mapping the development of the rat brain by gap-43 immunocytochemistry

Neuroscience Vol. 40, No. 1, pp. 277-287, 1991 Printed in Great Britain

0306-4522/91 $3.00 + 0.00 Pergamon Press plc

0 1991 IBRO

MAPPING THE DEVELOPMENT OF THE RAT BRAIN BY GAP-43 IMMUNOCYTOCHEMISTRY

J. W. DANI,*? D. M. ARMSTRONC*$ and L. I. BENOWITZ$~~**

*Department of Neurosciences, University of California at San Diego School of Medicine, La Jolla, CA, U.S.A.

§Department of Psychiatry and Program in Neuroscience, Harvard Medical School, Boston, MA, U.S.A

11 Mailman Research Center, McLean Hospital, Belmont, MA, U.S.A.

Abstract-Growth-associated protein-43 (GAP-43) is a phosphoprotein of the nerve terminal membrane which has been linked to the development and restructuring of axonal connections. Using a monospecific antibody prepared in sheep against purified GAP-43, we examined the temporal and spatial changes in the distribution of this protein from embryonic stage day 13 (E13) to adulthood. At stages in which neurons are still dividing and migrating, levels of GAP-43 are extremely low, as is seen in the cortical plate throughout the embryonic period. With the onset of process outgrowth, intense GAP-43 immunoreactivity appears along the length of axons: by E13, such staining is already strong in the brainstem, where it continues up through the first postnatal week and then disappears. In the neocortex, intense fiber staining first appears several days later but ends at the same time as in the brainstem. At the end of the period of intense axonal staining there is a brief interval in which high levels of GAP-43 immunostaining are seen in the neuropil.

In regions of the brain in which specific developmental events have been characterized anatomically and physiologically, the period of dense neuropil staining coincides with the formation of axonal end-arbors, the beginning of synaptogenesis, and the time at which synaptic organization can be modified by the impingent pattern of activity (i.e. the critical period). Over the next few days, staining in neuropil declines sharply in most regions except for certain structures in the rostra1 neuraxis which may be sites of ongoing synaptic remodeling.

The development of the nervous system involves a complex, precisely timed series of events that includes cell proliferation, migration, extension of processes, synaptogenesis, and cell death. One molecule that has been implicated in some of these events is the growth- associated phosphoprotein (GAP-43). Neurons growing either in vivo or in vitro show high levels of GAP-43 expression coincident with the beginnings of process outgrowth,‘7,23@‘,50 with the protein being conveyed in the rapid phase of transport down axonal, but not dendritic, processes.9~1s~26~57~s* During early phases of outgrowth, GAP-43 is localized on the inner face of axonal and growth cone membranes, becoming progressively concentrated at the more

‘/‘Present address: Department of Cellular Physiology, Stan- ford University School of Medicine, Stanford, CA, U.S.A.

$Present address: FIDIA Georgetown Institute of Neuro- science, 3900 Reservoir Road, NW, Washington DC, U.S.A.

**To whom correspondence should be addressed at: Laboratory for Neuroscience Research in Neurosurgery, Children’s Hospital/Enders 309, 300 Longwood Ave., Boston, MA 02115 U.S.A.

Abbreviations: E13-E21, embryonic days 13-21; GAP-43, growth-associated protein; P2-P16, postnatal days 2-16; TBS, T&buffered saline.

distal processes as maturation proceeds.25*37T”.42 In most neurons, levels then decline rapidly following the establishment of mature synapses.5~28~39,4’~42~s1 However, in certain parts of the adult CNS, particularly in higher limbic and integrative areas, moderately high levels of GAP-43 and its mRNA persist into adulthood,8,‘0,37*43*U,48 and may be related to per- sistent changes in synaptic efficacy, such as those that underlie long-term potentiation.**“sM Hence, GAP-43 may play a general role in all axogenesis and synaptogenesis, and may also allow a restricted subset of synapses to remain in a plastic state throughout life.‘3,56

By immunocytochemistry, striking changes in the concentration and localization of GAP-43 have been described during the postnatal development of several brain regions. In the primary optic pathway of the rat, hamster, and rabbit, for example, GAP-43 immunostaining is prominent along axons and their distal processes during axon elongation, shifts to developing synapses in the lateral geniculate nucleus and superior colliculus during the first few postnatal days, then drops to very low levels, becoming absent in mature optic fibers and only faint in the target neuropil. 37*39S40~42 Postnatal development in the neocortex, hippocampus, and cerebellum is likewise marked by a shift from intense axonal staining to a pattern of immunoreactivity in the neuropil at

217

278 J. W. DANI er ul

the time of synaptogenesis. and then a subsequent decline.8~J7~48

In view of these observations, it seems possible that developmental studies using GAP-43 immunostaining can reveal many hitherto unknown features of brain development. By identifying the times at which staining is intense in specific axons, shifts to the neuropil, and declines, it might be possible to define when various axonal systems are elongating and actively forming synapses. Moreover, delineation of those areas with high levels of GAP-43 throughout life may contribute to understanding where in the CNS synaptic remodeling may continue to occur in response to physiological factors or experience.

EXPERIMENTAL PROCEDURES

Fetal tissue was obtained from both timed and untimed pregnant Sprague-Dawley rats (Charles River, Wilmington, MA). The crown-rump length of each fetus was measured and used to determine the gestational age. Approximately four animals from each of the following time points were examined: embryonic day 13 (E13), Efi, E17; E18, E19, E20, E21. birth (PO). oostnatal dav 2 (P2). P4. P8. PlO. P12. P16, and’ adult.‘Tl&*brains of tie il3’;at kmdryos’were removed and immersion-fixed in 3.7% formalin made in 0.1 M phosphate buffer (pH 7.4) for 48 h at 4°C. All other fetal and postnatal rats were perfused transcardially with 3.7% buffered formalin (approximately 2ml/g body weight). Adult animals were perfused with 0.1 M phosphate-buffered saline until the blood cleared prior to aldehyde fixation. Brains were postfixed for 24 h at 4”C, transferred to a phosphate-buffered 15% sucrose solution until they sank, then embedded in Histo Prep (Fisher) and sectioned on the cryostat at a thickness of 15 pm in either the coronal or sagittal plane. Serial sections were collected on 3 x 1% gelatin (bovine, Type III, Sigma)-coated microscope slides. Embryonic tissue was collected on warm slides, while postnatal and adult tissue was collected on cold slides.

After sectioning, tissue was processed immediately for immunohistochemistry. The procedure consisted of extract- ing three times in 0.1 M Tris-buffered saline (TBS: 0.9% NaCl, 0.1 M Tris, pH 7.4) containing 0.25% Triton X-100, followed by a 30-min blocking step in 3% rabbit serum (G&co, made up in TBS),.two more extractions in Triton

X-100 (in TBS), and finally two TBS washes. The ,lntlhody to GAP-43 (Ref. 8) was diluted 1: 1500 in TBS containme 1% rabbit serum, 0.3% sodium azide. and then “puddled” onto each tissue section. This was allowed to incubate for approximately 48 hat 4°C in a humidified chamber. Control sections were treated similarly but without the addition ut the primary antibody; in previous studies, substitution of the primary antibody with preimmune serum was shown to give only diffuse, non-specific staining.8,?2,‘? Tissue sections were then washed twice in I % rabbit serum (in TBS), incubated 1.5 h in biotinytated rabbit anti-sheep IgG (as per manufacturer’s specifications: Vector Labs) at room temperature, washed twice in 1% rabbit serum in TBS, incubated for 1 hr in avidin-biotin complex (as per manufacturer‘s speclfica- tions: Vector Labs), and washed in TBS. Sections were then reacted with a 0.05% solution of 3,3’-diaminobenzidine. 0.01% H202 and 0.04% NiCl, in 0. t M Tris buffer ( pH 7.5). Incubation times varied from 6 to 20min depending upon the age of the rats (embryonic animals had the shortest incubation times while adults had the longest). Immuno- labeled sections were dehydrated through alcohols and xylene, and covered using Entellan (EM Science).

RESULTS

Overview of developmental changes

The overall changes in the distribution and intensity of GAP-43 immunostaining from El3 to adulthood are illustrated in Fig. 1. At the earliest time examined, axon fascicles coursing through the brainstem and basal forebrain show intense immunoreactivity. However, almost no staining at all is seen in the remainder of the telencephalon, particularly in the cerebral cortex, or around the ventricle of the superior colliculus, zones in which neuronal cell division is still actively taking place.

By E17, the picture in the telencephalon changes markedly, with intense fiber staining appearing in the intermediate layer. The ependymal layer continues to be unreactive, as does the caudate-putamen. Fibrous staining remains high throughout the basal forebrain and the brainstem. By E21, the final day of embryonic development, fiber staining in the superior colliculus

Abbreviation used in figures

: anterior commissure iml inner molecular layer (of dentate) basal forebrain I intermediate zone of the cortex

BS brainstem IC inferior colliculus BST bed nucleus of the stria terminalis LGN lateral geniculate nucleus Cb cerebellum LS lateral septum CP cortical plate M marginal layer CPU caudate-putamen MD mediodorsal nucleus (of thalamus) cx cortex mol molecular layer DG dentate gyrus ot optic tract E ependymal zone PO-P16 postnatal days O-16 ffX fimbria-fornix PC pyramidal cells (of hippocampus) fx fornix Pi pirifoxm cortex Gle external granule cell layer (Cb) SC superior colhculus Gli internal granule cell layer (Cb) Th thalamus HiF hippocampal formation wm white matter HyTh hypothalamus

Fig. 1. Parasagittal sections through the rat brain showing changes in GAP-43 immunostaining from El 3 to adulthood. Identification of structures and abbreviations used are based upon the atlas of Paxinos and

Watson.@ Scale bar is in millimeters.

GAP-43 in developing rat CNS 219

Fig. 1 (Part 1).

Fig. I (Part 21.

includes the superficiai retinor~ip~ent layer. The proliferative zone is an intensety i~~unor~act~ve cerebetfum shows unstained c&s in the external band containing the premj~atory di~~~~tiated granular iayer, where the granule celI precursors neurons, and below this, another layer of low are still proMkrating; however, at the base of this immunoreactivity containing several layers of as


yet undifferentiated Purkinje cells. Little staining is seen in the dorsal thalamus or in the cortical plate, though other fiber systems of the telencephalon, including the anterior commissure, fomix and the presumptive white matter of the neocortex, are densely stained.

By P4, this pattern has changed radically, particularly in the cortex. The entire cortical plate and the marginal layer now show intense immunostaining, while fibers in the white matter remain strongly reactive. Neuronal cell bodies stand out as being unstained, which is particularly evident in areas where somata are densely packed, such as the piri- form cortex, granule cell layer of the dentate gyrus, or the pyramidal cell field of the hippocampus. On P4, fibrous staining is evident in the ascending and/or descending fibers which course through the caudate-putamen interconnecting the cortex with the dorsal thalamus, and within the dorsal thalamus itself. The fiber staining in the brainstem remains high at this time point, including fibers coursing in the superficial, retinorecipient area of the superior colliculus and in the white matter of the cerebellum. The outermost layer of the cerebellar cortex still contains a layer of proliferating granule cell precursors (external granule layer), while the subjacent premigatory zone and molecular layer are intensely stained. A moderate level of staining is seen in the internal granule layer.

cortex, particularly in the marginal layer; in certain portions of the hippocampal formation (including the inner one-third of the dentate gyrus molecular layer, and the CA1 region of the hippocampal formation); and at the subcortical level, in a continuum of structures that includes the bed nucleus of the stria terminalis, lateral septum, and hypothalamus; in the dorsal thalamus, dark staining remains in the mediodorsal nucleus. The mesencephalon shows a much lower degree of staining compared with the earlier postnatal period, with only modest immunoreactivity in the superior colliculus and the inferior colliculus.

The brainstem and the cortex would appear to represent two contrasting extremes of brain development: whereas the former shows an intense staining of fiber fascicles early on but a nearly complete loss of immunostaining in the adult, the latter, by contrast, shows a relatively delayed appearance of fiber staining, but more immunostaining in the mature neuropil. For these reasons, we will review the development of these two areas.

Growth -associated protein -43 changes in the develop - ing brainstem

Four days later, on P8, the pattern of immunostaining has once again changed markedly: levels of GAP-43 immunoreactivity have declined consider- ably throughout the brain, as is particularly evident in the cortex. With the exception of the marginal layer, which remains darkly stained throughout life, the remainder of the cortical gray matter shows much lower levels of immunostaining than at day 4. Fiber staining has also diminished by day 8, as can be seen in the axon bundles passing through the caudate- putamen in the timbria-fomix, or in the cortical white matter. The cerebellum is still undergoing rapid differentiation, and intense immunostaining remains in the fibers of the white matter and in the molecular layer. Proliferative cells are still seen in the external granule layer, and this remains evident on PI6 as well.

From El3 to the first postnatal week, the pontine- mesencephalic level is dominated by intensely reactive fiber fascicles (Fig. 2). These are already numerous at the earliest time point examined and stand out against a background of light, diffusively stained cell bodies. This staining increases at E17, when numerous interlacing fascicles can be seen. By P4, the staining of fiber fascicles (arrows) relative to the neuropil has decreased; the latter shows a punctate pattern of staining surrounding cell bodies. This trend continues to P8, at which time the staining of fiber bundles (arrows) has become about equal to that of the neuropil, which continues to show a moderately intense level of punctate staining surrounding unreactive neuronal somata (asterisks). By adulthood, both the neuropil”and fiber staining have decreased greatly: unreactive fibers (arrows) are seen to stand out against the light, diffuse neuropil staining.

Developmental changes in the cortex

In the adult, the most striking change is the nearly On E13, the neocortex shows only very light stain- complete loss of staining in the brainstem, which now ing (Fig. 1). At higher magnification (Fig. 3) this shows only a light, diffuse staining of neuropil and an corresponds to a diffuse immunoreactivity in cell even lighter level of staining in fiber tracts. Most fiber bodies in the ependymal layer and a slightly darker tracts elsewhere in the brain display little staining, as staining in the cortical plate. Immunostaining in the in the fimbria-fomix, optic tract, anterior commis- cortical plate remains low throughout the rest of sure, or cortical white matter. The molecular layer of embyronic development, though by El 7 intense fiber the cerebellum shows a modest level of staining, staining becomes visible in the intermediate layer, although in some animals a more intense immuno- presumably reflecting thalamocortical afferents that reactivity is seen in this layer. The cerebellar granule course through this zone before ascending to form cell layer shows a much lower level of immunoreactiv- synapses with cortical neurons.36,61 By E21, staining ity, while the white matter is now essentially unreac- in the intermediate layer is intense, and defines what tive. At more rostra1 levels of the neuraxis, a fairly appear to be a number of different fiber systems at intense level of immunostaining remains in the neo- various depths of the presumptive white matter. Over

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Fig. 3. Changes in GAP-43 immunostaining in the cerebral cortex. Note the near absence of staining in the cortical plate (CP) throughout the embryonic period, the intense staining on P4, and the subsequent decline by P8. From P8 on, neuronal somata stand out as being unstained (asterisks). All sections were taken in cortex overlying the middle of the hippocampal formation. Sections in upper row were taken at 100x magnification, while those in the lower row represent portions of the upper sections at the

boundary of the marginal zone and the cortical plate taken at 400 x

the next few days, the pattern of staining in the cortex P8 is that the intensity of fiber staining continues to undergoes two striking transitions. On P4, immuno- decrease further, from the already modest levels seen stained fibers are seen coursing longitudinally and in the white matter and in radially coursing fibers on radially throughout the cortex (white arrows), and at day 8, to a near absence of staining in the gray and higher magnification, the cortical neuropil shows a white matter of the adult cortex. punctate staining in the marginal layer as well as in the cortical plate, with neuronal somata standing out DISCUSSION as being less intensely stained (asterisks). The overall level of staining falls sharply by day 8, at which time These results confirm and extend previous obser- the cortical neuropil shows a diffuse punctate staining vations on the developmental changes that occur in which is more concentrated in the marginal layer. the levels and distributional pattern of GAP-43. In At this time point and later, the negative staining terms of overall levels, GAP-43 appears to be most of neuronal somata (asterisks) becomes even more abundant midway during the first week of postnatal pronounced. An additional change that is seen after life in the rat, at which time intense immunostaining

284 .I. W. DANI et ul

is seen both in axons and in the neuropil throughout the brain. This accords well with the findings that the overall concentration of the protein, as visualized by two-dimensional gel electrophoresis, and of the mRNA, as visualized by Northern blots, peak at this same time.6,28 Previous studies on brain development using different antibodies to GAP-43 likewise confirm the intense staining seen in cortex, hippocampus, and elsewhere in the first postnatal week.2’~37~48 This peak is strikingly well-defined in time, especially in the cortex. In the first few postnatal days, prior to the invasion of thalamic afferents, levels of GAP- 43 in the cortical plate are very low. The peak that occurs at around P4 coincides with the time at which ascending fibers form collaterals which then begin to form synapses. 36161 Just a few days later (i.e. by P8) this immunostaining has declined markedly, although a modest, punctate concentration persists into adulthood, particularly in the marginal layer.8,37

In the visual system, neurons of the dorsal lateral geniculate nucleus are generated between El2 and E14, while cortical neurons destined from layer IV of the visual cortex are generated between El6 and El7 and migrate to their final position on El9 and E20.36 Axons from the dorsal lateral geniculate nucleus invade the telencephalon, coursing through the intermediate zone on E16. Invasion of the cortical plate does not occur to a significant extent until the last day of gestation. Projections to layers I and IV are observed by P4. In both the visual and primary somatosensory cortices of the rat, ascending thalamocortical axons “wait” in the subplate between PO and P2 before entering the cortical plate on P3.“,@ In more detailed studies of the developing rat somatosensory cortex, thalamic afferents intensely stained for GAP-43 were found to be organized in bundles as they invaded layer IV of the cortex on P3. These afferents presaged the formation of the “barrels” in which facial vibrissae are represented. GAP-43 staining within the neuropil of the barrels reached a maximum at P5 and then began to decline, so that by the second postnatal week, the centers of the barrels were devoid of GAP-43 immunostaining, whereas the staining in the “septa” surrounding the barrels increased.22 As shown here, the pattern of GAP-43 immunostaining attained in the second postnatal week remains essentially unchanged into maturity.*F2*

The brainstem demonstrates a rather different developmental sequence of GAP-43 from the cortex. Here, the staining of fiber fascicles begins consider- ably earlier, consistent with the earlier completion of cell birth, migration, and axogenesis at more caudal levels in the neuraxis.27 Among the earliest neurons of the brainstem to differentiate and send out projections are the monoaminergic cells. Serotonergic neurons of the raphe nuclei differentiate between El 1 and El5 with a dorsal peak at El4 and a medial peak at E13-E14.32 Projections from these neurons form

short bundles that primarily run rostrally with some caudal extensions as early as El2 (Ref. 47). Both dorsal and ventral groups of mesencephalic serotonin neurons travel parallel to the ventral surface of the brain at E13, giving rise to ascending axon bundles which follow the curvature of the midbrain flexure. Projections from serotonergic neurons located between the mesencephalic and pontine flexures reach the prosencephalon by E15. An increase in both size and density of serotonergic axon bundles is observed at E16, and by El8 this pathway appears fairly well-developed.54 Noradrenergic neurons of the locus coeruleus differentiate from El0 to El3 with a peak at E12, while dopaminergic neurons of the substantia nigra undergo their final cell division between El 1 and El5, peaking at El3 (Ref. 32). Catecholamine- containing neurons in the rostra1 part of the mesencephalon send axons rostrally toward the developing corpus striatum of the prosencephalic region around El 3 (Ref. 47). These project rostrolaterally into prosencephalon by El5, but do not yet reach the rudimen- tary neocortex cerebri. The identity of some of these early arriving fibers as being dopaminergic is confirmed by double-labeling studies using antibodies to GAP-43 and to tyrosine hydroxylase.‘,55 Between El5 and El6 catecholamine-containing fibers are observed penetrating the outer superficial layers of the cerebral cortex.53 Innervation of the developing neocortex proceeds in a ventral to dorsal and rostra1 to caudal direction. Noradrenergic axons are present in all three primordial cortical layers of frontal and lateral neocortex at El7 (Ref. 33). Collateralization occurs by E20 with development continuing to the third postnatal week. Similarly, the occipital neocortex is innervated by E20 with collateralization by birth, and the parietal neocortex is innervated with collateral formation at birth. Although we did not attempt to identify specific fiber systems of the brainstem, our results suggest that most of these follow a very similar developmental time-course with regard to GAP-43 immunostaining in their axons and terminals.

Changes in GAP-43 immunostaining during the postnatal development of the cerebellum have been described by others, and the present findings accord well with these.37s48 Extending this into the prenatal period, our El5 material shows an absence of immunostaining in the proliferative zone at the roof of the fourth ventricle, corresponding to the birth of the Purkinje and Golgi type II neurons.*’ By El 7, the first granule cell precursors can be seen in the external granule layer, and these undifferentiated cells likewise contain very little GAP-43. However, subjacent to the proliferative zone, intense immunostaining is seen in the premigratory zone and the developing molecular layer. This staining continues to late postnatal stages, as the rich network of parallel fiber terminals synapse onto the developing dendritic trees of Purkinje cells and other cells in the molecular layer. In the white matter of the cerebellum, immunoreactivity likewise


remains strong into the late postnatal period, but vanishes in the adult. Hence, the cerebellum follows the same general pattern seen elsewhere in the brain with regard to the absence of GAP-43 in neuroblasts and undifferentiated neurons, strong fiber staining during axonal outgrowth, and intense neuropil staining during synaptogenesis.

In the superior colliculus, as an example of a sensory area of the brainstem, neurogenesis begins at approximately El2 and continues from El8 to E20.i6 At El7 bundles of optic axons run tangential to the surface in a rostrocaudal direction. Distinct synapses are apparent under the electron microscope by El8 and El9 (Ref. 35). As the present studies demonstrate, past the first postnatal week, levels of GAP-43 show a far greater decline in the brainstem than in the cortex.8,28.37

Several lines of evidence suggest that GAP-43 may participate in the transduction of physiological activity to influence the functions, and perhaps the structure, of the nerve terminal membrane. GAP- 43 (i.e. B-50) is one of the principal substrates of protein kinase C in growth cones and in certain mature synapses,4,‘9s30@I and changes in its phosphorylation state occur in response to a number of physiological signals, including phorbol esters, changes in Ca2+ concentration, and membrane de- polarization.‘8~20~24~40 These phosphorylation changes are in turn associated with alterations in phospholipid metabolism,29 calmodulin binding,3 neurotransmitter release,” and with the long-lasting changes in synaptic efficacy that accompany long-term potentiation in the hippocampal formation.2,‘8~34 It has been proposed that either directly or indirectly, the final outcome of altering the phosphorylation state of the protein may be alterations in membrane fusion at specific sites of the axon.13,37 The present observations and others suggest that such events may occur along the entire length of the axon at early stages of elongation, but become restricted to the nerve endings at the time of synaptogenesis. 26,37,40,42,51 It is of interest that in several systems, peak levels of GAP-43 in the neuropil coincide with the time at which various physiological manipulations are able to alter the pattern of synaptic organization (i.e. the critical period), and it is tempting to propose that this protein, by virtue of its protein kinase C-regulated effects on membrane function, may contribute directly to these processes. For example, in the ascending trigeminal pathway, representation of individual vibrissae are sorted into distinct columns or barrels of the somatosensory cortex. Removal of one or more whiskers causes ascending projections from the remaining ones to occupy the region of the somatosensory cortex normally innervated by the missing one if surgery is done within the first postnatal week, but not later.7,6’ This coincides with the peak period of synaptogenesis in the cortex and with highest levels of GAP-43 in the neuropil.”

Likewise, in the developing hamster visual system, peak levels of GAP-43 are seen in the neuropil of the lateral geniculate nucleus and of the superior colliculus just when retinal axons begin forming synapses with their target neurons. It is during this period that retinotopic organization is being established and ingrowing afferents are able to occupy synaptic spaces that are made available by removing competing inputs.41,42 In the regenerating goldfish optic pathway, GAP-43 synthesis in growing retinal ganglion cells remains high throughout the period when retinotectal topography can be influenced by the pattern of physiological activity.i4 And in the cat striate cortex, levels of the protein likewise peak during the critical period for the tuning of ocular dominance columns and other feature detectors.‘2,38 While the critical period has only been defined for a small number of neural systems, we would propose that peak GAP-43 levels in synaptic endings may help define such properties throughout the CNS. Moreover, as previously suggested, 8,10,13,28,3’,43.45~52,56,59 regons where GAP-43

persists throughout life may be sites where such remodeling may continue to occur in response to physiological and behavioral stimuli” and may be important for learning and memory. In this regard, it is intriguing that in the brains of primates, including man, the associative neocortex and hippocampus retain high levels of GAP-43 and its mRNA throughout life. In contrast, in primary sensory, motor, and brainstem regions, where synaptic organization may remain relatively unmodifiable later in life, levels of GAP-43 are 10w.“,~~,~

CONCLUSION

Our results support the idea that probes for GAP-43 may afford a unique insight into the development of the nervous system. More than being a probe for developmental events, however, it is quite likely that GAP-43, through its regulation by protein kinase C, participates directly in processes essential for the growth of axons and the formation of synaptic relationships. Based upon the present results and others, we would propose that the changes in GAP-43 immunostaining that we are visualizing may reflect, in sequence, the elongation of axons, synaptogenesis, and the subsequent decline in synaptic plasticity at well-defined times in the development of various neural systems. Moreover, as has been suggested, the persistence of the protein in certain regions of the neuraxis may reflect synapses in which dynamic remodeling may remain possible throughout life, perhaps in relation to functional plasticity.

Acknowledgements-We are grateful to the National Institutes of Health (AG 05344 and AG 08206 to D.M.A. and NS 25830 to L.I.B.).

286 J. W. DANI (‘I 01

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(Accepted 27 April 1990)

mapping the development of the rat brain by gap-43 immunocytochemistry

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