Axon development in mouse cerebellum: Embryonic axon forms and expression of synapsin I

AXON

DEVELOPMENT

EMBRYONIC

AXON OF

IN MOUSE FORMS

AND

SYNAPSIN

I

C. A. Department

of Pharmacology,

CEREBELLUM: EXPRESSION

MASON

New York University School of Medicine. NY 10016. U.S.A.

550 First .4venuc.

Neu 1’orh.

Abstract---A fundamental question in central nervous system development is the timing of synaptogenesis in relation to invasion of targets by afferent axons. A related question is how growth cones transform into synaptic terminals. These two aspects of axon maturation were examined in developmg mouse cerebellum, by labeling single axons with horseradish peroxidase. to study their form and cytology. and by immunocytochemical staining of a synaptic vesicle antigen. synapsm I, a phosphoprotcln found on synaptic vesicles in all mature CNS synapses.” From embryonic day 16 to postnatal day 3. horseradish peroxidase-labeled afrerent axons extend well into the cerebellum and have simple forms. At embryonic day 16, axon growing tips are synapsin I-negative. Synapsin I is first expressed at embryonic day 17, and by embryonic day 18. fibers are stained throughout the cerebellum. Synapsin 1 expression coincides with a general increase in synaptic speciaitzations. although growing iips continue to have the cytology of growth cones. During the period that axons have primitive shapes, synapsin 1: is distributed throughout the terminal arbor, corresponding to the presence of small vesicles along neurite lengths, even at non-synaptic sites. After postnatal day 3. when synaptic terminals develop into stereotypic shapes and engage in characteristic synaptic relations, synapstn I is restricted to boutons. Thus, the synapse-specific protein synapsin I is expressed in fetal mouse brain, long before nerve endings have the structure and connections of adult brain. In cerebellar axons, the expression of this protcm follows axon arrival, coincides with the appearan.ce of elementary synapses, and accompanies the transformation of growing tips into stereotypic synaptic boutons. The time course of expression of synapsin 1, a phosphoprotein that may be involved in synaptic efficacy. suggests that transmitter release may influence early axon target cell interactions.

The events taking place after the initial entrance of axons into target regions as synaptogenesis begins are poorly understood. We have been interested in three aspects of these early events in developing central nervous system (CNS): the form and trajectory of immature axons and their growing tips after invasion of target regions. the onset of synaptogenesis. and the maturation of growth cones into synaptic endings. The cerebellum is a convenient model system because of the well-known adult circuitry and timetable of its cellular and synaptic development. Ulitil recently, analyses of synaptogenesis were limitc*d to observations with standard transmission electron microscopy coupled with special stains for synapses (e.g. ethanolic phosphotungstic acid”). silver -stained or Golgi material.” or thymidine studies.’ Here we combine two new appraoches to stud! early axonal connections. The first is to label individual axons with horseradish peroxidase (HRP) and .malyse their form. cytology and relations with surrc*unding cells in the light and electron microsc11pcs.21 %

The second

approach

involves

ilhh~-,~~~rri~t~~: &I(. cyclic guanosine

immunocytochem-

3’S’-monophosphatedependent protein kinase; E. embryonic ddy; HRP. horseradish peroxidase: NGS, normal goat serum; P. postnatal day: TBS. Tris-buffered saline.

istry with antisera to synapsin I, a phosphoprotein found in association with small clear synaptic vesicles in all mature CNS synapses.‘,‘“,” When antiserum to synapsin 1 is applied to sections cerebellum at adult or postnatal stages, the pattern of synaptic domains and the forms of boutons are demonstrated. One beauty of the cerebellum is that the different types of synaptic bouton are segregated into different cellular layers, such that in the mature cerebellum, mossy fiber glomeruli. for example. are clearly demonstrated in the granule layer. We have used antisera to synapsin I to investigate how the early patterns of synaptic connectivity differ from those of the adult. and to correlate these patterns with the forms and trajectory of developing axons as revealed by dense labeling with HRP.‘” Several questions were raised: ( 1) What is the precise time course of expression ofsynapsin I in cerebellum’? (2) Is the onset of synapsin I expression correlated with specific features of the projection and morphology of immature axons andior with the appearance of synaptic specializations~ and (3) Does the initial pattern of synapsin staining reveal features of early axon--target cell interactions‘!

of

EXPERIklENTAL

All experiments were carried derived from a timed-pregnancy

PROC‘EDI RES

out with C57BL;bJ mice hreeding colony in this

department. The morning on which noted was destgnated as day I (El )

d vaginal

plug

was

The procedures for filling axons. especially embryonrc axons, are identical to those previously described.” In brief. axons in embryonic and postnatal mice were labeled wtth HRP by inserting an HRP-coated micropipette into axon bundles in fresh slices of brain stem and cerebellum or into isolated half-brains. After 15 30 min, the tissues were unmersion-tixed III either I% parafomaldehyde/l% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) or in 3% glutaraldehyde in the same buffer. Vibratome sections of the slices or half brains were cut the next day at 75/tm and reacted with diaminobenzidine with cobalt chloride for light microscopy but without cobalt chloride for electron microscopy. Sections were prepared for electron microscopy by osmication, en bloc staining with uranyl magnesium acetate. dehydration in alcohols, and embedding in Epok (Fullaml in between two plastic slides. The correlative light and electron microscopic analysis is similar to previously described procedures.‘5.‘h,“’ Axons were drawn from the 75 pm wafer separated from the plastic slides and examined with a x 100 oil-immersion objective. Seven micron sections were cut from the original 75pm section, placed on glass slides, and those sections containing portions of the original axons were drawn, photographed, and remounted on epon stubs. Thin sections were then cut and placed on single-holed formvar-coated grids, stained with uranyl acetate and lead citrate, and examined in H JEOL IOOS electron microscope. Many of the preparations used for a previous analysis” were examined. Eight other embryonic cases and IO early postnatal cases were newly prepared for the present study.

Antiserum to synapsin I was kindly provided by Dr P. De Camilli, and was raised in rabbit against synapsin I purified from bovine brain.’ The cerebella and brainstem were studied from 27 embryonic (E) and 20 postnatal (P) mice, ranging in age from El3 to E19. and including PO, PI. P3, P7 and adult animals (P30-40). Embryos were obtained by anesthetizing pregnant mothers with sodium pentobarbitol, removing embryos from the uterine sac, and immediately fixing them by immersion into 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). After IO min. in embryos older than E16, the brain was exposed but left in the skull. Postnatal animals were perfused quickly with the same fixative. All tissues were left in fixative for I~-2 h. then rinsed in 3 4 changes of phosphate buffer over I h, and placed in 30% sucrose in 0.1 M sodium phosphate buffer. with sodium azide (0.1 mg/ml) added to prevent bacterial growth. Tissue blocks were frozen by placing them on a cryostat stub, covering them with Tissue-tek embedding compound, and allowing them to freeze within the cryostat chamber at -20’C. Sections were cut at 8810 pm, placed on gel-coated slides, air-dried and stored at 4’C until stained. Immunocytochemical staining for synapsin I was performed as follows: Sections were incubated (on slides) in 10% normal goat serum (NGS) in Tris-buffered saline (TBS) and 0.2% Triton-Xl00 for I h. The antiserum was diluted at I :I00 in TBS containing 1% NGS and 0.2% Triton and sections were incubated overnight at 4 C. After 3-4 washes in TBS over 30min. sections were then incubated for I h at room temperature with IIuoresceinconjugated goat anti-rabbit immunoglobulin G (Antibodies Incorporated) diluted at I: 100 in TBS with I% NGS and Triton. For control sections, preimmune serum was substituted for synapsin I antiserum. After an additional 3 rinses over 30 min. sections were mounted in 0. I % p-phenylenediamine in buffered glycerol, to reduce fading of

RESULTS Ason fiwm and cwttocts

prior

to .s,wap.psit~I 4’ ywtwiorl

When HRP was inserted into tiber tracts within the cerebellar anlage at El6, axons were labeled that project well within the cerebellum (Fig. 1). These axons have fine calibers, are relatively unbranched. and give off small tapered growing tips (2 Sl~rn long). or miniature growth cones (8.-lOLLm) with short filopodia. The axons that reach the future Purkinje cell and molecular layers run perpendicular to the external granule layer. Some axons arc ortented parallel to the external granule layer, and run in bundles. Other fibers that were traced from the incoming tracts, project within the inner medullar; areas and bifurcate to deliver branches to widespread portions of the anlage. It was not possible with the methods used to identify these fibers as mossy o: climbing fibers. Although axons are present within the cerebellum at El6, these axons do not express synapsin 1. In contrast, within the brainstem. tracts of fibers and small spherical structures arc synapsin-positive (Fig. 2~). At the ventrolateral base of the cerebellum, stained bundles of fibers run in a wide tract (Fig. 2b). These fibers may represent axons already prescut in cerebellum but in the process of transporting synapsin I. By E17, more medial regions of the cerebellum show staining. The immunostained structures in this region resemble bundles of fibers, rather than growing tips or boutons. Ultrastructural analysis of the neuropil at El7 demonstrates that few synapses are present (Fig. 7a,b). Growing tips as well as lengths of axon contain a mixture of large and small vesicles, and symmetrical densities are common, but contacts with both clusters of vesicles and densities are rare. A.ron form

and contucts

gftrr

.synapsin I e.rpression

By El8, fibers throughout the entire cerebellum show synapsin I reactivity, in four staining patterns. First, blotches of staining are prominent in the anterior medial region of the cerebellum. In phase views these stained blotches correspond to cell-free regions, and as at E17, probably represent bundles of fibers cut obliquely. Second, beneath the cxtcrnal granule layer, fine immunoreactive lengths of axon connected by small dots are oriented perpendicular to that layer (Fig. 3a,b), mirroring the form of axons that project into this region (Fig. 4). Third. within the “core” of the cerebellum. consisting medially of the deep nuclei, and throughout of the future granule cell layer and white matter tracts, larger spherical structures are stained. Some of these boutons may represent Purkinje cell axons terminating in the deep

Synapsin I in immature

cerebellar

axon5

Fig. I HRP-labeled cerebellar axons at El 6. Although many of these axons project far into the ccrebcllar anlage and nearly reach the external granule layer (egl), they have simple unbranched forms and hear small growing tips (small arrows), some of which have the form of miniature growth cones (large arrows). Such axons are synapsin l-negative. Inset. arrow indicates of placement of axons. Bar : IO ~(m

nuclei. Fourth. oval structures are stained. some of which have processes (Fig. 3c.d). These may be migrating cells. Even though synapsin I is expressed in axons at El8. the form and trajectory of these axons changes little from the form at El6 or 17 (Fig. 4). Growing tips are small and simple, especially on the fibers located beneath the external granule layer (Fig. 4). As

at El6, the staining pattern vvtth synapsin I rctlects the placement and dimensions of these growmg ups. Ultrastructural analysis of growing tips at EIX reveals that at least two populations of vesicles are present within them, small clear synaptic vesicles and larger clear vesicles (Fig. 5). Surveys of the cercbellum in unlabeled preparations show that morphological synapses. defined here by the occurrence

El8 -

Fig. 4. HRP-labeled axons at El& Although synapsin I is expressed at this time, there is no significant change since El6 (Fig. 1) in the form and projection of axons, or in the shapes of growing tips, with the exception of more extensive branching of some arbors (arrow). The ultrastructure of smaller growing tips and a larger growth cone-like form (arrows a+) is shown in Fig. 6. Inset, arrow indicates location of axons in cerebetlar a&age. Bar = 10 pm.

of a pre- and postsynaptic density with concomitant aecumuiation of vesicles, are more prevalent after El8 (Fig. 6c,d). These synapses are primarily asymmetrical. Before E18, junctions are symmetrical, but vesicle clusters are not associated with them, even though vesicles are scattered nearby (Fig. 6a,b). At this age, vesicles are common in axon segments between growing tips (see Fig. 4C in Ref. $5, and

Fig. Sb), where membrane specializations are often absent. For this reason, the pattern of synapsin expression continues to match the forms of axons and the shapes of their boutons (Fig. ga,b). Between the onset of synapsin expression and P3, the form and trajectory of axons remains relatively constant (Fig. 7). At PO, isolated cells beneath the external granule cell layer are synapsin-positive, and

Fig. 2. Sqnapsin staining in the cerebellar anlage and brainstem at E16. (a) Phase micrograph of a parasagittdl section through the lateral cerebellar anlage. (b) Fluorescence micrograph of inset * in (a). showing bundles of synapsin I-immunoreactivity in fibers entering the cerebellum. The remainder of the ccrebelium is synapsin I-negarive. (cf Fluorescence rnicr~~~raph of inset ** in (a). In contrast to the ccrebcllum, much of the brainstem expresses synapsin. in the form of small dots (small arrows). vJ~ich may represent etthcr growth cones or synaptic swellings. and bundles of fibers (large arrow). (a) * 75; (b. c) x 2x0.

1323

Fig. 3. Synapsin staining in medial and lateral cerebellum at E18. (a, b) Low and high power micrograph:~ of a parasagittal section of medial cerebellum, demonstrating fine fibers with small stained growing tips oriented perpendicular to the external granule layer (egl). Compare the form of the axons revealed by immunostain~ng (arrow, b) to the form obtained by HRP tilling (Fig. 4). (c, d) Low and high power micrographs of a more lateral parasagittai section showing stained perikarya of neurons (medium arrow). The elongated form (large arrow) suggests that they are migrating neurons, positioned among the line stained axons (double arrow). (a, c) x 280; (b. d) x 560.

1324

Fig. 5. Ultrastructure of HRP-labeled growing tips of embryonic axons at El8. Micrographs (a-c) correspond to arrows (a-c) in Fig. 4. All three growing tips, despite their differences in shape, contain both large (LV) and small (SV) clear vesicles. Note that the growth cone in Fig. 5a, shown in (a), is apposed to the process of a cell. In the labeled profile in (b), an accumulation of large clear vesicles occurs at a varicosity of the axon (arrow). Small synaptic-sized vesicles are found along neurite lengths in both this labeled profile and an adjacent one (asterisk). (a-c) x 20,000. 1325

Fig. 6. Ultrastru~ture of elementary contacts in cerebellum at Et6 and E18. (a, b) At E16, symmetrical membrane tbicke~ings resembling puncta adberentia (small arrows) are common. Although synaptic-sized vesicles (SV) are present, they do not necessarily gather at such junctions. (c, d) After E 18,when synapsin I is expressed, asymmetricai junctions (targe arrows) with an associated accumulation of synaptic vesicles. are more frequent. In (d), note fusion of a synaptic vesicle with the membrane (long arrow). (a) x 25,000: (b-d) x 26,000.

Fig. 8. Synapsin staining of cerebellum at PO. (a, h) Low and high power fluorescence microgr~phs of two different parasagittal sections demonstrating fine stained axons with small growing tips (arrows). As at El& synapsin I is distributed in the entire terminal arbor and corresponds to the form HRP-labeled axons (compare to Fig. 7). (c, d) Low and high power microyraphs of a section showing synapsin I-positive growing tips encircling labeled perikarya (arrows). (e. f) As late as PO, there is some immunoreactivit) of fibers within incoming tracts to the cerebellum (brackets in e, phase micrograph of area in brackets shown in f). Cff Neurite lengths (sin& arrow) as well as small el~)ngated structures (double arrow) :trc stained. (a. c. e) x 280; (h. d, f) x SAO.

Fig :. 8

I: 127

Fig. 10. Synapsin staining in cerebellum at P3. P7. and in the adult. (a) In lateral or less mature ospectr of the cerebellum at P3, synapsin staining does not reveal segregation of cell types and thus synaptic domains, as it does more medially. (b) Medially, the Purkinje cell layer (PL) and the contacts onto Purkinje somatic spines are shown, and the emerging granule layer (CL) with larger mossy-like boutons is also demonstrated. Synapsin I-positive structures have increased since PO. (c) At P7, note that form and size of stained boutons in the Purkinje (PL) and granule (GL) layers are now quite different and compare to the form and projection of the immature climbing and mossy fibers in Fig. 9. (d) Adult cerdbellum, demonstrating that all categories of synaptic boutons are synapsin I-positive, including parallel fiber synapses (arrow), in molecular layer (ML), and basket cell axon synapses around Purkinje somata (B, arrow). In contrast to early postnatal periods, interbouton iengths of axon are not stained. WM, white matter. (a d) x280.

1328

Synapsin

I in immature cerebellar axons

PO

F’IP. 7. HRP-labeled fibers at PO. The form of cerebellar axons is still primitive. and consists unbranched axons with simple small growing tips (arrows). Bar = IOpm.

brightly stained boutons impinge on them (Fig. 8c.d). Within the axon tracts entering the cerebellum from brainstem, not only are fibers stained. but small elongated structures are synapsin I-positive (Fig. 8e.f). These could represent focal accumulations of synaptic vesicles, transient synapses. or growth cones of late arriving axons which contain synapsin I.

01‘ relatIveI]

After P3. the stereotypic terminal boutons 01 climbing and mossy fibers are more evident on HRPlabeled axons (Fig. 9). At this point, the pattern of :synapsin I staining changes dramatically, and only boutons are immunoreactive. Cell layers. and the partitioning of different types of synaptic bouton

C. ;\. Masol

GL

MFFig. 9. HRP-labeled cerebellar afferents at P7. Three types of afferents are common in the Purkinjc ccl1 (PL) and granule cell (GL) layers. Typical mossy fibers (MF) have a slightly oblique course and large irregular boutons (large arrow). Immature climbing fibers (CF) primarily project onto Purkinje cells. Climbing fibers go through an exuberant stage whereby they send branches with small growing tips (small arrows) onto more than one Purkinje cell soma (CF,), and then focus with a nest of growing tips (CF,) onto individual cells. Many fibers (MF-CF) have mossy-like morphology in the granule layer and send transient collaterals or long filopodia that bear small climbing-fiber-like growing tips (double arrows) onto Purkinje somata. This demonstrates that the form of the developing growing tips or synaptic boutons is dictated by the cellular and synaptic domain of these individual layers. Synapsin I staining reveals this difference in boutons form and placement (Fig. IO) but does not indicate that individual axons can hacc both forms.

(and thus synaptic arrangements) in those cell layers become more obvious (Fig. 10). At P3, the more lateral cerebellar regions resemble embryonic and early postnatal ages. lacking the differential pattern of regionalization or sizing of stained boutons (Fig. IOa). Medially, however, fine axons beneath the external granule layer are more distinct than those in the layer below (Fig. lob). Below the immature Purkinje cell layer, the emerging granule cell layer contains larger representing stained structures, immature mossy fiber boutons (Fig. lob). This pattern is more striking at P7 (Figs 9 and IOc), when the climbing fiber input to Purkinje somata and emerging apical dendrites is graphically demonstrated, in agreement with the original immunostaining results of De Camilli et ~1.~One feature of the staining pattern at P7, is that the zone immediately below the Purkinje cell layer is synapsin I-negative while the rest of the granule layer contains synapsin I-positive large boutons of mossy fibers (Fig. 10~). Synapsin I staining is also absent in the upper molecular layer where there are parallel fiber synapses onto Purkinje dendrites. Both negatively

staining zones might contain the most recently arrived granule cells and their parallel fibers, respectively, that may not yet express synapsin I. This is consistent with the apparent lag in synapsin expression after axon arrival, as observed in fetal periods. In the adult (Fig. IOd), the entire molecular and granule layers are positive for synapsin 1. and fluorescent dots, corresponding to basket cell axon boutons surround Purkinje somata. After P3, axon tracts are devoid of staining. While synapsin I staining demonstrate the development of synaptic domains and the form of boutons, it does not indicate the involvement of individual axons in more than one domain. A large number of mossy-like axons project line collaterals into the Purkinje cell layer (Fig. 9, MF-CF). The small tapered boutons on these collaterals resemble immature climbing fiber endings (Fig. 9, CF), and associate with Purkinje somatic protrusions.26 The pattern of synapsin I staining also illustrates that the form of different types of bouton in the Purkinje and granule layers is dictated by the postsynaptic dendrites in each of these layers.

Synapsin I in immature cerebellar axons DISCUSSION

Patterns of‘ synapsin I immunostaining By combining HRP labeling with immunostaining to the synaptic vesicle antigen synapsin I, several features of afferent input and synapse maturation were demonstrated. The afferents that project into the cerebellar anlage by El6-17 do not express synapsin 1. The expression of synapsin I at El 8 is accompanied by an Increase in contacts that resemble immature synapses, yet growing tips maintain the cytology of both growth cones and synaptic terminals for at least a week. lmmunocytochemical staining with antisera to synapsin I is a useful way to chart maturation of synaptic and cellular organization in developing CNS, such as the changes in the size and shape of growing tips and terminal arbors, and the emergence of synaptic domains m different cell layers. However, antisera to synapsln I cannot at present be used as a marker for individual synaptic terminations in developing brain, since it is not known whether synapsin I reactivity in a growmg axon represents a synaptic specialization. The staining might simply reflect the presence of the antigen alone, or the antigen associated with vesicle membranes, even when the vesicles are not clustered at a synaptic site. More precise localization of the antigen must be carried out at the ultrastructural level in individual developing axons and nerve terminals during synaptogenesis in situ. In fetal and neonatal ages, entire terminal arbors are synapsin I-positive, in contrast to later postnatal and adult periods, when only synaptic boutons express synapsin. There are two possible and related explanations for these staining patterns. First, free synapsln I may be transported from the cell bodies of origin, and thus appears along the length of the axon. Second. synapsin I might be associated with synaptic vesicles, even before they accumulate at a synaptic site. In axon growing tips in the kitten lateral geniculate nucleus and in the mouse cerebellum, small clear synaptic vesicles (40-60 nm) are found in axon lengths in between developing boutons or growing tips. for an extended period of time after axons have invaded the target region.” ” This is an additional indication that synapsin I reactivity in developing brain may not necessarily indicate a morphological synapse. The patterns of immunostaining with antisera to synapsln I do not provide direct information on axon-target selection. Extrapolations must be made in concert with other data. One possibility is that there is a burst of synapsin I expression when a correct choice in cell selection is made.” The other possibility, one that is suggested by these data, is that thcrc is a gradual addition of synapsin I to axon endings, in the manner that other proteins, for example, cytoskeletal elements. are transported to axon terminals.

1331

Correlations with cerebellar development HRP labeling has demonstrated that afferent axons project into the emerging cerebellar cortex well before many cellular targets, particularly granule neurons. are born or positioned.3” Fibers have been seen within cerebellum as early as El3 with silver staining.“.“ with immunostaining and antisera to the 160 kD component of the neurofilament triplet (Mason and Liem, unpublished observations), and in Nisslstained material.‘.‘.” However, these methods do not show the polarity of the axon, making it difficult to assess whether fibers are afferents or efferents. Recent studies using new tracing methods shah that in the neonatal rat, olivary (climbing fibers), and pontine and spinal afferents (mossy fibers) are present within the cerebellum.4,36 In the opossum, climbing fibers invade the cerebellum several days before Purkinje cells form a monolayer.” Thus, it is likely that both mossy and climbing fibers have begun to enter the cerebellum at the earliest times axons were labclcd with HRP in this study, or at E16. Both HRP labeling and synapsin I staining show that fibers projecting to beneath the external granule layer are afferents. The initial appearance of synapses in cerebellum as defined by aldehyde-osmium or ethanolic phosphotungstic acid begins at El9-22 in rat cerebellum. primarily in the future molecular layer.?’ Afferent input to Purkinje cells has been recorded as early as PO in rat,” and activity is generally detectable at P3.7.40From the data presented here and previously.” it appears that even though fetal and neonatal axon arbors are primitive and the growth-cone-like shape and cytology of their growing tips persist, the axons that would mediate these responses have synaptic vesicles and synapsin I. Other studies employing synapse- or enzj’me-spec@ antisera Synapsin I is currently one of several known synaptic vesicle antigens.‘~‘0~“~‘427The 65 kD protein characterized by Matthew et al.” appears much earlier than synapsin I in chick brain.6 In rat retina. monoclonal antibodies to this protein (SV 48) that stain the inner and outer plexiform layers, also stain the nerve fiber layer from El7 to P3, possibly reflecting transport from the cell body towards axon endings. However, as in cerebellum, this period does not correlate strictly with synaptogenesis.” In the cat visual system, the arrival and waiting of retinal afferents at the outer limits of the lateral geniculate nucleus and of geniculocortical afferents to the visual cortex coincides with synapsin I expression.8 The invasion of synapsin I-positive fibers into the fetal lateral geniculate nucleus matches onset of electrical activity, before the segregation of retinal afferents into their eye-specific layers.jJ Synapsin I staining also occurs in the incipient cortical white matter for a period of days, supporting other evidence that synapses are present here and in the

C’ A. MAWPU

1332

cortical layer above the white matter prior to synaptogenesis in the remainder of the cortex (Chun and Shatz, personal communication). In Rhesus monkey, the expression in Purkinje cells of cyclic guanosine 3’,5’-monophosphate-dependent protein kinase (cGK), an enzyme that catalyses phosphorylation of specific substrate proteins, correlates with the onset of rapid dendritic growth and the appearance of the first axosomatic synapses, presumably from climbing fibers.“’ These synapses were detected ultrastructurally and with antisera to synapsin I. Whether there are climbing fiber contacts onto Purkinje cells prior to synapsin I expression cannot be construed from that study. Wassef and Sotelo” showed that during late embryonic development in rat, cGK-immunoreactive clusters of Purkinje cells are intermixed with negative clusters until P3, at which time all cells are positive. It would be of interest to determine if the onset of synapsin I expression in rat also showed compartmentalization. Relecancr

of‘ synapsin

I expression

The significance of the onset of synapsin I expression is still in question. Although synapsin I is known to be a substrate for phosphorylation by cyclic adenosine 3’,5’-monophosphateor Ca’ +/calmodulindependent protein kinases,i6 its role in synaptic function has not been clearly defined. Recently, synapsin I has been shown to be a spectrin-binding protein related to erythrocyte membrane skeletal protein 4. I .5 Both spectrin (fodrin) and synapsin are present in synaptosomes, and spectrin is a component of the presynaptic compartment. One hypothesis is that calcium entry into the nerve terminal activates calcium/calmodulin-dependent phosphorylation of synapsin I. The protein would then dissociate from the vesicle membrane, allowing the vesicle to fuse more easily with the terminal membrane”,‘* and/or increase the availability of releasable synaptic vesicles. Another issue is when a growth cone shifts from a growing mode to a transmitter secreting mode, and how synapsin I expression corresponds to this event. Patch clamp studies on developing neuromuscular

junctions in vitro have shown that freel! moving growth cones can release acetylcholine.“‘” (.;rowth cones have a mixture of large and small vcs~ch. al-Id there are indications that in rirro they contain synapsin I.‘.” Because both types of vesicles exist in cerebellar growing tips even after expression of synapsin I, immature tips of axons in targets might have characteristics of both growth cones and synaptic boutons. in terms of growing and secretory ability. The small immunoreactive structures in tracts leading into cerebellum suggest that some growth cones during the outgrowth phase contain synapsin I. but this must be confirmed by electron microscopy immunocytochemistry. It is unclear. houcver. why some axons would express synapsin I during outgrowth and others would do so only after invading targets. The synthesis of neurotransmitters and the presence of a pre- and postsynaptic speciahzation are not necessarily coupled.‘2~2”~‘q’”Therefore, presence ot synapsin I and small vesicles might ensure that transmitter in an immature axon ending is available for release, raising the possibility that growth cones and immature growing tips release substances onto potential target cells even before conventional morphological synapses are assembled. The present study demonstrates that synapsin I is expressed in axon growing tips as components of synaptic specializations are assembled, but before synaptic terminals and postsynaptic dendrites are mature. Thus, expression of synapsin I is likely to be one of a sequence of steps, rather than a terminal step in the construction of a synapse. These data should provide a basis for more detailed immunocytochemical studies on individual axons, that would define how synapsin I expression relates to the switch in immature axon endings from a growing mode to a synaptic mode. Acknowledgements--I am indebted to P. De Camilli tar the antisera to synapsin I and for many useful discussions, I thank E. Gregory for superb technical assistance and M. E. Hatten, R. Liem and R. Llinas for helpful comments on the manuscript. This work was supported by National Institutes of Health Research Grant NS- 16951 and a Research Career Development Award, and an Irma T. Hirsch1 Career Scientist Award.

REFERENCES 1. Altman J. (1982) Morphological development of the rat cerebellum and some of its mechanisms. In The C’erehellumNew Vistas (eds Palay S. L. and Chan-Palay V.), pp. 8-46. Springer-Verlag, New York. Altman J. and Bayer S. A. (1978a) Prenatal development of the cerebellar system in the rat. I. Cytogenesis and histogenesis of the deep nuclei and the cortex of the cerebellum. J. camp. Neural. 178, 23348. Altman J. and Bayer S. A. (1978b) Prenatal development of the cerebeilar system in the rat. II. Cvtogenesis and histogenesis of the inferior olive. pontine gray, and the precerebellar reticular nuclei. J. camp. Neurbl. 178, 49 76. Arsenio-Nunes M. L. and Sotelo C. (1983) Development of the spinocerebellar system in the oostnatal rat J. c’ornn. Neural. 237, 29 l-306. 5. Baines A. J. and Bennett V. (1985) Synapsin I is a spectrin-binding protein immunologically related to erythrocyte protein 4.1. Narure 315, 410-413. 6. Bixby J. L. and Reichardt L. F. (1986) The expression and localization of synaptic vesicle antigens at neuromuscular junctions in vitro. J. Neurosci. 5, 3070-3080. 7. Crepe1 F. (1972) Maturation of the cerebellar Purkinje cells. I. Postnatal evolution of the Purkinje cell spontaneous firing in the rat. Expl Brain Res. 14, 463 -471.

Synapsin

I in immature

cerebellar

axons

1.333

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