THE JOURNAL OF COMPARATIW NEUROLOGY 28851-58 (1989)

Immunohistochemical Localization of GAP43 in the Developing Hamster

Retinofugal Pathway

KENNETH L. MOYA, SONAL JHAVERI, GERALD E. SCHNEIDER, AND LARRY I. BENOWITZ

Department of Brain and Cognitive Sciences, Whitaker College, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (K.L.M., S.J., G.E.S.); Program in Neuroscience,

Department of Psychiatry, Harvard Medical School, Mailman Research Center, McLean Hospital, Belmont, Massachusetts 02178 (L.I.B.)

ABSTRACT Metabolic labeling studies have shown that the developing hamster reti-

notectal pathway is marked by a high level of synthesis and axonal transport of the neuron-specific phosphoprotein GAP-43, which then decline sharply with synaptic maturation. To understand better the relationship of GAP-43 to specific developmental events, we used a monospecific antibody to examine the location of this protein in the optic tract and retinal target areas at various stages. In late embryonic and in neonatal hamsters, dense GAP-43 immuno- staining was seen along the entire extent of the optic tract axons, including fascicles coursing over and through the lateral geniculate body (LGB) and within the upper layers of the superior colliculus (SC). The retinal origin of many of these fascicles was confirmed by their rapid disappearance after removal of the contralateral eye. During the first postnatal week, immuno- staining in the fiber fascicles showed a marked decline, though the protein was still present throughout the neuropil of the LGB and SC. In the second post- natal week, the neuropil staining also diminished, and by 12 days after birth, both structures showed only light immunoreactivity. The high levels of GAP- 43 in embryonic and neonatal optic tract axons coincide temporally with axon elongation, initial target contact, and collateral formation by the retinofugal fibers, whereas subsequent concentration of the protein in the neuropil sug- gests its involvement in the elaboration of terminal arbors and synaptogene- sis.

Key words: visual system, GAP-43 (B-SO), developmental neurobiology, immunocytochemistry

The developmental events of the hamster retinofugal pathway have been described in some detail. Retinal gan- glion cell axons emerge from the eye on E l l and elongate rapidly in fascicles through the optic nerve and over the sur- face of the thalamus, many reaching their central targets by embryonic day 13 (E13; PO = the day of birth [E16]). Dur- ing the first 2 postnatal weeks, these axons send branches into the target neuropil and form end arbors. By the begin- ning of the third week, the eyes have opened and the mor- phology of retinal axon arbors resembles that seen in the adult (Frost et al., '79; Jhaveri et al., '83; Sachs and Schneider, '84; Jen et al., '84; Schneider et al., '85; Woo et al., '85).

In order to understand the biological mechanisms that underlie these morphogenetic events, we have investigated developmental changes in the synthesis and transport of membrane proteins that are destined for retinal terminals and presumably participate in such events as membrane addition, growth cone motility, responsiveness to extrinsic signals, cell-cell recognition, and adhesion (Moya et al., '88). Among the prominent molecular events noted in these stud- ies was a marked change in the synthesis and axonal trans- port of the growth-associated protein GAP-43, a neuron-

Accepted May 9,1989.

0 1989 ALAN R. LISS, INC.

52 K.L. MOYA ET AL.

GAP-43 IN DEVELOPING OPTIC PATHWAY 63

Fig. 2. High-magnification views of GAP-43 localization in dorsal nuclei of the lateral geniculate body (LGBd) of P5 (A) and P8 (B) pups. Note the immunoprecipitate in axons and neuropil on P5. By P8 most of the protein is present only in neuropil regions, with only faint staining of axons. Medial is to left, dorsal is up for both figures. Scale bar = 50 Fm.

specific phosphoprotein of the nerve terminal membrane that has been associated with axonal development and regeneration in other systems as well (Skene and Willard, '81a,b; Benowitz et al., '81; Benowitz and Lewis, '83; Red- shaw and Bisby, '84; Katz et al., '85; Kalil and Skene, '86; Meiri et al., '86, '88; Moya et al., '87a, '88; Perry et al., '87). In most neuronal populations, synthesis of GAP-43 decreases markedly with maturation, although the protein persists in synapses of certain brain areas throughout life (Jacobson et al., '86; Benowitz et al., '88; McGuire et al., '88). In one such region, the hippocampus, changes in the phosphorylation of GAP-43 have been correlated alterations in synaptic func- tion or structure that underlie with long-term potentiation (Lovinger et al., '85; Nelson and Routtenberg, '85). It is pos- sible that GAP-43 may likewise play a role in mediating activity-dependent changes in synaptic organization during development and regeneration (Benowitz and Schmidt, '87; Meiri et al., '88).

In the hamster retinofugal pathway, isotope labeling studies reveal that the synthesis and axonal transport of GAP-43 are high in neonatal animals and then decline sharply in the second postnatal week (Moya et al., '88). However, it is possible that the actual concentration of the protein in terminals could follow a different time course, depending upon its rate of turnover. In this study, we used a monospecific antibody against GAP-43 to determine the levels of the protein along the retinofugal pathway and to analyze developmental changes in its distributional pattern. Our results demonstrate that the intracellular localization

Fig. 1. Immunohistochemical localization of GAP-43 in the lateral geniculate body during development. Densely stained fiber fascicles are observed in the optic tract and coursing through the neuropil of the lat- eral geniculate body (LGB) at E15, P2, and P5 (A-C). At P5 and P8 the staining appears mottled as immunoreactivity is absent from cell bodies (C and D). At P12 (E), staining has diminished and by PI9 (F) it has taken on the adult pattern (not shown), with low levels in the OT and LGB. Arrow indicates the intergeniculate leaflet. Scale bar = 100 Fm. Abbreviations: LGNd, LGNv = dorsal and ventral nuclei of the lateral geniculate body; OT = optic tract.

of the protein shows a striking shift with development: it is abundant throughout retinal axons during elongation and early stages of arborization, shifts away from axon trunks but is still present in the target neuropil as further elabora- tion of axon arbors occurs within terminal zones, and finally diminishes to very low levels as mature terminal arbors become established.

MATERIALS AND METHODS The selectivity and specificity of the anti-GAP-43 anti-

body used in these studies have been described (Benowitz et al., '88; Moya et al., '88). For the present studies, 2-3 ham- sters at embryonic day 15 (day of conception = EO), P2, P5, P8, P12, PI9 and in adulthood were deeply anesthetized with an overdose of Chloropent (Fort Dodge Laboratories) or Nembutal (Abbott Laboratories) and transcardially per- fused with buffered 4 % paraformaldehyde. Brains were removed, postfixed for 2-3 hours, and cryoprotected in buff- ered 30 % sucrose. Frozen 40 wm transverse or sagittal sec- tions were cut into phosphate-buffered saline with 0.5% NaN, (PBSN, pH 7.4). Endogenous peroxidase activity was neutralized by incubating sections in 0.3 % H202 in metha- nol for 30 minutes. Sections were washed in 3 changes of PBSN for 30 minutes and nonspecific binding was blocked by incubation in 20% normal rabbit serum. After washing again, the sections were incubated for 3 days at 4°C in immune serum, (or preimmune serum for control sections) a t a dilution of 1:2,000 in PBSN with 0.4% Triton X-100 (TX-100, Sigma). The tissue was washed in PBS TX-100 and incubated with biotinylated secondary antibody (Vec- tor Labs) at a dilution of 1:250 for 1 hour, rinsed, and incu- bated with an avidin-biotin complex conjugated to horse- radish peroxidase (Vector Labs, ABC Kits). After 2 rinses in 50mM Tris buffer (pH 7.5), the peroxidase was visualized by using DAB as the chromogen.

To determine the origin of immunopositive fibers in the superior colliculus, P1 and P4 animals were anesthetized by hypothermia and the right eye was removed. Animals were allowed to recover under a heat lamp and returned to the

GAP-43 IN DEVELOPING OPTIC PATHWAY

nest. After 24 hours, an interval adequate to allow consider- able retinal axon degeneration to take place in young ham- sters (Schneider, '73; Leonard, '74), they were anesthetized and perfused. Immunohistochemistry was carried out as de- scribed above.

55

greater in medial portions of the SC than in lateral regions (see also Fig. 4, normal side), and this gradient became even more pronounced at P5. By P8, fascicles in the lateral part of the SO showed virtually no staining, while a few lightly stained bundles were still present in the medial half of this layer (Fig. 3D). This staining gradient was most obvious in the caudal half of the SC.

In order to demonstrate that the darkly stained fiber fas- cicles in superficial layers of the neonatal SC were of retinal origin, GAP-43 immunoreactivity was examined 24 hours after unilateral eye enucleation in P1 and P4 animals. Densely stained fiber fascicles were found to be eliminated in the SGS and SO of the colliculus contralateral to the eye removal, whereas fiber bundles remained evident in the ipsi- lateral SC, which receives most of its retinal projection from the remaining intact eye (Fig. 4). However, in deeper layers of the SC, which do not receive retinal projections, GAP-43- positive fiber fascicles were unaffected on both sides. In the LGB staining was similarly lighter on the side contralateral to the removed eye than on the ipsilateral side. A number of densely stained fibers did remain in the OT and LGB, how- ever, which presumably include fibers from the ipsilateral eye along with axons of nonretinal origin (e.g., cortical, tec- tal, etc., en route to the LGBd and other thalamic and sub- thalamic structures).

RESULTS The LGB and SC showed striking developmental changes

in the localization and intensity of GAP-43 immunoreactiv- ity. A t E15, the protein could be visualized along the entire extent of optic tract (OT) fibers coursing over the surface of the thalamus (Fig. 1A). By this time, the first retinal axons have already reached the SC, although later-arriving axons are still elongating in the OT and no major axon collateral- ization has yet occurred (Jhaveri et al., '83; Schneider et al., '85). Darkly stained fiber bundles were also evident coursing transversely within the dorsal nucleus of the LGB, where the deeper-running retinal axons of the internal optic tract are located (Schneider and Jhaveri, '83). A similar pattern of axonal staining was seen on P2 and P5 (Fig. lB,C), and immunopositive reaction product was also evident through- out the retinal target zones (Fig. 2A). By P8, densely stained fiber fascicles were less prominent, although immunoreac- tivity remained distributed throughout the target neuropil; this staining showed a mottled appearance, consistent with a localization in presynaptic terminals and an absence of reaction product in postsynaptic cell bodies (Figs. lD, 2B). During the second postnatal week, the expression of GAP- 43 diminished so that the LGB was only lightly stained on P12 and at later ages (Fig. 1E,F). A band of dark immuno- reactivity differentiating the intergeniculate leaflet was ap- parent on P2 and became more pronounced at P5 and P8 before diminishing to low levels in more mature animals.

In the SC, the time course and pattern of GAP-43 immu- noreactivity was similar to that observed in the LGB. Densely stained fiber fascicles were observed in the superfi- cial layers of the SC a t E15, P2, and P5, appearing as dark clusters (Fig. 3A-C). Sagittal sections through the neonatal SC (data not shown) verified that this pattern of staining was composed of longitudinal, GAP-43-positive fascicles coursing rostrocaudally through the upper layers of the SC in a pattern similar to the known distribution of retinal fibers a t this age (Woo et al., '85; see Edwards et al., '86, for studies in the mouse). By P5, the optic fiber layer (SO) had differentiated and immunoreactive axons were now re- stricted primarily to this stratum. On P8 the fascicles could be only faintly visualized in the SO (Fig. 3D). GAP-43 immunoreactivity throughout the neuropil of the SC re- mained apparent a t P8 but declined markedly by P12 (Fig. 3E). Control sections reacted with nonimmune serum showed only light and nonspecific staining (Fig. 5).

During the first postnatal week, the staining pattern of fiber fascicles in the SC showed a mediolateral gradient (Fig. 3B,C). At P2, immunoreactivity in axon bundles was

Fig. 3. Immunohistochemical localization of GAP-43 in the superior colliculus. Darkly stained fiber fascicles appear as punctate clusters in the superficial layers of the colliculus where retinal fibers course; these are prominent at E15, P2, and P5 (A-C). At P8 fewer fascicles are visualized with the antibody (D). Note that a mediolateral gradient of these fascicles is apparent in the neonate and has diminished greatly by P8. At P12 and P19 (E and F) the staining of the neuropil has also diminished to near adult levels. Scale bar = 100 pm. Abbreviations: SGS = superficial grey layer; SO = optic fiber layer.

DISCUSSION Our studies show that GAP-43 is present at high levels in

the hamster primary visual pathway during early stages of development but then declines sharply as this pathway ma- tures. Overall, the temporal changes in GAP-43 localization are in agreement with the previously described time course of GAP-43 synthesis and transport (Moya et al., '88), indi- cating that considerable amounts of the protein are present during axon elongation, target contact, establishment of retinotectal topography, and the initiation of end-arbor spe- cializations. The subsequent disappearance of GAP-43 coin- cides with the formation of adult-like terminal arbors, increased number of retinofugal synapses, and eye opening (Sachs and Schneider, '84; Schneider and Jhaveri, '84; c.f. Lund and Lund, '72, for studies of synaptogenesis in the rat; and Sachs et al., '86, for studies in the mouse).

Up until P5, GAP-43 immunoreactivity is dense along the entire extent of axons, reflecting either high levels of the protein being transported to nerve terminals or its deposi- tion along the axonal membrane during the early phase of axon outgrowth, as suggested previously (Perry et al., '87). High concentrations of GAP-43 along the axonal trunk dur- ing the initial stages of fiber development have been noted in other studies as well (Meiri et al., '88; McGuire et al., '88; G o s h et al., '88). From in vitro observations, it has been argued that axons elongate by membrane addition in termi- nal areas (Bray and Chapman, '85), but it is clear that axon maturation in vivo must also involve membrane insertion along the length of the fiber to support the marked increase in axon diameter (Black and Waxman, '86). The presence of GAP-43 throughout the axonal membrane at early stages enable this protein to participate in the expansion of axons, the continued lengthening of fibers that occurs postnatally as the brain increases in size, or the sprouting of collaterals.

The dense GAP-43 immunoreactivity seen along retinofu- gal axon fascicles a t early stages of development diminishes by P8. This decline coincides temporally with a previously noted shift in the pattern of retinal axon growth, from one

56 K.L. MOYA ET AL.

Fig. 4. Densely stained fascicles in the superficial superior colliculus are of retinal origin. P1 pups had one eye enucleated and were perfused on P2 and prepared for immunohistochemistry. On the side contralat- eral to the enucleation, fiber fascicles in the superficial SC are eliminated, while the SC receiving projections from the remaining eye is comparable to normal, intact controls. Scale bar = 200 pm.

marked by rapid, fasciculated elongation, the formation of short multiple side-branches, and rudimentary terminal ramifications, to a second mode characterized by slower growth and the elaboration of adult-like arbors in target areas (Jhaveri et al., '83; Schneider et al., '85, '87; Sachs et al., '86). Coincident with this shift is a corresponding decrease in the plastic response of retinofugal axons to injury: retinal axons regenerate and functionally reinner- vate normal tectal targets if the brachium of the SC is cut before P4 but fail to grow across the cut if surgery is done after this time, although the axons can still sprout collater- als proximal to the lesion for another 10 days or so (Schneider, '73; So and Schneider, '78; Frost et al., '79; So et al., '81; Schneider et al., '85).

While it is clear from the eye enucleation cases that most of the darkly stained fascicles in the upper layers of the neo- natal SC are of retinal origin, immunoreactivity observed at later ages in the SGS and SO may also include GAP-43 in axons of nonretinal origin, e.g., cortical axons which first enter the SO on P5 (Ramirez et al., '86). In the upper layers, however, few darkly stained axon bundles survive contralat- eral eye enucleation done on P4 (data not shown) although fibers in the deep tectal layers retain their immunoreactiv- ity. Thus, the later-arriving cortical axons may not express high levels of GAP-43 as they elongate into the SC, or they may not be as fasciculated nor form bundles as large as the retinofugal axons.

At the early time points, a mediolateral gradient was observed in the staining of fascicles in the upper layers of the SC. Axons in the medial portion of the SC retain their immunoreactivity longer than those in the lateral colliculus, suggesting that the latter fibers (originating in the upper retina) mature earlier than axons from the lower retina. Although a mediolateral gradient in the maturation of reti- notectal axon arbors has not previously been reported, there is considerable evidence that the development of the ventral retina lags behind the dorsal half during embryonic life (Sil- ver and Robb, '79; Edwards et al., '86). Fiber-tracing studies

have shown that ganglion cell axons from the upper (and temporal) retina elongate and mature before those originat- ing from the lower retina (Wikler et al., '85; Edwards et al., '86), a finding likely to be the basis of the observed gradient of GAP-43 immunoreactivity.

The immunoreactivity seen in the target neuropil be- tween P2 and P12 most likely represents GAP-43 within growth cones and presynaptic terminals (Gispen et al., '85; Verhaagen et al., '88; Goslin et al., '88). The apparent shift in GAP-43 localization, from the protein being present along the entire axonal process to becoming restricted only to the terminal region, coincides with the time of terminal arbori- zation in target areas, the formation of large numbers of synaptic connections, and the establishment of retinal to- pography (Schneider and Jhaveri, '84). GAP-43 then de- clines in the neuropil of primary visual targets during the second postnatal week when mature-looking retinal arbors form. Similarly, in the rat SC, GAP-43 immunoreactivity was also reported to become progressively more restricted to the neuropil during the time of synaptogenesis before diminishing (McGuire et al., '88). When the eyes open at P14 in the hamster, pupillary responses to light can be evoked, indicating that at least some retinal axons are capa- ble of conveying visual information at this time. In the regenerating goldfish optic pathway, the correlation be- tween GAP-43 expression and the time course of activity- dependent tuning suggests that the presence of the protein in nerve terminals may contribute to the process whereby patterned visual stimulation influences the organization of synaptic relationships (Benowitz and Schmidt, '87).

GAP-43 is associated with the internal face of the plasma membrane (De Graan et a]., '85; Basi et al., '87) and presum- ably exerts its influence on portions of the axonal membrane through its regulation of calmodulin levels (Alexander et al., '89), phosphoinositide metabolism, and subsequent protein kinase activation in response to physiological stimuli (Zwiers et al., '80; Aloyo et al., '83; Meiri et al., '88; Van Hooff et al., '88; see Nishizuka, '86, for review). The idea

GAP-43 IN DEVELOPING OPTIC PATHWAY 57

Fig. 5. Transverse section through the superior colliculus of a P2 pup processed with preimmune serum. The tissue is devoid of all specific staining. Scale bar = 200 pm.

that it may play a universal role in the growth process per se early in development is supported by its abundance in vir- tually all growing axons which have been studied (Kalil and Skene, '86; our unpublished data), its enrichment in growth cones (DeGraan et al., '85; Katz et al., '85; Meiri et al., '86; Skene et al., '86), and the suggestion that transfection of PC12 cells with the cloned GAP-43 gene enhances process outgrowth (Neve et al., '88). In certain areas of the brain, where synaptic relationships can presumably be modified throughout life, GAP-43 may contribute to the mechanism whereby the pattern of physiological activity continues to influence synaptic structure or function (Lovinger et al., '85; Nelson and Routtenberg, '85; Benowitz and Routtenberg, '87; Snipes et al., '87; Neve et al., '87; Benowitz et al., '88).

ACKNOWLEDGMENTS This work was supported by NIH Grants EY05504,

EY05690, EY00126, and EY02621. We are grateful to Bobby Dolan and Reha Erzurumlu for their help in the preparation of the manuscript.

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