differential synaptic vesicle protein expression in the barrel field of developing cortex

THE JOURNAL OF COMPARATIVE NEUROLOGY 375:321-332 (1996)

Differential Synaptic Vesicle Protein Expression in the Barrel Field

of Developing Cortex

OLIVIER STETTLER, BERTRAND TAVITIAN, AND KENNETH L. MOYA INSERM U334 (O.S., B.T., K.L.M.) and CNRS URA 1285 (K.L.M.),

Service Hospitalier Frederic Joliot, Departement de Recherche Medicale, Commissariat a 1’Energie Atomique, 9140 1 Orsay, France.

ABSTRACT The distribution of four proteins associated with synaptic vesicles, SV2, synaptophysin,

synapsin I, and rab3a, was investigated during postnatal development of the posteromedial barrel subfield (PMBSF) in the rat somatosensory cortex. A distinct progression in the appearance of the different synaptic vesicle proteins within the PMBSF was observed. SV2, synapsin I, and synaptophysin revealed the organization of the barrel field in the neonate. This early demarcation of the cortical representation of the vibrissal array coincides with the earliest known age for the emergence of the cytoarchitectonic organization of this region. In contrast, rab3a did not delimit the barrels until the end of the 1st postnatal week, coincident with the known onset of adult-like physiological activity and the loss of plasticity in afferents to this region. In addition, the appearance of the different synaptic vesicle proteins occurred earlier within the PMBSF than in the adjacent extra-barrel regions of the cortex.

These results show that the molecular differentiation of synaptic fields across the cortex is not a homogeneous and synchronous process in terms of synaptic vesicle protein expression. Because these proteins act together in mature synapses to ensure the regulated release of neurotransmitters, our results suggest that this temporo-spatial asynchrony may underlie different potentials for synaptic activity and thus contribute to the development of cortical maps. D 1996 Wiley-Liss, Inc.

Indexing terms: PMBSF, rab3A, SV2, synapsin I, synaptophysin

Processing of information in the brain depends on the regulated release of neurotransmitters at synaptic contacts organized in a precise circuitry. Thus, two crucial steps in cerebral development are the establishment of a meaning- ful circuitry and the maturation of an integrated molecular synaptic machinery. In the rodent cerebral cortex, synaptogenesis is largely a postnatal phenomenon. Although some synaptic specializations appear as early as the late embry- onic stage in the rat brain (Konig et al., 1975), synapses are still few and poorly differentiated at birth (Jones and Revell, 1970). During the first 2 postnatal weeks, the number of synapses dramatically increases in combination with an increase in presynaptic vesicular content and a thickening of postsynaptic membranes (Kristt and Mol- liver, 1976; Blue and Parnavelas, 1983). In previous studies, we noted that the arrival of the synaptic vesicle protein rab3a in terminal fields of the rat hippocampus and cerebral cortex was delayed with respect to the known onset of synaptogenesis (Stettler et al., 19941, raising the question as to whether there are different maturational steps of synapse formation.

The rat barrel field is well suited to study the maturation of cortical synapses as the developmental progression of its connections is well characterized and its macroscopic organization facilitates the localization of the same regions in different animals (Chapin and Lin, 1990). In this highly differentiated synaptic field of the somatosensory cortex, disjunctive groups of neurons and axons faithfully replicate the punctate array of mystacial vibrissae and perioral sinus hairs of the contralateral snout (Woolsey and Van der Loos, 1970; Welker and Woolsey, 1974; Jhaveri and Erzurumlu, 1992). Merents that arise in the ventrobasal thalamic complex (VB) terminate within barrels, resulting in focal sets of connections (Jensen and Killackey, 1987), whereas the interbarrel region, i.e., the septa, receives inputs from

Accepted June 23, 1996. Olivier Stettler is now at The Randall Institute, King’s College, London,

Address reprint requests to Kenneth L. Moya, INSERM U334, SHFJ, WC2B 5RL, UK.

CEA, 4 place du general Leclerc, 91401 Orsay cedex, France. E-mail: moya(@uriens.shfj.cea.fr

o 1996 WILEY-LISS, INC.

322

the posteromedial thalamic nucleus (Koraleck et al., 1988; Nothias et al., 1988). Functionally, each barrel processes peripheral information from one vibrissa via the brainstem trigeminal complex and the VB nuclei (Kossut, 1992). The size of the territory devoted to the whisker representation is highly dependent upon sensory experience during development and is subjected to important plastic modifications following peripheral manipulation (Schlaggar et al., 1993; Killackey et al., 1994; Zheng and Purves, 1995).

Molecular studies have characterized a number of con- stituents of the presynaptic machinery such as synaptic vesicle (SV) proteins and proteins of the presynaptic membrane, which act in concert to ensure the vesicular release of neurotransmitters (for a recent review, see Sudhof, 1995). We directly compared the ontogenic pattern of four major SV proteins, synaptophysin, SV2, synapsin I, and rab3a, in the rat barrel field cortex. We show that they follow distinct developmental time-courses in their localization to this well-characterized terminal field, suggesting the existence of a temporal control of cell signaling in the developing rat brain. At a spatial level, this temporal progression is not synchronized between all regions of layer IV in the somatosensory cortex and transiently reveals the architectural organization of the whisker-related cortical map.

0. STETTLER ET AL.

MATERIALS AND METHODS Tissue preparation

All experimental procedures were in strict accordance with the recommendations of the EU (86/609/EEC) and of the French National Committee (87/848) for the use and care of laboratory animals. Sprague-Dawley rats (Iffa- Credo, France) of various postnatal ages (see Table 1; the first 24 hours after birth is designated as PO), as well as adult rats (more than 4 months old), were terminally anesthetized with an overdose of pentobarbital and transcar- dially perfused with buffered 4% paraformaldehyde. For tangential sections, cerebral cortices were removed and flattened according to Strominger and Woolsey (1987). Cortices were gently compressed between two histological glass slides separated 1-2 mm by glass spacers. For immunohistochemistry, the cortices held between the slides were immersed overnight in sucrose (4%) and paraformaldehyde (4%) in PBSN [lo0 mM phosphate buffer, pH 7.4, 155 mM NaCl, 0.5% sodium azide (NaN3)1. The flattened cortices were separated from the slides and placed in 30% buffered sucrose for cryoprotection. Serial 50-pm tangential sections were cut with a freezing sliding microtome (Microm HM440E) and collected in 0.1 M phosphate buffer. For coronal sections, perfused brains from adult animals were postfixed 1 hour in buffered paraformaldehyde, cryopro- tected, and frozen. Frozen 40-pm coronal sections were then collected in PBSN.

Whisker ablation Litters of rat pups at postnatal day 0 (PO) were anesthe-

tized with fluothane, and the follicles of all whiskers in row C were electrolytically cauterized. The cauterized whisker and its follicle were then removed with a fine forceps. Pups were allowed to recover and returned to the nest. On P7 or P8 (Table 11, animals were terminally anesthetized and perfused with 4% buffered paraformaldehyde. The cortical hemisphere contralateral to the lesion was removed and tangential sections obtained as above.

TABLE 1. Summary of the Number of Cases Examined a t the Indicated Ages With the Indicated Antibodies'

Cytochrome Am Synaptophysin SV2 Rab3a Synapsin I oxidase

P3 3 2 2 2 P4 2 2 1 -

2 P5 1 1 P6

P7 1 1 2 1 PR 1 PI0 1 1 1 1

1 PI2 1 2 P14 1 1 1 1 PI8 1 1 1 - Adult (tangential) 1 2 2 1 Adult (coronal) 1 1 1 1 2 Vihrissect. 2(P7l 2 (P7) 2 (P71 2 (P7l

- - - - -

- - -

-

2 (P8)

'For the vihressectomized animals, the number of animals examined IS followed by their age a t time of death in parentheses.

Immunohistochemistry We used our standard procedure for revealing immunore-

activity as described previously (Moya et al., 1992; Stettler et al., 1994). Briefly, brain sections were incubated with 0.3% H20z in methanol to neutralize endogeneous peroxi- dase activity, After rinsing in PBSTN [lo0 mM phosphate buffer, pH 7.4, 155 mM NaCl; 0.4% Triton X-100; 0.5% sodium azide (NaN,)], nonspecific binding was blocked in PBSTN with either 20% normal goat serum (NGS, for rab3a and synapsin I immunohistochemistry) or with 20% normal horse serum (NHS, for SV2 and synaptophysin immunohistochemistry). The sections were then incubated with the antibodies against rab3a (3Ap3 at 1:600; Moya et al., 1992), synapsin I (1:100, a gift from Dr. T. Petrucci. Petrucci et al., 19911, synaptophysin (1:600, Sigma), or SV2 (clone 10H tissue culture supernatant diluted 150; a gift from Dr. K. Buckley, Buckley and Kelly, 1985) in PBSTN with 1% NGS or NHS. The sections were rinsed and then incubated with biotinylated anti-rabbit (Vector Labs) for rab3a and synapsin I (1:250 in PBSTN, 1% NGS), or with biotinylated anti-mouse for SV2 and synaptophysin (1:250 in PBSTN, 1% NHS). After extensive rinsing, immunoreactivity was revealed using avidin-biotin-horseradish peroxi- dase (ABC Kit, Vector Labs) and diaminobenzidine as the chromagen. The treated sections were mounted on slides and covered.

Control experiments in our developmental studies con- sisted of incubating sections in the absence of the primary antiserum. In other experiments, we preadsorbed the 3Ap-3 serum (anti-rab3a) with a &fold molar excess of purified rab3a protein (a gift from Dr. F. Darchen). Under such control procedures, virtually all immunoreactivity was abol- ished (see Stettler et al., 1994).

RESULTS Synaptic vesicle protein distribution in the PMBSF at the time of barrel appearance

By postnatal day 2 (P21, the thalamic afferents originating from the ventrobasal complex form a vibrissa-specific pattern in the S1 cortex of the rat (Erzurumlu et al., 1990). Because 1 day later barely discernable cellular agregation demarcating the cytoarchitectonic boundaries of cortical barrels are detected for the first time by Nissl staining (Rice et al., 19851, we use P3 to designate the day when barrels

SV PROTEINS IN DEVELOPING CORTEX 323

Fig. 1. Tangential sections through flattened cerebral cortices of rat pups in the early postnatal period. Orientation of the section is indicated in the top left by the perpendicular arrows (a, anterior; 1, lateral). Rows of barrels are labeled A-E in the top right. At P3, SV2 immunohistochemistry reveals the barrels whose arrangement faithfully replicates the whisker representation. A moderate labeling is also observable in the neuropil adjacent to the barrel field, whereas between the barrels (i.e. the septa), immunostaining is less prominant. At P4,

anti-synaptophysin stains the neuropil across the barrel region and is intensely localized to barrels. Synapsin I reveals barrels at P3 (bottom left), whereas outside the barrel field, synapsin I is unevenly distributed. The immunolabeling produced by the rab3a antibody at P4 displays a different pattern (bottom right ), the protein being detected essentially in the cell bodies of the granular layer IV. No barrels are detectable with anti-rab3a at this age. Scale bar = 400 Fm.

have formed. At this age, the posteromedial barrel subfield (PMBSF), representing the mystacial vibrissae, appears immunoreactive for the SV proteins SV2 and synapsin I in sections through the upper layers of flattened cortices (Fig. 1).

MablOH directed against SV2 stains the neuropil of the presumptive layer IV and defines the complete whisker representation on tangential sections through the barrel field of the somatosensory cortex at P3 (Fig. 1). The patches of increased SV2 immunoreactivity are restricted to the cores of barrels and reflect the peripheral array of the mystacial vibrissae. The density of immunoreactivity in barrel centers is similar to other areas of the cortex but stands out against a lesser staining in the septa at this age. A higher magnification of this region (Fig. 3) shows that SV2 staining densely surrounds nonimmunoreactive cell bodies, within both the focal patches and the septa. This distribution seen at P3 persists at older ages (see below).

In all three pups examined at P3, synaptophysin is distributed heterogeneously in the somatosensory cortex. One day later on P4, when the prospective PMBSF becomes clearly detectable by Nissl staining (Rice et al., 19851, the entire vibrissal array is visualized with the synaptophysin antibody (Fig. 1). The neuropil of barrel centers is more immunoreactive than the septa which appear similar in intensity to surrounding areas of the S1 cortex. At high

magnification (Fig. 3) the fine distribution of synaptophysin is comparable to that observed for SV2, i.e., it is present as dense deposits silhouetting blank cell profiles.

Anti-synapsin I stains patches corresponding to the barrel field on P3 (Fig. 1). These round patches are arranged in a vibrissa-related array and are separated by an area of pale staining. In the surrounding nonbarrel cortex, there is an evenly distributed neuropil staining whose intensity appears lower than the intensity of the patches. In addition to neuropil labeling, anti-synapsin I darkly stains some scattered cell bodies throughout the presumptive layer IV. Some of these labeled cells are arranged in large clusters evocative of still-migrating cells. At higher magnification, synapsin I-positive barrel-like structures are readily observable (Fig. 4).

Interestingly, the synaptic vesicle proteins synapsin I, SV2, and synaptophysin clearly delimit barrels in the PMBSF in the early neonate brain, whereas the timing and pattern of rab3a are notably different. Unlike other components of the synaptic vesicle membrane, rab3a is concen- trated in cell bodies in the neonatal brain and shifts to nerve terminals at the end of the 1st postnatal week (see Stettler et al., 1994). The image of rab3A at P4 (Fig. 1) is typical of our results for the five cases examined at P3-P5 and shows heterogeneously stained cell bodies in the presumptive

324 0. STETTLER ET AL.

Fig. 2. Patterns of SV protein distribution in the barrel field during the 2nd postnatal week reveal important differences in the relative intensity of SV protein labeling between the barrels and the adjacent cortex. At P7 and P10 the level of synapsin I and rab3a immunoreactivity in barrels stands out against the lower labeling in septa and in other regions of the cortex. At P10 and P14, SV2 and synaptophysin

immunoreactivity increase considerably both in septa and in the adjacent cortex, rendering the barrels more difficult to discern than in neonates. The dashed lines and the “V” in the top left panel show the limits of the whisker barrel field. Scale bar = 400 pm for P7 and P10. 650 K r n for P14.

layer IV, forming aggregates across the cortex. Neuropil staining is not evident, and no barrel organization is discernable with anti-rab3a at these ages.

Synaptic vesicle protein distribution after the 1st postnatal week

After the 1st postnatal week, the immunoreactivity for the synaptic vesicle proteins SV2, synapsin I, synaptophysin, and rab3a delimit the barrels of the PMBSF corresponding to the vibrissal representation (Fig. 2). However, in contrast to early postnatal ages (P3-P5), the barrels of the anterior snout and the jaw representation are also visualized with each individual synaptic vesicle protein antibody at this age (see Fig. 5).

During the 2nd postnatal week (PlO), SV2 staining has intensified throughout the cortex, and at P12 the contrast between barrels and septa is far less striking than that observed in P3 pups (compare Fig. 2 with Fig. 1). Despite this, many barrels are outlined by a thin, poorly immunoreactive circular band at P10. A high-power photomicrograph of this SV2 pattern at P12 is presented in Figure 3 and shows SV2 staining surrounding blank cell profiles in septa and in barrel centers. At this magnification the labeling engulfing cell profiles is clearly less intense in the side than in the center of barrels and in the septa. Dense deposits of SV2 staining are also distinguishable in barrel centers.

In 1-week-old rats the synaptophysin-labeled vibrissa- related barrels appear somewhat more distinguishable than at P4 (see Fig. 5). Densely immunoreactive barrels in individual rows stand out against a lower immunolabeling in the septa and in the nonbarrel cortex. Two weeks after birth, however, barrels are less distinguishable owing to the markedly increased immunoreactivity throughout the cortex during the 2nd postnatal week (Fig. 2).

By P7, the patches of synapsin I immunostaining are larger compared to earlier ages and they fill the neuropil of individual barrels (Fig. 2). The labeling within the centers of barrels stands out against the lower immunoreactivity in septa and in other regions of the cortex, rendering the barrels sharply demarcated in sections of flattened cortices. High magnification of the whisker barrels at P7 shows a fairly even distribution of the few remaining synapsin I-immunoreactive cell bodies, and the barrels are defined by a synapsin I neuropil staining (Fig. 4). A gradient is apparent such that the immunoreactivity appears to be less pronounced at the periphery (i.e., in the wall) than in the center (i.e., the hollow) of the individual barrel.

At P5 and P6, barrels are difficult to discern by rab3a immunoreactivity; however, they become more readily apparent at P7, coincident with the shift of rab3a from a cellular to a neuropil distribution (Stettler et al., 1994). At P10, barrel centers are defined by a rab3a neuropil staining


Fig. 3. High magnification of the whisker barrel field show similar subcellular distributions for SV2 (upper) and synaptophysin (lower) at the early (P3, P4; left) and the late (P7, P12; right) postnatal period. Both proteins form dense deposits of immunostaining which often

silhouette blank cell profiles. Note that the morphology of immunoreactive barrels changes from a rounded shape in P3-P4 neonates to an oval shape in P7 and P12 pups. Scale bar = 100 pm.

against a much lighter background in the surrounding cortex (Fig. 2). High magnification shows that in young animals, numerous immunoreactive cell bodies are dis- persed throughout the barrels, which are readily apparent

due to the moderate and diffuse neuropil-like staining (Fig. 4). The contrast in rab3a immunoreactivity is greatly increased in barrels by P10 (Fig. 2); however, by P18 the general increase in the level of staining renders the barrels


Fig. 4. Synapsin I (upper) and rab3a (lower) in developing barrels. Synapsin I shares some similarities with rab3a in its subcellular distribution during development. At P3, numerous synapsin I-positive cell bodies are discernable throughout the immature layer IV, and this protein is also distributed in the neuropil at this stage and has started to reveal barrel rows. At P'7 a cellular rab3A staining is evident; the

neuropil-like distribution reveals barrels at this age. Note the low density of rab3A-stained cells and the lower neuropil staining in the septa. At P18, rab3a immunoreactivity forms dense deposits of immunostaining throughout the neuropil with a preferential distribution within barrels. Scale bar = 100 bm.

difficult to delineate, and blank profiles of cell bodies are silhouetted by the rab3a immunoreactivity at this age (Fig. 4).

In the mature brain, the distribution of the synaptic vesicle proteins synapsin I, SV2, synaptophysin, and rab3a

has undergone marked changes. In tangential and coronal sections through the adult somatosensory cortex, none of the antibodies used here can define barrels in layer IV (not shown; see also Moya et al., 1992; Stettler et al., 1994).


Lesion-induced changes We examined the changes in SV protein distribution that

accompany peripheral disruption of the thalamocortical somatosensory pathway. In the rat, removal of a vibrissae row at birth prevents the clustering of thalamocortical projections in discrete clusters matching barrels (Killackey et al., 1976). Cauterization of row C whisker follicles in pups on PO alters the patchy distribution of SV proteins in the PMBSF corresponding to the lesioned whiskers at P7 and P8 (Fig. 5 ) . In this region, SV protein-stained barrels in row C are fused, resulting in an elongated band of immunoreactivity. The width of this SV immunoreactive band is considerably reduced compared with the width of a normal barrel row, and barrels from adjacent rows appear somewhat enlarged in comparison to their normal size in nonlesioned animals.

DISCUSSION Cortical synapse formation as revealed by SV protein

immunohistochemistry is not homogeneous but follows a radial progression (Chun and Shatz, 1988; Devoto and Barnstable, 1989; Voigt et al., 1993). Synapses are first formed in the outer marginal zone and the deeper subplate, and then expand into the cortical plate. This spatially and temporally ordered input formation has been compared to the inside-out progression of migrating cortical neurons and would imply that synaptogenesis occurs later in the later-differentiated layers (Voigt et al., 1993). However, little is known about synapse differentiation within a given cortical layer such as layer IV, which contains a complex assembly of distinct synaptic fields (Wise and Jones, 1976; Olavarria et al., 1984; Agmon et al., 1993). Here, we have examined the differentiation of the rat barrel field in terms of the molecular composition of presynaptic vesicles. Our results reveal an asynchronous molecular differentiation of terminals in the tangential plane of somatosensory cortex, coincident with the development of somatotopic representation.

Intraregional progression of SV protein distribution

It is a common observation that synaptic vesicles as well as SV proteins are progressively localized from cell bodies to terminals during development (Scarfone et al., 1991; Ovtscharoff et al., 1993; Stettler et al., 1994). In vitro, SV proteins accumulate in axons as soon as they develop, but become restricted to synaptic terminals later, following contacts with dendrites of other neurons (Fletcher et al., 1991, 1994; Phelan and Gordon-Weeks, 1992; Basarsky et al., 1994). However, different SV proteins appear to be sorted by different mechanisms in neurons (Mundigl et al., 19931, and temporal differences in the subcellular distribution of various SV proteins along the developing axon have been reported. For example, antibodies to synaptophysin and rab3a stain cell bodies and processes of cultured hippocampal neurons soon after plating, but synaptophysin becomes progressively relocalized to discrete areas along neurites before any changes in rab3a are observed (Motoike et al., 1991). Synaptophysin is an early marker for differen- tiating spinal cord neurites, and it has been reported to segregate more rapidly than synapsin I in vivo in peripheral extensions of developing vestibular neurons (Bergmann et al., 1991; Scarfone et al., 1991). In the present studies, antibodies to SV2 and synaptophysin intensely labeled the

entire layer IV of rat somatosensory cortex by the 3rd postnatal day, forming dense deposits surrounding cell bodies. This pattern resembles the previously described distribution of these proteins in presynaptic specializations during early CNS development (Voigt et al., 1993; Okada et al., 1994). In contrast, at P3, rab3a was localized to cell bodies whereas synapsin I was present both in cell bodies and in the neuropil. By the beginning of the 2nd postnatal week the immunoreactivity for all SV proteins studied here had attained a terminal distribution in barrels, although in several nonbarrel and/or interbarrel regions of cortex, synapsin I and rab3a had yet to completely attain this pattern. The present findings concern in vivo development and indicate that individual SV proteins do not follow the same time-course for their accumulation in synaptic endings. Thus, in terms of the subcellular distribution of molecular components, the maturation of the synapses in the somatosensory cortex is not a synchronous and rapid phenomenon.

Synaptophysin and SV2 are integral membrane glycopro- teins (Buckley and Kelly, 1985; Buckley et al., 1987). Synaptophysin has been implicated in the fusion of vesicles with the presynaptic membrane, an essential step in neurotransmitter release (Edelmann et al., 1995). Indirect evidence suggests that, in immature neuronal cells, the subcellular distribution of synaptophysin reflects the localization of vesicles or other organelles which might undergo fusion (Fletcher et al., 1991; Matteoli et al., 1992). SV2 has 12 transmembrane domains and shares strong similarities with a number of prokaryotic and eukaryotic transporters, suggesting that this protein is a vesicular transporter, although its substrate has not been identified (Bajjalieh et al., 1992; Feany et al., 1992).

Synapsin I and rab3a are nonintegral, membrane- associated SV proteins, and although neither appears to be essential for exocytosis per se (Rosahl et al., 1993, 1995; Geppert et al., 19941, they exert important regulatory roles during the vesicular release of transmitters (Johannes et al., 1994; Rosahl et al., 1995; Li et al., 1995). The synapsins seem to be required for the sustained release of neurotransmitters during repetitive stimulation at the synaptic release site (Pieribone et al., 19951, and they contribute to short- term synaptic plasticity and synaptogenesis (Rosahl et al., 1995; Chin et al., 1995). Small GTP-binding proteins of the rab family regulate the one-way movement of vesicles at every step of the secretory pathway (for review, see Zerial and Stenmark, 1993). Rab3a associates with synaptic vesicles late in the secretory pathway (Matteoli et al., 19911, and it dissociates from vesicle membranes upon Ca2+- triggered exocytosis (Fisher von Mollard, 1991; however, see Bielinski et al., 1993). Recent evidence has accumulated indicating that rab3a is involved in the regulation of vesicle docking in preparation for exocytosis, and in particular during repetitive stimulation (Geppert et al., 1994; Holz et al., 1994; Johannes et al., 1994).

In the mature synapse, all of the various SV proteins are thought to be required to support controlled neurotransmitter release (Sudhof, 1995). The results from our developmental studies suggest that SV proteins can be divided into at least two classes based on their timing of localization to terminals in vivo: 1) a class of synaptic proteins present at the axon periphery early during synaptogenesis such as SV2 and synaptophysin; 2) another class of proteins compris- ing synapsin I and rab3a, whose restriction to the terminal is more progressive and coincides with an increased physiological activity.


Synaptic activity is weak and poorly drivable in rat somatosensory cortex during the 1st postnatal week (Vilagi et al., 1992), a time when high levels of synaptophysin and SV2 are present. During this period synapsin I increased progressively and labeled the barrels more intensely than outlying regions of layer IV. Within the vibrissae barrels, an intense staining for rab3a became apparent only at P6, and this date marks both an increase in physiological activity within these structures in response to peripheral sensory stimulation (Armstrong-James, 19751, and the end of the critical period (see Fox and Zahs, 1994). In comparison, evoked synaptic response in the barrel field at the beginning of the 1st postnatal week is highly labile and often strongly depressed after a small number of stimuli (Agmon and O’Dowd, 1994). The absence of rab3a from barrels at this stage is probably physiologically relevant, because neurotransmitter release during repetitive trains of stimuli is strongly depressed in nerve terminals deficient in rab3a in the adult (Geppert et al., 1994). In vitro, Basarsky and co-workers (1994) have shown that spontaneous synaptic currents (defined as the ability to release transmitters) are sometimes observed in immature hippocampal neurons at a time when rab3a and synapsin I are still restricted to the cell soma. Moreover, these authors demonstrated that evoked transmitter release and a more prominent spontaneous activity became detectable at a time when rab3A and synapsin I were localized to synapses. Thus, both in vitro and in vivo evidence suggests that some SV proteins are localized to axon endings before or during the very early steps of synaptogenesis, whereas others accumulate in presynaptic terminals when an increased exocytotic capabil- ity requires a rapidly recruitable vesicle pool near the active zone. Our results point to the localization of rab3a to terminals as one of the last molecular steps in the maturation of the presynaptic machinery.

Interregional differences in SV proteins In addition to the temporal sequence of synaptic protein

disposition within a terminal field (i.e., the center of individual barrel and the marginal zone), we also observed clear differences in the intensity of neuropil staining between adjacent cortical regions. Synapsin I and rab3a accumulate preferentially in the neuropil of the barrels of the PMBSF before they increase in the terminal fields of interbarrel and nonbarrel regions (Stettler et al., 1994; present results). This interregional ontogenic pattern is maintained even though these two proteins do not delimit the neuropil of the barrels at the same time (P3 for synapsin I and P7 for rab3a, see Figs. 1,3). This differs from the patterns observed for synaptophysin and SV2. In the case of synaptophysin, a homogeneous immunoreactivity is seen throughout layer N at P3, and barrels become discernable with an increase in synaptophysin immunoreactivity at P4. SV2 is intense throughout layer IV at P3, and the barrels are visible because of a lesser immunoreactivity in septa. Thus, the synaptic localization of SV proteins appears not only as a temporally ordered process but also as a process undergoing spatial controls during cortical development.

In this regard, it is interesting to note that we observed differences between the mystacial and facial barrel regions. Vibrissal barrels were the first to be visualized at P3-P5 by SV protein immunohistochemistry followed by barrels in the jaw and snout region after the 1st postnatal week, suggesting fine spatial control within the face-mystacial

barrel territory. This in turn is consistent with a differential development across the entire barrel field (see McCand- lish et al., 1989).

Synapsin I, SV2, and synaptophysin form a vibrissa- related immunoreactive pattern shortly after axons are known to have reached their target territory in layer IV on P2-P2.5 (see Jhaveri and Erzurumlu, 1992). In the developing S1 cortex, these afferents consist principally of fibers arising in the VB or originating in the raphe nuclei. By P3-P4 these two fiber systems develop into dense clusters of axonal arbors in a barrel-like arrangement (D’Amato et al., 1987; Agmon et al., 1993). Thus, the barrel distribution of SV proteins observed in newborn rats reflects their localization to thalamocortical and/or tegmentocortical terminals. The fusion of the SV protein-delimited barrels after vibrissal lesions clearly indicates that the proteins are localized to remodelled subcortical afferents; however, this manipulation does not allow us to distinguish between the thalamocortical and tegmentocortical fiber systems, as both fiber populations are altered by vibrissectomy (Van der Loos and Woolsey, 1973; Killackey et al., 1976; Erzurumlu et al., 1990; Rhoades et al., 1990). It is nonetheless likely that an important part of the staining for SV proteins in barrels is contributed by VB afferents, because the serotonin- ergic innervation from the raphe nuclei declines markedly in barrels after P10 in rat (D’Amato et al., 1987), whereas an intense staining for SV proteins persists at these loci even at P18.

Possible implications As a consequence of the heterogeneous and asynchronous

distribution of SV proteins in the developing S1 cortex, not all parts of the neuropil in layer IV in this region show the same SV protein composition at a given early postnatal age (summarized in Fig. 6). By P7 the neuropil which receives VB afferents (i.e., the barrels) presents high levels of the four SV proteins examined. In contrast, at this age the neuropil of the interbarrel region and of the cortex surrounding the barrel field, which receive posteromedial thalamic afferents and commissural afferents, respectively, displays much higher levels of synaptophysin and of SV2 than of rab3a and synapsin I. It is only after the 2nd postnatal week that levels of immunostaining for all of the SV proteins tested become comparable throughout layer IV, rendering the somatotopic pattern less visible. A possible interpreta- tion of this developmental schedule is that the changes in SV protein composition in the neuropil reflect a sequential maturation of the afferents that project to distinct regions of S1 cortex. By P7, the barrels present a more complete set of SV proteins than adjacent areas, and interestingly, barrels are the most active zones in layer IV at this age (Riddle et al., 1993). This raises the intriguing possibility that if there is a critical combination of SV proteins required for the regulated and efficient transmission of

Fig. 5. Cauterization of row C whisker follicles at PO produces a specific reorganization of SV protein distrihution in the corresponding row C of barrels of the controlateral S1 cortex. At this site (denoted by arrows), the patchy distribution of rab3A (upper), synaptophysin (middle), and synapein I (lower) immunostainingis replaced by a fused band ofimmunoreactivity in the correspondingbarrel row. Note that in the cases presented in the middle and lower panels, an individual barrel in row C can be distinguished. This is likely due to an incomplete destruction of the corresponding follicle. J, V, and-, indicate barrels of the lowerjaw, of the whiskers, and of the antenor snout, respectively. Scale bar = 300 pm.

Figure 5


Fig. 6. Summary of the spatial and temporal changes in SV protein distribution in the developing somatosensory cortex. Upper: The intensity of shading indicates the level of the SV protein in the neuropil of barrels (circles) in layer IV of S1 cortex (ovals). Cell body staining is indicated by stippling. Lower: some of the major known morphogenetic and physiologic events are indicated (see text for references). SV2 and synaptophysin preferentially delimit the barrels during the 1st postnatal week (1st PNW) which coincides with a period of intense synaptogenesis throughout the cortex and also with the time when afferents of barrels arborize collaterals. Synapsin I delimits barrels and stains numerous cell bodies at this time, whereas rab3a only stains cell bodies. During the 2nd postnatal week (2nd PNW), interegional labeling differences are reduced for SV2 and synaptophysin, and barrels can be distinguished because of a lesser staining in septa. Synapsin I and rab3a are preferentially localized in the neuropil of barrels. I n the adult, the immunostaining for SV proteins throughout layer IV is homogeneous and is low compared to the level of immunostaining attained by SV2 and synaptophysin in the developing brain. The period when rab3a first stains barrels at the end of the first PNW coincides with the end of the critical period and with the beginning of adult-like activity. SYP, synaptophysin; SYN 1, synapsin I; TC, thalamocortical; 5HT, serotonin- ergic.

neuronal information, the temporal and spatial differences in SV proteins could pattern the level of afferent activity across the neonatal somatosensory cortex.

The contribution of physiological activity in the development of functional maps has long been described in mam- mals, especially in the visual cortex (for review, see Shatz, 1990). Although the blocking of electrical activity does not prevent the formation of the overall barrel architecture (Chiaia et al., 1992). this map can be modified in an activity-dependent manner following peripheral lesions

(Schlaggar et al., 1993). Moreover, an increase in metabolic activity, which is thought to reflect synaptic activity, in developing barrels correlates with an increased growth of the neuropil in this region compared to other adjacent neuropils (Zeng and Purves, 1995). Taken together these data outline a role for synaptic signaling in the differential shaping of cortical circuitry devoted to sensory representation (see Purves et al., 1992). In this context, our findings suggest that differences in synaptic signaling in the S1 cortex might arise from an earlier and more complete molecular differentiation of the secretory machinery in the barrels compared to adjacent synaptic fields. This in turn could provide competitive advantages that help to stabilize the cortical map.

ACKNOWLEDGMENTS We thank Dr. Kathleen Buckley for anti-SV2 mablOH,

Dr. Tamara Petrucci for anti-synapsin I, Dr. Ahmed Zahr- aoui for anti-rab3a, Dr. FranGois Darchen for the rab3a protein, and Dr. Emmanuel Brouillet and Marie-Caroline Guyot for help with histochemistry. We gratefully acknowl- edge the constant support of Dr. Luigi DiGiamberardino. This work was supported by the CEA, INSERM, and CNRS.

LITERATURE CITED Agmon, A., and D.K. O’Dowd (1994) Immature thalamocortical synaptic

responses in neonatal mouse barrel cortex. Soc. Neurosci. Abstr. 20: 1700.

Agmon, A,, L.T. Yang, D.K. O’Dowd, and E.G. Jones (1993) Organized growth of thalamocortical axons from the deep tier of terminations into layer IV of developing mouse barrel cortex. J. Neurosci. 125365-5382,

Armstrong-James, M. (1975) The functional status and columnar organization of single cells responding to cutaneous stimulation in neonatal rat somatosensory cortex S1. J. Physiol. 246.501-538.

Bajjalieh, S.M., K. Peterson, R. Shinghal, and R.H. Scheller (1992) SV2 2 brain synaptic vesicle protein homologous to bacterial transporter Science 257:1271-1273.

Basarsky, T.A., V. Purpura, and P.G. Haydon (1994) Hippocampal synaptogenesis in cell culture: developmental time course of synapse formation, calcium influx, and synaptic protein distribution. J. Neurosci. 11:6402- 6411.

Bergmann, M., G. Lahr, A. Mayerhofer, and M. Gratzl(1991) Expression of synaptophysin during the prenatal development of the rat spinal cord: correlation with basic differentiation processes of neurons. Neuroscience 42569-582.

Bielinski, D.F., H.Y. Pyun, K. Linko-Stentz, I.G. Macara, and R.E. Fine (1993) Ral and Rab3A are major GTP-binding proteins of axonal rapid transport and synaptic veisicles and do not redistribute following depolarization stimulated synaptosomal exocytosis. Biochim. Biophys. Acta. 1151246-256.

Blue, M.E., and J.G. Parnavelas (1983) The formation and maturation c.f synapses in the visual cortex of the rat. 11. Quantitative analysis. J. Neurocytol. 12:697-712.

Buckley, K.M., and R.B. Kelly (1985) Identification of a transmembrane glycoprotein specific for secretory vesicles of neural and endocrine cells. J. Cell Biol. 100:1284-1292.

Buckley, K.M., E. Floor, and R.B. Kelly (1987) Cloning and sequence analysis of cDNA encoding p38, a major synaptic vesicle protein. J. Cell Biol. 105:2447-2456.

Chapin, J.K., and C . 3 . Lin (1990) The somatosensory cortex of the rat. I n B. Kolb and R.C. Tees (eds): The Cerebral Cortex of the Rat. Cambridge: The MIT Press, pp. 341-380.

Chiaia, N.L., S.E. Fish, W.R. Bauer, C.A. Bennett-Clarke, and R.W. Rhoades (1992) Postnatal blockade of cortical activity by tetrodotoxin does not disrupt the formation of vibrissa-related patterns in the rat’s somatosensory cortex. Dev. Brain Res. 66.244-250,

Chin, L.S., A. Ferreira, K.S. Kosik, and P. Greengard (1995) Impairment of axonal development and of synaptogenesis in hippocampal neurons of synapsin I-deficient mice. Proc. Natl. Acad. Sci. U S A . 929230-9234.

SV PROTEINS IN DEVELOPING CORTEX

Chun, J.M., and C.J. Shatz (1988) Redistribution of synapticvesicle antigens is correlated with the disappearance of a transient synaptic zone in the developing cerebral cortex. Neuron 1:297-310.

D’Amato, R.J., M.E. Blue, B.L. Largent, D.R. Lynch, D.J. Ledbetter, M.E. Molliver, and S.H. Snyder (1987) Ontogeny of the serotonergic projection to rat neocortex: transient expression of a dense innervation to primary sensory areas. Proc. Natl. Acad. Sci. U.S.A. 84:43224326.

Devoto, S.H., and C.J. Barnstable (1989) Expression of the growth cone specific epitope CDAl and the synaptic vesicle protein SVP38 in the developing mammalian cerebral cortex. J. Comp. Neurol. 290:154-168.

Edelmann, L., P.I. Hanson, E.R. Chapman, and R. Jahn (1995) Synaptobre- vin binding to synaptophysin: a potential mechanism for controlling the exocytotic fusion machine. EMBO J. 14t224-231.

Erzurumlu, R.S., S. Jhaveri, and L.I. Benowitz (1990) Transient patterns of GAP-43 expression during the formation of barrels in the rat somatosensory cortex. J. Comp. Neurol. 292443-456,

Feany, M.B., S. Lee, R.H. Edwards, and K.M. Buckley (1992) The synaptic vesicle protein SV2 is a novel type of transmembrane transporter. Cell 70:861-867.

Fischer von Mollard, G., T.C. Siidhof, and R. Jahn (1991) A small GTP- binding protein dissociates from synaptic vesicles during exocytosis. Nature 349:79-81.

Fletcher, T.L., P. Cameron, P. De Camilli, and G. Banker (1991) The distribution of synapsin 1 and synaptophysin in hippocampal neurons developing in culture. J. Neurosci. 11:1617-1626.

Fletcher, T.L., P. De Camilli, and G. Banker (1994) Synaptogenesis in hippocampal cultures: evidences indicating that axons and dendrites become competent to form synapses at different stages of neuronal development. J. Neurosci. 11:6695-6706.

Fox, K., and K. Zabs (1994) Critical period in sensory cortex. Curr. Opin. Neurobiol. 4: 112-119.

Geppert, M., V.Y. Bolshakov, S.A. Siegelbaum, K. Takei, P. De Camilli, R.E. Hammer, and T.C. Siidhof (1994) The role ofRab3A in neurotransmitter release. Nature 369:493-497.

Holz, R.W., W.H. Brondyk, R.A. Senter, L. Kuizon, and I.G. Macara (1994) Evidence for the involvement of Rab3A in Ca”-dependent exocytosis from adrenal chromaffin cells. J. Biol. Chem. 269:10229-10234.

Jensen, K.F., and H.P. Killackey (1987) Terminal arbors of axons projecting to the somatosensory cortex of the adult rat. I. The normal morphology of specific thalamocortical afferents. J. Neurosci. 7:3529-3543.

Jhaveri, S., and R. Erzurumlu (1992) Two phases ofpattern formation in the developing rodent trigeminal system. In S.C. Sharma and A.M. Goffinet (eds): Development of the Central Nervous System in Vertebrates. New York Plenum Press, pp. 167-178.

Johannes, L., P.-M. Lledo, M. Roa, J.-D. Vincent, J.-P. Henry, and F. Darchen (1994) The GTPase Rab3a negatively controls calcium- dependent exocytosis in neuroendocrine cells. EMBO J. 13.2029-2037.

Jones, D.G., and E. Revel1 (1970) The postnatal development of the synapse: a morphological approach utilizing synaptosomes I. General features. Z. Zellforsch. 1 11: 179-194.

Killackey, H.P., G. Belford, R. Ryugo, and D.K. Ryugo (1976) Anomalous organization of thalamocortical projections consequent to vibrissae removal in the newborn rat and mouse. Brain. Res. 104:309-315.

Killackey, H.P., N.L. Chiaia, C.A. Bennett-Clarke, M. Eck, and R.W. Rhoades (1994) Peripheral influences on the size and organization of somatotopic representations in the fetal rat cortex. J. Neurosci. 3:1496- 1506.

Konig, N., G. Roch, and R. Marty (1975) The onset ofsynaptogenesis in rat temporal cortex. Anat. Embryol. (Berl.) 148:73-87.

Koralek, K.A., K.F. Jensen, and H.P. Killackey (1988) Evidence for two complementary patterns of thalamic input to the rat somatosensory cortex. Brain Res. 463t346-351.

Kossut, M. (1992) Plasticity of the barrel cortex neurons. Prog. Neurohiol. 39:389-422.

Kristt, D.A., and M.E. Molliver (1976) Synapses in newborn rat cerebral cortex: A quantitative ultrastructural study. Brain Res. 108: 180-186.

Li, I., L . 3 . Chin, 0. Shupliakov, L. Brodin, T.S. Sihra, 0. Hvalby, V. Jensen, D. Zheng, J.O. McNamara, P. Greengard, and P. Andersen (1995) Impairment of synaptic vesicle clustering and of synaptic transmission, and increased seizure propensity in synapsin I-defficient mice. Proc. Natl. Acad. Sci. U.S.A. 92t9235-9239.

Matteoli, M., K. Takei, R. Cameron, P. Hurlbut, P.A. Johnston, T.C. Siidhof, R. Jahn, and P. De Camilli (1991) Association of Rab3A with synaptic vesicles a t late stages ofthe secretorypathway. J. Cell Biol. 115:625-633.

331

Matteoli, M., K. Takei, M.S. Perin, T.C. Sudhof, and P. De Camilli (1992) Exo-endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons. J. Cell Biol. 11 72349-861.

McCandlish, C., R.S. Waters, and N.G.F. Cooper (1989) Early development of the representations of the body surface in SI cortex barrel field in neonatal rats as demonstrated with peanut agglutinin binding: evidence for differential development within the rattunculus. Exp. Brain Res. 77:425-431.

Motoike, T., K. Sano, S. Tsuneishi, H. Nakamura, Y. Kushima, H. Hatanaka, and Y. Takai (1991) Expression and localization ofsmg p25A (=rab3A) in cultured rat hippocampal cells. Neurosci. Lett. 134:109-112.

Moya, K.L., B. Tavitian, A. Zahraoui, and A. Tavitian (1992) Localization of the rus-like rah3A protein in the adult rat brain. Brain Res. 590:118- 127.

Mundigl, O., M. Matteoli, L. Daniell, A. Thomas-Reetz, A. Metcalf, R. Jahn, and P. De Camilli (1993) Synaptic vesicle proteins and early endosomes in cultured hippocampal neurons: differential effects of brefeldin A in axon and dendrites. J. Cell Biol. 122:1207-1221.

Nothias, F., M. Peschanski, and J.M. Besson (1988) Somatotopic reciprocal connections between the somatosensory cortex and the tbalamic Po nucleus in the rat. Brain Res. 447:169-174.

Okada, M., A. Erickson, and A. Hendrickson (1994) Light and electron- microscopic analysis of synaptic development in macaca monkey retina as detected in immunocytochemical labeling for the synaptic vesicle protein SV2. J. Comp. Neurol. 339535-558.

Olavarria, J., R.C. Van Sluyters, and H.P. Killackey (1984) Evidence for the complementary organization of callosal and thalamic connections within the rat somatosensory cortex. Brain Res. 291:364-368.

Ovtscharoff, W., M. Bergmann, B. Marqueze-Pouey, P. Knaus, H. Betz, D. Grabs, I. Reisert, and M. Gratzl (1993) Ontogeny of synaptophysin and synaptoporin in the central nervous system: differential expression in striatal neurons and their afferents during development. Dev. Brain Res. 72:219-225.

Petrucci, T.C., P. Maccioci, and P. Paggi (1991) Axonal transport kinetics and posttranslational modification of synapsin I in mouse retinal ganglion cells. J. Neurosci. 11.2938-2946.

Phelan, P., and P.R. Gordon-Weeks (1992) Widespread distribution of synaptophysin, a synaptic vesicle glycoprotein, in growing neurites and growth cones. Eur. J. Neurosci. 4:1180-1190.

Pieribone, V.A., 0. Shupliakov, L. Brodin, S. Hilfiker-Rothenfluh, A.J. Czernik, and P. Greengard (1995) Distinct pools of synaptic vesicles in neurotransmitter release. Nature 375493497.

Purves, D., D.R. Riddle, andA.3. LaMantia (1992) Iterated patterns ofbrain circuitry (or how the cortex got its spots). Trends Neurosci. 15:362-368.

Rhoades, R.W., C.A. Bennett-Clark, N.L. Chiaia, F.A. White, G.J. Mac- Donald, J.H. Haring, and M.F. Jacquin (1990) Development and lesion- induced reorganization of the cortical representation of the rat’s body surface as revealed by immunocytochemistry for serotonin. J. Comp. Neurol. 293:190-207.

Rice, R.L., C. Gomez, C. Barstow, A. Burnet, and P. Sands (1985) A comparative analysis of the development of the primary somatosensory cortex: interspecies similarities during barrel and laminar development. J. Comp. Neurol. 236:477-495.

Riddle, D.R., G. Gutierrez, D. Zheng, L.E. White, A. Richards, and D. Purves (1993) Differential metabolic and electrical activity in the somatic sensory cortex of juvenile and adult rats. J. Neurosci. 10:41934213.

Rosahl, T.W., M. Geppert, D. Spillane, J. Herz, R.E. Hammer, R.C. Malenka, and T.C. Siidhof (1993) Short-term synaptic plasticity is altered in mice lacking synapsin I. Cell 75:661-670.

Rnsahl, T.W., D. Spillane, M. Missler, J. Herz, D.K. Selig, J.R. Wolff, R.E. Hammer, R.C. Malenka, and T.C. Siidhof (1995) Essential functions of synapsin I and I1 in synaptic vesicle regulation. Nature 375:488-493.

Scarfone, E., D. Demgmes, and A. Sans (1991) Synapsin I and synaptophysin expression during ontogenesis of the mouse peripheral vestibular system. J. Neurosci. 11:1173-1181.

Schlaggar, B.L., K. Fox, and D.D.M. O’Leary (1993) Postsynaptic control of plasticity in developing somatosensory cortex. Nature 364~623-626.

Shatz, C. (1990) Impulse activity and the patterning of connections during CNS development. Neuron 517455756.

Stettler, O., K.L. Moya, A. Zahraoui, and B. Tavitian (1994) Developmental changes in the localisation of the synaptic vesicle protein rab3A in rat brain. Neuroscience 62587-600.

332

Strominger, R.N., and T.A. Woolsey (1987) Templates for locating the whisker area in fresh flattened mouse and rat cortex. J. Neurosci. Methods 22.1 13-118.

Siidhof, T.C. (1995) The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature 375645-653.

Van der Loos, H., and T.A. Woolsey (1973) Somatosensory cortex: structural alterations following early injury to sense organs. Science I79:395-398.

Vilagi, I., I. Tarnawa, and I . Banczerowski-Pelyhe (1992) Development of long-term potentiation in the somatosensory cortex of rats of different ages. Neurosci. Lett. 141t262-264.

Voigt, T., A. De Lima, and M. Beckmann (1993) Synaptophysin immunohistochemistry reveals inside-out pattern of early syuaptogenesis in ferret cerebral cortex. J. Comp. Neurol. 330t48-64.

0. STETTLER ET AL.

Welker, C., and T.A. Woolsey (1974) Structure of layer IV in the somatosensory neocortex of the rat: description and comparison with the mouse. J. Comp. Neurol. 158:437454.

Wise, S.P., and E.G. Jones (1976) The organization and postnatal development of the commissural projection of the rat somatic sensory cortex. J. Comp. Neurol. 168t313-344.

Woolsey, T.A., and H. Van der Loos (1970) The structural organization of layer IV in the somatosensory region (SI) of mouse cerebral cortex. Brain Res. 17:205-242.

Zerial, M., and H. Stenmark (1993) Rab GTPases in vesicular transport. Curr. Opin. Cell Biol. 5t613-620.

Zheng, D., and D. Purves (1995) Effects of increased neural activity on brain growth. Proc. Natl. Acad. Sci. U S A . 92:1802-1806.

differential synaptic vesicle protein expression in the barrel field of developing cortex

Documents