74, Academic, New York, 1982, pp. 163-186. 12 Cunningham, T.J., Sprouting of optic projections after cortical lesions, Anat. Rec., 172 (1972) 298. 13 Cusick, C.G. and Lund, R.D., Modification of visual callosal projections in rats, J. Comp. Neurol., 212 (1982) 385-398.

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