Protein-Synthetic Machinery at Postsynaptic Sites During Synaptogenesis: a Quantitative Study of the Association Between Polyribosomes and Developing Synapses

The Journal of Neuroscience February 1986, 6(2): 412423

Protein-Synthetic Machinery at Postsynaptic Sites During Synaptogenesis: A Quantitative Study of the Association Between Polyribosomes and Developing Synapses

Oswald Steward and Paula M. Falk DeDartments of Neurosuraerv and Phvsioloav. Universitv of Virginia School of Medicine, Ch’arlottesville, Virginia 22306

Previous studies have revealed dramatic accumulations of poly- apses. We propose that these elements produce protein(s) that ribosomes under growing spine synapses, suggesting a critical are critically involved in the formation of the synaptic contact. role for protein synthesis at the postsynaptic site during synaptogenesis. The present study quantitatively analyzes the dis- Despite the obvious importance of synaptogenesis in neural tribution of polyribosomes under synapses during develop- development, relatively little is known about exactly what cel- mental synaptogenesis in the rat’s dentate gyrus. The middle lular and molecular processes are involved in the making of a molecular layer of the suprapyramidal blade of the dentate gy- synapse. Synapse formation clearly requires the elaboration of rus was examined electron-microscopically at 1,4,7,10,15,20, several types of structural specializations, including axons, den- and 28 d of age. At each age, we evaluated (1) synapse density drites, pre- and postsynaptic contact regions, and, in many neu- (the number of synapses/100 pm2 of neuropil), (2) the width of rons, dendritic spines; however, most of our knowledge about the molecular layer, (3) the proportion of spine synapses with the cellular mechanisms of neuron growth pertains to growing underlying polyribosomes, and (4) the number of polyribosome- axons. An area of particular interest about which relatively little containing synapses/1000 pm* of neuropil. From the first two is known involves the construction of the portion of the post- measures, an estimate was obtained of the total extent of syn- synaptic cell that is specialized for the receipt of the synapse. aptogenesis, taking into account both the increase in synapse Neurons of the CNS receive most of their synaptic contacts density and the increase in total area of neuropil. upon postsynaptic membrane specializations (the postsynaptic At 1 d of age, very few synapses were found in the molecular density). These membrane specializations are thought to rep- layer of the dentate gyrus, and those that were present were resent the active zone of the synapse, and presumably contain quite immature in appearance. Synapse density increased about a variety of molecules involved in synaptic function, including 140-fold between 1 and 28 d of age, from an average of 0.36 neurotransmitter receptors, ion channels, molecules to immo- synapses1100 pm* at 1 d of age to 49 synapses/100 pm* at 28 d bilize the membrane proteins to keep them localized at the of age. An inverse relationship was found between synapse den- synaptic site, regulatory enzymes such as kinases and adenylate sity and the proportion of synapses with polyribosomes. Be- cyclase, molecules to define the chemical identity (specificity) tween 1 and 7 d of age, about 60% of the spine synapses had of the contact, and adhesion molecules (for studies of the com- one or more polyribosomes under the spine base. Thereafter, position of the postsynaptic density, see Carlin et al., 198 1; the proportion of spines with polyribosomes decreased as syn- Cohen et al., 1977; Feit et al., 1977; Grab et al., 1981a, b; Kelly apse density increased. Similarly, the proportion of shaft syn- and Cotman, 1978). Furthermore, many neurons in the verte- apses with underlying polyribosomes was greatest between 1 brate CNS elaborate microspecializations of dendritic form at ahd 7 d postnatal, and decreased thereafter. While the propor- each site of synaptic contact (dendritic spines). tion of synapses with polyribosomes was greatest between 1 and The remarkable feature of many CNS neurons is that they 7 d, the actual number of polyribosome-containing synapses/ elaborate a multitude of specializations for synaptic contact all 1000 I.cmZof neuropil was negligible at 1 d, increased to a peak along their dendritic processes, sometimes with different types at 7 d of age, and then decreased as synapse density increased. of contacts on different portions of the dendritic tree. For ex- Qualitatively, the most dramatic accumulations of polyribo- ample, Purkinje cells of the cerebellum construct tens of thou- somes were also found at 7 d of age. sands of contact sites along their dendrites, some specialized for We conclude that spine-associated polyribosomes represent a the receipt of parallel fibers and others for the receipt of climbing structural specialization of dendrites at sites of synapse con- fibers. The situation for pyramidal cells of the cerebral cortex struction and as such may represent a marker for growing syn- or the hippocampus is even more complex, since these cells may receive many different afferent types, each terminating on a particular portion of the dendritic tree. In these cases, not only Received Apr. 11, 1985; revised Aug. 23, 1985; accepted Aug. 26, 1985. must the neuron construct thousands of synaptic sites; it must Thanks to S. L. Vinsant and F. L. Snavely for their excellent technical help in somehow organize its receptive surface so as to accommodate the early stages of the electron microscopic analysis. Thanks also to R. A. Ogle for help with the illustrations, and to M. P. Janssen for secretarial help. This work different types of synapses (presumably with different require- was supported by National Institutes of Health Grant NS 12333 to O.S. OS. was ments for receptors, etc.) at different locations. How such mi- the recipient of a Research Career Development Award (NS 00325) during the crospecializations are constructed remains largely a mystery, time that much of this work was undertaken. A preliminary account of some of although until recently it was generally assumed that the ma- these results has been published (Steward and Falk, 1985). Correspondence should be addressed to Oswald Steward, Departments of Neu- terials for the contact site were synthesized in the neuronal cell rosurgery and Physiology, University of Virginia School of Medicine, Charlottes- body and delivered to the synaptic site by some sort of targeted ville, VA 22908. transport system. Copyright 0 1986 Society for Neuroscience 0270-6474/86/020412-12$02.00/O Our recent discovery of protein-synthetic machinery (poly-

412 The Journal of Neuroscience Polyribosomes Under Developing Synapses 413 ribosomes) at the base of dendritic spines of CNS neurons (see then sectioned on an Oxford Vibratome. Sample blocks of the dentate Steward, 1983b; Steward and Levy, 1982) has provided a new gyms from approximately one-quarter the way along the septotemporal way of thinking about how synapses might be constructed and axis ofthe dentate gyrus were dissected freehand, osmicated, and embed- maintained. Specifically, regulation of postsynaptic contact re- ded in plastic using routine procedures (Steward and Vinsant, 1983). gions could involve a local synthesis of some protein(s) at the Thin-sections were cut perpendicular to the layer of granule cell bodies in order to obtain sections that extended from the cell bodylayer to the synaptic site, rather than depending entirely on transport of zone containing the tips ofthe granule cell dendrites (the outer molecular material synthesized in the cell body. In this regard, it is of layer). Both suprapyramidal and infrapyramidal blades of the dentate particular interest that the incidence of polyribosomes under gyrus were evaluated qualitatively, and representative photomicro- spines increases dramatically during synapse growth induced, graphs were taken at 10,000 x . in mature animals, by denervating lesions (Steward, 1983a). For the quantitative analyses, a sample of 42-45 photographs were These data led us to propose that local synthesis at the post- taken in the middle molecular layer of the suprapyramidal blade of the synaptic site might be particularly important during synapse dentate gyrus in each animal (initial magnification, 10,000 x ; final mag- growth (Steward, 1983a, b; Steward and Fass, 1983). nification, 27,000 x). For all animals, except those prepared at 1 and 4 While the studies cited above clearly suggested a role for the d of age, the sampling procedure was similar to that described in Steward and Levy (1982). The middle molecular layer was scanned for spines polyribosomes in lesion-induced reinnervation, the precise na- that were visible in their entirety (from their point of emergence on the ture of their role remained unclear. Synapse replacement fol- parent dendrite to the region of synaptic contact), and photomicro- lowing lesions involves processes that may not be important graphs, centered on such identified spines, were taken. Each photograph during normal development, such as those involved in the deg- represented a cross-sectional area of about 55-60 wm2, providing an radation of old sites of contact left vacant by the removal of average sample area of 2321 pm2 of neuropil in each animal (range, narmal innervation. It was therefore of interest to evaluate 2 103-25 14 pm2). For the animals prepared at 1 and 4 d of age, synapses whether polyribosomes were also a prominent feature of growing were rare, and those that were present were often found in clusters; thus synapses in the developmental period. Furthermore, it was of for these animals, rows of photographs were taken from the middle third interest to determine when, in the synaptogenic period, the po- of the molecular layer in order to sample the same total area of neuropil as above. lyribosomes were most prevalent. Particularly interesting was From these collections of photomicrographs, we evaluated the num- the question of whether polytibosomes were most prominent at ber of spine and shaft synapses. Synapses were defined as appositions the time of initial contact formation, or during some later phase between a presynaptic process with at least three synaptic vesicles and of the differentiation of the contact zone. In the present study, a postsynaptic element with a definable postsynaptic specialization (see the relationship between polyribosomes and developing syn- Steward and Vinsant, 1983). In the molecular layer of mature rats, the apses is evaluated in the dentate gyrus of the rat, using quan- vast majority of synapses are on well-developed spines (Steward and titative electron-microscopic techniques. Vinsant, 1983). In the very young animals, however, very few well- The dentate gyrus offers several advantages for studies of developed spines were observed. Rather, synapses tended to be found developmental synaptogenesis, owing to its synaptology and on very stubby protrusions from the dendrite. It seems likely that these relatively late development. The dendrites of the granule cells stubby protrusions are the precursors of spines, since they represent the predominant form in the young animals, and in mature animals most and their associated afferents are found in a neuropil layer (the synapses are on spines. In addition, these stubby protrusions usually molecular layer), where each of the major afferents terminates contain flocculent material resembling that found in mature spines. in a discrete lamina. Throughout the neuropil, most of the syn- Thus, any synapse that was associated with even a slight irregularity of aptic connections terminate on dendritic spines with character- the smooth profile of the dendritic shaft and contained such flocculent istic mushroom or lollipop shape in mature animals (Steward material was scored as a spine synapse. Synapses not associated with and Vinsant, 1983). Most of the differentiation of granule cells irregularities of the dendritic contour were scored as shaft synapses. occurs postnatally. Approximately 90% of the granule cells After determining the number of synapses in each collection of pho- undergo their final mitosis after birth (Bayer, 1980; Schlessinger tomicrographs, the total cross-sectional area covered by the photomi- et al., 1975). From birth until about 20 d of age, granule cells crographs was determined. From these values, the number of spine and shaft synapses/100 pm2 was determined for each animal. We also mea- elaborate their dendritic processes, and come to receive afferent sured the width of the molecular layer of the dentate gyrus (from the innervation (Fricke and Cowan, 1977; Loy et al., 1977; for a top of the granule cell layer to the hippocampal fissure) in order to review, see Cowan et al., 1980). Thus, from birth until about provide an estimate of the increase in the area of neuropil. These values, 20 d of age, the dentate gyrus is a site of formation of an enor- together with the estimates of synapse density, could then provide an mous number of synaptic connections. Early in this period, estimate of the total extent of synaptogenesis in the molecular layer virtually any synapse observed must be either in the process of across the developmental period. formation, or newly formed. This is an optimal setting for study- For the analysis of the proportion of spine synapses with underlying ing cellular specializations associated with growing synapses. polyribosomes, we determined the relative incidence of polyribosomes We report that developing synapses have even more dramatic under “identified spine bases.” Following conventions that we had es- tablished previously (Steward, 1983a; Steward and Levy, 1982), “iden- accumulations of polytibosomes than synapses growing in re- tified spine bases” refer to intersections between a main dendrite and sponse to lesions; the number of polyribosomes under synapses a spine neck. Spine necks were identified as such on the basis of (1) decreases with synapse maturation. A preliminary account of being continuous with a spine head that received a presynaptic contact some of these results has been published (Steward and Falk, upon an area of membrane specialization (the entire profile of the spine 1985). must be visible in the section) or (2) containing a well-defined spine apparatus. At all ages-but particularly in the young animals-most of Materials and Methods the spines were included in the sample on the basis of criterion (1). A Sprague-Dawley-derived rats of both sexes were prepared for EM at 1, spine was considered positive for polyribosomes if the clusters were 4, 7, 10, 15, 20, and 28 d of age (two animals at each interval, except found under the base of the spine. However, if microtubules or other for 1,4, and 15 d, when three animals were analyzed). The day of birth organelles were found between the polyribosomes and the spine base, is considered day 0. The animals were deeply anesthetized with sodium the polyribosomes were considered to lie within the dendrite proper, pentobarbital and perfused transcardially with mixed aldehyde fixatives and the spine was scored as polyribosome-free. warmed to 30-37°C. Most of the animals were perfused with the same Polyribosomes were defined as collections of three or more particles fixative that we routinely use with mature animals (2% paraformalde- of the size and electron density of ribosomes, with an interparticle hyde/2% glutaraldehyde in 0.13 M cacodylate buffer, pH 7.2), although spacing typical of polyribosomes. It is important to note that this is a at both 7 and 10 d of age, one animal was perfused with 1% paraformal- morphological identification, which is not unequivocal. Nevertheless, dehyde/l.5% glutaraldehyde in 0.13 M cacodylate buffer. The brains because of their uniform size, the particles were clearly distinguishable were removed, and postfixed in the perfusion solution for about 1 hr, from other similar electron-dense granules, including glycogen; if they 414 Steward and Falk Vol. 6, No. 2, Feb. 1986 are not polyribosomes, they are an unidentified cytoplasmic component. ribosomeswere not as dramatic as at later ages(see below). Since previous studies have shown that the particles are sensitive to There was a tendency for the synapsesto occur in clusters- ribonuclease treatment (Steward, 1983b), their identification as poly- some photographic fields had several synapses,while the vast ribosomes seems reasonably secure. majority had none. We also counted the number of polyribosome-containing spine heads that were not connected with a dendritic shaft in the plane of section, In addition to the synapses,there were unapposedpostsyn- and the number of shaft synapses with underlying polyribosomes. From aptic densities,some of which had underlying accumulations of these,we calculatedthe numberof polyribosome-positivesynapses/ polyribosomes(see Fig. 1, A and D). In addition, specializations 1000 pm* of neuropil. The valuesobtained for synapsedensity, the resemblingpostsynaptic densities were sometimesfound at sites relative incidenceof polyribosomesunder spines,and the numberof of contact between two dendritic processes(identified by the polyribosome-containingsynapses/ 1000 pm* of neuropilwere com- presenceof polyribosomes).These dendrodendritic contactswere paredwith valuesfor adult animalsobtained from materialprepared also present in 4 d old animals (see Fig. 20), but were not for other studies(Steward, 1983a; Steward and Levy, 1982;Steward apparent in animals more than 4 d of age. Some caution must and Vinsant, 1983). be exercisedin identifying all polyribosome-containing profiles as dendrites, since one profile was observed which contained Results vesiclesof the size of synaptic vesicles, yet polyribosomeswere present on the presynaptic side. It could not be determined Qualitative observations whether this unique profile was a dendrodendritic chemical syn- Our qualitative evaluations focused on the question of when, apseor an axodendritic synapse,where the presynaptic member in the processof synapse formation, the polyribosomes were contained polyribosomes. most evident. Some of the issueswere whether polyribosomes At 4 d of age, the infrapyramidal blade of the dentate gyrus were present at future synaptic sitesprior to the differentiation still did not exhibit a definable molecular layer. The molecular of morphologically identifiable synaptic junctions, and whether layer of the suprapyramidal blade contained dendrites (identi- the polyribosomes were most prominent under the youngest- fied by the presenceof ribosomes),microtubule-containing pro- appearing synapses.Thus, the distribution of polyribosomes cessesthat were probably axons, and presumedgrowth cones under noninnervated synaptic specializations and under syn- (seeFig. 2). At this age, there were fewer of the vesicle-filled aptic specializations apposedto apparent growth cones was of moundsthan at 1 d of age,although the presumedgrowth cones particular interest. Also of interest was whether the number of filled with filamentous material, and the processesfilled with polyribosomes increasedduring the maturation of the contact endoplasmicreticulum were still quite common (seeFig. 2A). region. The number of synapseswas obviously considerably higher than At 1 d of age, the gradient of differentiation in the dentate at 1 d of age, although the distribution was still patchy; again, gyrus was particularly apparent. In the suprapyramidal blade, several synapseswere often found in a given photographic field, there was already a discernible cell layer and thin molecular while other fields contained none. The synapsesthat were pres- layer. In contrast, the infrapyramidal blade was much lessma- ent at 4 d of age were more often on profiles that clearly pro- ture, and was characterized by a loosely organized collection of truded from the dendritic shaft (seeFig. 2); the identification of cells, with no evidence of a molecular layer. Within the molec- such profiles as spineswas thus lessambiguous than was the ular layer of the suprapyramidal blade, relatively few synaptic caseat 1 d of age. A few profiles could be found in which the contacts were found. Rather, the layer contained numerousmi- spine had a shapesimilar to that in adult animals, with a thin crotubule-containing processes,and presumedgrowth cones(see neck and enlargedhead (Fig. 2C). Fig. 1). Someof the microtubule-containingprocesses were clearly While synapseswere more numerousthan at 1 d of age, most dendrites, since they could be seento emergedirectly from the still appearedquite immature, in that the presynaptic element neuronal somata of the granule cell layer. These contained ac- often had few vesicles, and the vesiclesthat were present were cumulations of polyribosomes. Other processeswere lesseasy often not accumulated near the contact region (Fig. 2E). Some to identify, since they contained neither polyribosomesnor syn- apparent spineswere also apposedto the enlargedfilamentous aptic vesicles. Many of thesemay be axons. In addition to the profiles thought to represent growth cones (Fig. 2A). Vesicles microtubule-containing profiles, there were numerousprocesses larger than typical synaptic vesicles were also common (Fig. filled with a filamentous material with few other intracellular 2E). In addition, the postsynaptic membrane specializationwas organelles(Fig. 1A). These appearedto be identical to the types often quite thin. All of thesefeatures are characteristic of grow- of growth cones that have been identified in a number of brain ing synapsesin other brain regions (Bloom, 1972; Foelix and regions (Hinds and Hinds, 1976; Skoff and Hamburger, 1974; Oppenheim, 1974; Hinds and Hinds, 1976; Vaughn and Sims, Tennyson, 1970; Vaughn and Sims, 1978). Most of thesecould 1978). Although a few profiles could be found that resembled not be identified as axonal or dendritic, except when they could poorly developed spines,without identifiable presynaptic con- be seen in continuity with ribosome-containing processesor tacts, these were not common. Unapposed postsynaptic den- when they emerged from processeswith identifiable synaptic sitieswere also found, but thesewere lesscommon than at 1 d relations with other elements.In addition, some processesex- of age. The accumulations of polyribosomes under synapses hibited bulbous enlargementswith endoplasmic reticulum (see tended to be more dramatic than at 1 d of age, in that more Fig. 1, A and B). These processesresembled the growth cones rosetteswere present per synapse(see Fig. 2, A, B, F, and G); described by Del Cerro and Snider (1968) and Kawana et al. nevertheless,the accumulationswere still not as dramatic as at (197 1). There were also numerousvesicle-filled mounds,similar 7 d of age. While more polyribosomeswere also present in the to those frequently found on growth conesin culture (Pfenninger dendrite proper than with mature animals, the majority of the and Rees, 1976). dendritic polyribosomesappeared to lie under spinesor mounds The few synapsespresent at 1 d of age were typically found in the dendrite, resembling the basesof spines (seeFig. 2B). on stubby protrusions from the dendrites(see Fig. 1C). Because Thus, as is also true in mature animals (Steward and Levy, there are few shaft synapsesin the mature dentate gyrus, and 1982), the localization of polyribosomesunder spinesappeared becausemost of these stubby protrusions contained flocculent to be selective. No quantitative evaluation of the degreeof this material similar to that found in mature spines,we have iden- selectivity was undertaken, however. tified these as very immature spine synapsesfor quantitative At 7 d of age,both supra- and infrapyramidal bladespossessed purposes. Many of thesepresumed spines were marked by ac- a definable molecularlayer. The infrapyramidal bladewas char- cumulations of polyribosomes,although the collections of poly- acterized by considerableextracellular space,with scatteredax- The Journal of Neuroscience Polyribosomes Under Developing Synapses

Figure 1. The molecular layer of the suprapyramidal blade of the dentate gyrus of 1 d old rats. A, Two types of profiles previously identified as growth cones (gc) (i.e., enlarged profiles with vesicles resembling endoplasmic reticulum and filamentous enlargements with few other cytoplasmic specializations). Slanted arrow indicates polyribosomes under membranous densities resembling postsynaptic densities. Straight arrow indicates presumed empty psd’s. B, Two vesicle-filled growth cones (gc) with polyribosomes near but not within the growth cone proper. Dendrite (den), identified by presence of polyribosomes. Slanted arrow, Polyribosomes under what may be an immature synaptic contact (the presynaptic terminal is indicated by t.9. C, One of the rare mature-appearing synapses at 1 d of age. D, Two apparently noninnervated psd’s with underlying polyribosomes. Calibration bar, 1 Wm. 416 Steward and Falk Vol. 6, No. 2, Feb. 1986

Figure 2. The molecular layer of the suprapyramidal blade of the dentate gyms of 4 d old rats. A, An apparent spine in contact with a growth cone (gc). B, The very stubby spines characteristic of this age. C, An example of one of the more rare elongated spines. The arrow with asterisk in D indicates a dendrodendritic contact. E-G, Examples of spine synapses with polyribosomes. s = spine; other symbols and abbreviations as for Figure 1. Calibration bar, 1 pm.

onal and dendritic processes,some of which had the character- icles, and often containing larger vesicles. istics of growth cones(Fig. 3, A and B). Very few synapseswere The suprapyramidal blade appearedmore mature than at 4 present,however, and thosethat were appearedquite immature, d of age, with a larger number of synapses,and an increasein again containing few vesiclesof the size of typical synaptic ves- the apparent degreeof differentiation of the synaptic contact The Journal of Neuroscience Polyribosomes Under Developing Synapses

Figure 3. Examples of developing spine synapses from the molecular layer ofthe dentate gyms at 7 d of age.A and B were taken in the infrapyramidal blade, while C-E were taken in the suprapyramidal blade. Note the accumulations of polyribosomesunder and within the spines,and the fact that the polyribosomes seem to be preferentially localized under the spines (see particularly A). Portions of the dendritic segments illustrated in A and B resemble growth cones, with large vesicles of smooth endoplasmic reticulum. Abbreviationsand symbolsas in Figure 1. Calibration bar, 1 pm. region (Fig. 3, C-F). For example, many synaptic contactswere with narrow necks and enlargedheads (see Fig. 3C for an ex- associatedwith profiles that were unambiguousspines, although ample). most spines still appeared more stubby and lesswell-defined As is evident in Figure 3, C-E, accumulations of polyribo- than in more mature animals. Only a few spines were found somesunder the site of synaptic contact were particularly strik- 418 Steward and Falk Vol. 6, No. 2, Feb. 1986

Figure 4. Developingspine synapses from the molecularlayer of the dentategyms at 10(A andB), and 15(C-E) d of age.Note that the neuropil appearssomewhat more mature,both in termsof numbersof synapsesand in termsof the shapeof the spines.Symbols and abbreviations asin Figure 1. Calibrationbar, 1 pm.

ing in the 7 d old animals. Most of the polyribosomesappeared one cluster (seebelow for quantitative considerations).The se- to lie within the stubby protrusion of the spine, out of the cyl- lectivity of the localization of the polyribosomes under spines inder of the dendrite. The polyribosome clusterswere both dra- was still quite apparent (Fig. 34. matic, with many well-defined rosettesunder individual spines, The general picture of the molecular layer at 10 d of agewas and ubiquitous, in that most spinesappeared to contain at least similar to that at 7 d, except that synapsesappeared to be some- The Journal of Neuroscience Polyribosomes Under Developing Synapses what more mature (Fig. 4, A and B), and even qualitatively it was clear that there were more of them. However, most spines were still quite stubby, and very few had the thin necks and large heads typical of mature spines. Polyribosomes remained prominent, but spines with multiple clusters of ribosomes were encountered less frequently. By 15 d of age, the synapses appeared even more mature (Fig. 4, C-E). Some spines had well-defined narrow necks and enlarged heads-the lollipop or mushroom shape characteristic of mature spines. The lollipop shape did not appear to be as well developed as in mature animals, however, in that many spines had necks almost as wide as the heads. Furthermore, most spines appeared to be shorter than those of mature animals. The clusters of polyribosomes under spines were less dramatic than at 7 and 10 d of age. Few profiles had the sorts of multiple rosettes that were characteristic of the younger ages, although, qualita- Days Post-Natal tively, it was clear that the proportion of spines with polyribosomes was still higher than in mature animals. In addition, polyribosomes were common in the spine head, which is rarely seen in mature animals (Steward, 1983a). The trends toward synapse maturation continued between 15 and 20 d of age. The spines in the neuropil of 20 d old animals appeared somewhat longer and more mature (in terms of the development of adult-like shape), although the neuropil was still easily distinguishable from the adult. By 28 d of age, however, the neuropil appeared qualitatively mature.

1 4 7 10 15 20 28 ‘+A Quantitative assessment of synaptogenesis Days Post-Natal Our quantitative evaluations focused on the suprapyramidal blade. As illustrated in Figure 5A, synapse density in the suprapyramidal blade was quite low at 1 d of age; indeed, an average of only 0.36 synapses were present per 100 pm* of neuropil. Synapse density increased in an almost linear fashion between 1 and 15 d of age, and continued to increase at a rate that was only slightly slower between 15 and 28 d of age. By 28 d of age, the average synapse density in the middle molecular layer of the suprapyramidal blade of the dentate gyrus was 49 synapses per 100 pm2. This represents a 136-fold increase in synapse density between postnatal days 1 and 28. Interestingly, synapse density was slightly higher at 28 d of age than in adult animals (see Fig. 5). The number of shaft synapses was quite low throughout the postnatal period (see Fig. 5A, broken line). From the increase in synapse density across the postnatal Days Post-Natal period, the rate of synapse addition during each interval was calculated by determining the difference in synapse density be- Figure 5. Synaptogenesisin the suprapyramidalblade of the dentate tween each age (how many new synapses were added) and di- gyrus.A, Synapsedensity per unit area.Total spinesynapses are indi- catedby the solid lineand shaft synapses by the brokenline. B, Thickness viding this number by the number-of days in the interval. Thus, of the molecular layer of the suprapyramidal blade at the site from between 1 and 4 d of age, synapse density increased from 0.36 which counts of synapses were made. C, Estimated number of synapses to 7.4 synapses/100 pm2 (7.1 synapses were,added per 100 pm* per IO-pm-wide strip of the molecular layer (in order to estimate the in 3 d). The rate of synapse addition was thus 2.4 synapses/100 overallextent of synaptogenesis,taking into accountboth the increases wm2/d. Similar calculations revealed rates of synapse addition in synapse density per unit area and the increases in the volume of the of 2.2 synapses/ 100 PrnVd between 4 and 7 d, 2.0 synapses/ 100 neuropil). Vertical bars, The range of values at each age. A, Values from PrnVd between 7 and 10 d, 2.4 synapses/ 100 pm2/d between 10 adult animals. and 15 d, 1.4 synapses/100 pm2/d between 15 and 20 d, and 1.3 synapses/ 100 pm2/d between 20 and 28 d. Thus the number misleading picture would be presentedregarding the time of of new synapses added per 100 pm2 was remarkably constant most active synaptogenesis.For this reason, we attempted to over much of the developmental period. obtain a more complete picture of the extent of the increasein synapsesover the postnatal interval by correcting for increases Increases in the thickness of the molecular layer in neuropil volume. Simple measures of synapse density do not provide a complete A rough indication of the increasein the neuropil can be picture of synaptogenesis, since there is a considerable increase obtained simply by measuringthe thickness of the molecular in the volume of the molecular layer over the postnatal interval. layer of the suprapyramidalblade at the point of sampling.This For the purposes of the present study, the principal concern was is obviously not an accurate indication of the overall increase when synaptogenesis occurred; less important was the total ex- in the volume of the molecular layer, sincethere is an increase tent of synaptogenesis. Nevertheless, volumetric increases could in septotemporallength, mediolateralbreadth, and dorsoventral interact with measures of synapse density in such a way that a height. Nevertheless,volumetric measurementsare not useful 420 Steward and Falk Vol. 6, No. 2, Feb. 1986

1.0

0.8 I

I1 I 1 4 7 10 15 20 28 “A Days Post-Natal 1 4 7 10 15 20 28 ‘A Figure6. Proportion of the total number of observed synapses whose Days Post-Natal connection with the dendritic shaft can be seen in the plane of section. This ratio provides a quantitative but indirect measure of the increase in the length and decrease in diameter of dendritic spines, since the probability of finding a spine head in continuity with the dendritic shaft decreases as the spine length increases and spine neck diameter decreases. Verticalbars, The range of values at each age. A, Values from adult animals. becauseof the gradients in maturation; unlessone sampledsev- eral areasfor estimatesof synapsedensity, correcting values for total volumetric increaseswould be misleading.The increasein the height of the molecular layer provides an estimate of the volumetric increaseat the samplingsite, which can then aid in 1 4 7 10 15 20 28 determining, at least roughly, what the actual increasein the Days Post -Natal number of synapsesat that point in the neuropil must be. The measuredthickness of the molecular layer in each animal (Fig. b 5B) was used to determine the area (in pm2) of a lo-pm-wide 60~ strip from the top of the granule cell layer to the distal tips of 1 T the granule cell dendrites (the hippocampal fissure). We then multiplied the area thus obtained in each animal by the mea- suredsynapse density obtained from the sameanimal, providing an estimate of the total number of synapsesin a lo-pm-wide strip across the molecular layer (see Fig. 5C’). As is evident, correcting for the increasesin the thickness of the molecular layer doesnot alter the conclusionsregarding the time of most active synaptogenesis. A quantitative measureof spine elongation As was noted above, an aspect of maturation that was quali- . tatively obvious wasan increasein spinelength. In the youngest 1 4 7 10 15 20 28 “A animals, synapseswere found on blunt protrusions from the Days Post-Natal dendrite, whereasmore and more mature-appearingspines were found assynaptogenesis proceeded. This increasein spinelength Figure 7. Three measures of the prominence of polyribosomes under synaptic contacts across the postnatal interval. A, Incidence of polyri- was reflected by an increasein the proportion of synapseson bosome-containing spine bases (the proportion of spine bases with un- spine heads that did not exhibit a continuity with a main den- derlying polyribosomes). B, Number of polyribosome-containing spines dritic shaft in the section plane (seeFig. 4). One way to obtain (spines with polyribosomes in either the head or neck region) per area a quantitative indication of this spineelongation isto determine of neuropil at each age. C, Total number of polyribosome-positive the proportion of synapseson “connected spines” (thosein con- synapses per area of neuropil across the postnatal interval (spine syn- tinuity with a dendritic shaft) acrossthe postnatal interval (see apses with polyribosomes at the base or within the spine head or neck, Fig. 6). At 1 d of age, 80% of the synapseswere on spineswhose and shaft synapses with underlying polyribosomes). Verticalbars, The connection with the main dendritic shaft could be seenin the range of values. A, Values from adult animals. planeof section. The proportion of synapseson connectedspines decreasedover the postnatalinterval, reachinga plateauof about 20% by 15 d of age, which was maintained through day 28. Quantitative analysesof polyribosomesassociated with There was an additional slight decrease in the proportion of developingsynapses connected spine headsafter 28 d of age. These data provide a Figure 7A illustratesthe incidenceof polyribosomesunder iden- quantitative, but indirect, indication of the time courseof elon- tified spinesacross the developmental period and in adult rats. gation of dendritic spinesacross the postnatal period. However, As is evident, the proportion of spine synapseswith underlying it is likely that once spine length has reached a certain value, polyribosomeswas highest at 1, 4, and 7 d of age;at all three further increaseswould not affect this parameter. Thus, there ages,about 60% of the identified spine baseshad underlying could be a further elongation of spinesafter 15 d of age,which polyribosomes.The incidence of polyribosomes decreasedbe- would not be reflected by this measure. tween 7 and 10 d of age,and again between 10 and 15 d, and The Journal of Neuroscience PolyribosomesUnder Developing Synapses 421 then remained at a plateau until 28 d of age.Interestingly, while with polyribosomes in the area of neuropil sampled. As illus- synapsedensity had reached adult levels by 20 d of age, and trated in Figure 7C, the number of polyribosome-positive syn- wasactually higher than adult levels at 28 d of age,the incidence apsesper area of neuropil was negligible at 1 d of age, despite of polyribosomes at 20 and 28 d of agewas considerably higher the fact that the majority of the synapsespresent had polyri- than in mature animals. Thus, the incidence of polyribosome- bosomes.The number of polyribosome-positive synapsesin- containing spines was highestwhen most of the synapseswere creaseddramatically after day 1, reacheda peakat 7 d postnatal, presumably newly formed or under construction, but was also and subsequentlydecreased to a plateau that was maintained higher than adult levels throughout the period when synapse between 15 and 28 d of age.Again, there wasa further decrease numbers were being adjusted. in this measurebetween 28 d and adulthood. In our previous study of synapsesgrowing in responseto lesionsin adult animals (Steward, 1983a), the increasesin poly- Discussion ribosomesunder spinebases were accompaniedby increasesin The present results strongly support our previous suggestion, polyribosomeswithin spineheads and necks,suggesting a move- basedon studiesof synapsegrowth following lesions,that polyri- ment of the protein-synthetic machinery out into the spineprop- bosomesare particularly prominent at the postsynaptic site dur- er during synapseconstruction. This sort of analysis was com- ing synaptogenesis(Steward, 1983a,b; Steward and Falk, 1985; plicated in the present study by the fact that spineswere quite Steward and Fass, 1983; Steward, in press).Prior to discussing short in the very young animals; thus, distinguishing the point the possiblerole of the polyribosomes,it is necessaryto briefly of transition between the dendritic shaft and the spine wasdif- discussthe quantitative methods upon which the conclusions ficult. Nevertheless, when polyribosomes that appeared to lie are based. within the spineproper (either within the head or the neck) were It is, first, important to note that the measuresof synapse separately counted, there was a clear peak in the number of density that are herein presentedare not necessarilyrepresen- polyribosome-containing spines per area of neuropil at 7 d of tative of synapsedensity throughout the dentate gyrus; the gra- age(see Fig. 7B). Theseresults must be interpreted cautiously, dient in maturation between supra- and infrapyramidal blades since polyribosomes at the base of the spine would lie much makesit difficult or impossibleto obtain an overall picture (for closer to the synaptic site in short spineseven if there were no a discussionof this problem, seeCowan et al., 1980). For ex- migration out into the spine proper. Thus, certain sectioning ample, synaptogenesisin the infrapyramidal blade is clearly anglesmight yield profiles in which the polyribosomesappeared severaldays behind that in the suprapyramidal blade (compare to lie within a spine head even though the polyribosomes were the present data with that of Crain et al., 1973). The fact that actually still at the intersection between the spinebase and the the data may not be representative for the entire dentate gyrus dendritic shaft. Even if the polyribosomes do migrate out into doesnot affect our conclusions,since the key issueis the rela- the growing spine,the actual distanceof the polyribosomesfrom tionship betweenthe time courseof synaptogenesisat the sam- the main shaft of the dendrite is not great in short spines.Never- pling site and the prevalence of polyribosomes in the synapses theless,these results provide additional evidence for a special that are found at that site. relationship between the polyribosomes and the synaptic site A secondlimitation that shouldbe recalledhas been discussed during synapseconstruction. in previous studies:The quantitative analyseswe have utilized We were very interested in determining whether polyribo- provide only a relative measureof the incidence of polyribosomeswere also present under shaft synapsesduring the de- somesunder spines(Steward, 1983a; Steward and Falk, 1985; velopmental period. To addressthis question, we adopted a Steward and Levy, 1982).Even with the smallestspines, a single very rigorous criterion for shaft synapses.As was noted above, thin-section doesnot reveal all of the area comprisingthe spine many early forming synapseswere found on very blunt protru- base;thus, some of the spinesthat do not have polyribosomes sions from dendrites. Thesewere classifiedas spinesfor quan- in a given section neverthelesshave underlying polyribosomes titative purposeswhenever there was even the slightest indi- that can be revealed by a serial section reconstruction of the cation of a protrusion at the site of the postsynapticspecialization, entire spinebase (see Steward, 1983a;Steward and Levy, 1982). or at either side. Synapseswere classifiedas shafts only if the For this reason, measuresof the incidence basedon singlesec- profile ofthe dendrite wasentirely flat at and around the synaptic tions (what we have termed “relative incidence”) consistently region. Thus, while some of the synapsesclassified as spines underestimate the actual incidence. The .extent of the under- might actually be shaft synapses,the opposite error is mini- estimate in the developing animals is not known; particularly mized. Becauseof this rigorous criterion, very few shaft synapses in the youngestanimals, spinesare stubby, and their basessub- were identified throughout the developmental period (seeFig. sumea large area.At the earliestages, it may well be that every 5A, broken line); nevertheless,the sametrends seemedto apply spinehas underlying polyribosomes. regarding the higher incidence of polyribosomes during synapse While the quantitative measurementshave limitations, we do formation. For example, combining the data from all 1, 4, and not feel that theselimitations compromisein any way the con- 7 d old animals yielded a total of 50 shaft synapses;16 of these clusionsthat the proportion of synapseswith polyribosomesis (32%) had underlying polyribosomes. A total of 71 shaft syn- highest at l-7 d, during the initial stagesof synaptogenesis, apseswere found in the 1O-l 5 d old animals; 11 of these(15%) whereasthe number of polyribosome-containing synapses/area had underlying polyribosomes. Finally, a total of 80 shaft syn- of neuropil is greatestat about 7 d. A discussionof the signif- apseswere found in the animals sacrificed at 20-28 d of age; icanceof theseobservations requires a considerationof the other five of these (6%) had underlying polyribosomes. events taking place in the dentate gyrus at this time. If the proportion of synapseswith polyribosomes remains constant between 1 and 7 d of agewhile the number of synapses The developmentof the dentate gyms increases,there must be an increase in the total number of A fairly complete picture is now available regarding many as- synapseswith polyribosomes per area of neuropil during this pectsof the development of the dentate gyrus (for a review, see interval. This is of interest, since the impression gained from Cowanet al., 1980).These data can bereviewed only very briefly qualitative observation was that the most dramatic accumula- here. Granule cells of the dentate gyrus are generated over a tions of polyribosomes were at 7 d of age.To directly determine prolonged period from embryonic day 14 through about the the total number of synapseswith associatedpolyribosomes per secondpostnatal week (Bayer, 1980; Schlessingeret al., 1975). area of neuropil, we determined the number of polyribosome- While the infrapyramidal blade is developmentally much less containing spinebases and spineheads, as well asshaft synapses mature than the suprapyramidal blade throughout early devel- 422 Steward and Falk Vol. 6, No. 2, Feb. 1986 opment, there is a continuing addition of cells to both blades contact region itself (i.e., the production of receptors,ion chan- (Bayer, 1980). This meansthat even though the neuropil of the nels, structural proteins of the postsynaptic membrane special- suprapyramidal blade is well developedby 7-10 d, a substantial ization, proteins involved in the stabilization of the membrane number of granule cells are just beginning to grow dendrites proteins, or any of a number of enzymes that are found at the into the neuropil layer. Interestingly, the peak of neurogenesis postsynaptic site, such as calmodulin or protein kinase); the occurs toward the end of the first postnatal week (days 5-8; see building of the dendritic spine, and perhapsthe elaboration of Schlessingeret al., 1975). This is at about the same time that moleculesthat induce or facilitate some aspectof this final dif- the greatest number of polyribosome-containing synapsesare ferentiation, and which, themselves,are not part of the synaptic found. contact. Only limited information is available regarding the devel- The hypothesis that the proteins produced by the polyribo- opment of the granule cell dendrites. The existing data, based somespromote the growth ofthe axon, or attract it to the contact on Golgi-stained material, suggestthat the granule cells elabo- site, was initially proposed becausethe increasesin polyribo- rate their characteristic dendritic treesbetween birth and 20 d somesfollowing lesionsoccurred at the initial phaseof the pre- of age (Fricke, 1975; reviewed in Cowan et al., 1980). There synaptic growth response,prior to the time of significant axonal wasno distinction betweenthe supra-and infrapyramidal blades ingrowth (Steward, 1983a).This hypothesiswould therefore pre- in the quantitative summary of increasesin dendritic length dict that axon ingrowth ought to be associatedwith the peak in over the developmental period. Given the substantial differ- polyribosomes at 7 d of age. We did not measurethe rate of encesin the maturation of supra- and infrapyramidal blades, ingrowth of presynaptic processes,since these could not be iden- the combination of data from both bladesmakes it impossible tified with certainty until they formed contacts; however, the to relate the available Golgi data to the present electron-micro- constant rate of synapseaddition over the postnatal interval scopic evidence, except to say that dendritic growth seemsto would suggesta constant rate of axon ingrowth. In addition, occur over roughly the sameperiod as the increasesin synapse there is ample evidence for a substantialavailability of afferents density. even at 1 d of age(for a review, seeCowan et al., 1980). Thus, Despite the relative immaturity of the dentate gyrus at birth, the present data argue against the possibility that the polyri- at least someof the afferents appear in the neuropil very early: bosomesproduce a factor to promote axon ingrowth. fibers of the dentate commissural system can first be detected If the polyribosomes were set out by the postsynaptic cell to in the neuropil the day after birth (Cowan et al., 1980) and the induce or promote initial contact formation, one would also projection from the entorhinal cortex appearsto develop some- expect the number of polyribosome-positive spinesto be closely what earlier (Fricke and Cowan, 1977; Loy et al., 1977). The related to the number of recently formed contacts. However, presenceof theseprojections at an early period suggeststhat at the rate of synapseaddition appearsto be constant from 1 to least somepresynaptic fibers are present before very much den- 15 d of age(averaging about two synapsesadded per 100 pm2/ dritic growth has occurred, and long before the peak in poly- d), whereasthe number of polyribosome-positive synapsesis ribosome-containing spines. greatestat 7 d of age. Given the problems with asynchronous Becausesynaptogenesis is ongoing while some granule cells growth of the granule cells during the postnatal interval, these are just beginning to grow dendrites into the molecular layer, observationscertainly do not rule out the possibility that polyri- the age of given dendrites cannot be determined, and any es- bosomesare set out by the postsynaptic neuron to induce or timation of the probable age of given synaptic contacts is quite promote contact formation, but the observations are not con- questionable.Presumably, very immature synapsescoexist with sistent with the predictions of the hypothesis either. ones that are several days old. Nevertheless,if synapsedensity The hypothesis that the polyribosomes produce proteins in- increasesover days, and if there is no compressionof the neu- volved in the growth of the dendritic spine is not supported, ropil during the same interval, then the increase in synapse sinceit would predict that polyribosomeswould not be present density indicates the number of synapsesadded. In fact, given at nonspine synapses.While the data in this regard are not that there is a simultaneousgrowth of the neuropil, the increase compelling, owing to the small number of shaft synapsesin the in synapsedensity provides a minimum estimateof the number molecular layer of the dentate gyrus, polyribosomeswere more of new synapsesin a given area of neuropil. It is therefore prominent under the shaft synapsesfound during the period probably safeto conclude that, between 1 and 15 d of age,each that they were most prominent under spines.In addition, we 100 pm2 of neuropil contains at least 2-2.5 synapsesthat have have recently found that polyribosomesare associatedwith non- been formed within the preceding 24 hr. spine synapseson axon initial segments(0. Steward and C. E. Ribak, unpublishedobservations). These results do not disprove Possible role of the polyribosomes the hypothesisthat the polyribosomesproduce proteins related The present data do not by themselves reveal what role the to the construction of the spine,but they do make the alternative spine-associatedpolyribosomes play; they do, however, permit possibilitiesmore attractive. more informed speculation than has heretofore been possible. We are left with the hypothesis that the polyribosomes and A priori, the participation of the polyribosomes in dendritic the proteins that they produce contribute to the differentiation elongation seemsunlikely; they are associatedwith synapses, of the synapse;the protein(s) could be a component of the syn- and are essentially absent from dendritic growth cones.Thus, aptic region itself, or could be necessaryfor differentiation of attention is focused on the synaptic site. Synapse formation the synapse,but not be a component (i.e., someretrograde factor during development involves a number of sequentialprocesses, that might stabilize the presynaptic terminal; seeChangeux and beginning with the growth of the axon and dendrite into ap- Danchin, 1976). The construction of mature synapsescan be position, and ending with the elaboration of all of the structural viewed as a seriesof stages,with each stagerepresenting a de- specializationsof the contact region, including the postsynaptic cision point at which the construction of the synapsecan either membrane density and the microspecialization of form (the be continued or aborted (seeBurry, 1982). For example, it has spine).The polyribosomes and the proteins they produce could beensuggested that synapsesinitially are labile, and progressto be involved in (1) inducing or promoting the growth of the axon stablecontacts as a consequenceof functional interactions with or attracting it to the postsynaptic site; (2) the formation of the the target (seeChangeux and Danchin, 1976). The duration of initial contact (i.e., recognition or adhesion); or (3) the differ- the labile phasecould depend on a number of factors and need entiation of the mature synaptic contact. The latter is by far the not come at a specifiedtime after the initial contact. Thus, the broadest of the possibilities, involving the construction of the peak in synapse-associatedpolyribosomes at 7 d could herald The Journal of Neuroscience Polyribosomes Under Developing Synapses 423 a stage of synapse maturation; perhaps this stage is brought Grab, D. J., R. K. Carlin, and P. Siekevitz (1981a) Function of cal- about by some change in the functional activity of either pre- modulin in postsynaptic densities I. Presence of a calmodulin-acti- or postsynaptic elements, or perhaps it is related to more general vatable cyclic nucleotide phosphodiesterase activity. J. Cell Biol. 89: effects (hormones, or experience). In this regard, it is of consid- 433-439. Grab, D. J., R. K. Carlin, and P. Siekevitz (198 1b) Function of cal- erable interest that there is an increase in polyribosome-con- modulin in postsynaptic densities II. Presence of a calmodulin-acti- taining spine synapses in the visual cortex in animals that had vatable protein kinase activity. J. Cell Biol. 89: 440-448. been exposed to an enriched environment during development Greenough, W. T., H.-N. S. Hwang, and C. Gorman (1985) Evidence (Greenough et al., 1985). for active synapse formation or altered postsynaptic metabolism in As we noted previously (Steward and Falk, 1985) the fact visual cortex of rats reared in complex environments. Proc. Natl. that polyribosomes are also present under spine synapses in Acad. Sci. USA 82: 4549-4552. mature animals does not contradict the suggestion that they are Hillman. D. E. (1979) Neuronal shane narameters and substructures involved in synapse growth. There is a growing consensus that as a basis of neuronal form. In The kekosciences Fourth Study Pro- there is a natural turnover of synapses in mature animals, which gram, F. 0. Schmitt and F. G. Worden, eds., pp. 477-498, M.I.T., Cambridge, MA. may be particularly important for replacing or repairing syn- Hinds, J. W., and P. L. Hinds (1976) Synapse formation in the mouse apses damaged by injury or disease (Cotman et al., 198 1). Fur- olfactory bulb. II. Morphogenesis. J. Comp. Neurol. 169: 41-62. thermore, it has long been suspected that the storage of expe- Kawana, E., C. Sand& and K. Akert (1971) Ultrastructure of growth riential information may also require synapse growth (Tanzi, cones in the cerebellar cortex of the neonatal rat and cat. Z. Zellforsch. 1893; and see Cotman et al., 198 1). If polyribosomes under the 115: 284-298. synapse are a hallmark of growing synapses, and if there is Kelly, P. R., and C. W. Cotman (1978) Synaptic proteins: Character- normally an ongoing synapse turnover in mature animals, it is ization of tubulin and actin and identification of a distinct postsyn- possible that polyribosome-containing spines in mature animals aptic density polypeptide. J. Cell Biol. 79: 173-183. mark synapses under construction. If this is true, a much needed Loy, R., G. Lynch, and C. W. Cotman (1977) Development ofafferent lamination in the fascia dentata of the rat. Brain Res. 121: 229-243. means would be available for identifying growing synapses in Pfenninger, K. H., and R. P. Rees (1976) From the growth cone to mature nervous systems. the synapse: Properties of membranes involved in synapse formation. In Neuronal Recognition, S.H. Barondes,ed., pp. 13l-l 78, Chapman References and Hall, London. Bayer, S. A. (1980) Development of the hippocampal region in the Schlessinger, A. R., W. M. Cowan, and D. I. Gottlieb (1975) An rat: I. Neurogenesis examined with 3H-thymidine autoradiography. autoradiographic study of the time of origin and the pattern of granule J. Comp. Neurol. 190: 87-114. cell migration in the dentate gyrus of the rat. J. Comp. Neurol. 159: Bloom, F. E. (1972) The formation of synaptic junctions in developing 149-176. rat brain. In Structure and Function of Synapses, G. D. Pappasand Skoff, R. P., and V. Hamburger (1974) Fine structure of dendritic and D. P. Purpura, eds., pp. 101-120, Raven, New York. axonal growth cones in embryonic chick spinal cord. J. Comp. Neurol. Burry, R. W. (1982) Development of apparent presynaptic elements 153: 107-148. formed in response to polylysine coated surfaces. Brain Res. 247: l- Steward, 0. (1983a) Alterations in polyribosomes associated with den- 16. dritic spines during the reinnervation of the dentate gyrus of the adult Carlin, R. K., D. J. Grab, and P. Siekevitz (198 1) Function of cal- rat. J. Neurosci. 3: 177-188. modulin in postsynaptic densities III. Calmodulin-binding proteins Steward, 0. (1983b) Polyribosomes at the base of dendritic spines of of the postsynaptic density. J. Cell Biol. 89: 449-455. central nervous system neurons: Their possible role in synapse con- Changeux, J.-P., and A. Danchin (1976) Selective stabilisation of de- struction and modification. Cold Spring Harbor Symp. Quant. Biol. veloping synapses as a mechanism for the specificity of neuronal 48: 745-759. networks. Nature 264: 705-7 12. Steward, 0. (in press) Protein synthesis under dendritic spine synapses Cohen, R. S., F. Blomberg, K. Berzins, and P. Siekevitz (1977) The during lesion-induced synaptogenesis: Evidence for regulation of rein- structure of postsynaptic densities isolated from dog cerebral cortex nervation by the target cell. Exp. Brain Res. (Suppl.). I. Overall morphology and protein composition. J. Cell Biol. 74: 18 l- Steward, O., and P. M. Falk (1985) Polyribosomes under developing 203. spine synapses: Growth specializations of dendrites at sites of syn- Cotman, C. W., M. Nieto-Sampedro, and E. W. Harris (1981) Synapse aptogenesis. J. Neurosci. Res. 13: 75-88. replacement in the nervous system of adult vertebrates. Physiol. Rev. Steward, O., and B. Fass (1983) Polyribosomes associated with den- 61: 684-784. dritic spines in the denervated dentate gyrus: Evidence for local reg- Cowan, W. M., B. B. Stanfield, and K. Kishi (1980) The development ulation of protein synthesis during reinnervation. In Molecular In- of the dentate gyrus. In Current Topics in Developmental Biology, 15: teractions Underlying Higher Brain Functions, J.-P. Changeux, J. Neural Development, Part 1: Emergence of SpeciJicity in Neural His- Glowinski, M. Imbert, and F. E. Bloom, eds., pp. 131-136, Prog. togenesis, pp. 103-157,Academic, New York. Brain Res. 58. Crain, B. J., C. Cotman, D. Taylor, and G. Lynch (1973) A quantitative Steward, O., and W. B. Levy (1982) Preferential localization of polyri- electron microscopic study of synaptogenesis in the dentate gyrus of bosomes under the base of dendritic spines in granule cells of the the rat. Brain Res. 63: 195-204. dentate gyrus. J. Neurosci. 2: 284-29 1. Del Cerro, M. P., and R. S. Snider (1968) Studies on the developing Steward, O., and S. V. Vinsant (1983) The process of reinnervation cerebellum. Ultrastructure ofthe growth cones. J. Comp. Neurol. 133: in the dentate gyrus of the adult rat: A quantitative electron micro- 241-362. scopic analysis of terminal proliferation and reactive synaptogenesis. Feit, H., P. Kelly, and C. W. Cotman (1977) Identification of a protein J. Comp. Neurol. 214: 370-386. related to tubulin in the postsynaptic density. Proc. Natl. Acad. Sci. Tanzi, E. (1893) J Fatti i le induzioni nell’odiema istologia de1 sistema USA 74: 1047-1051. nervoso. Riv. Sperim. Freniatria Medic. Leg. 19: 4 19-472. Foelix, R. F., and R. Oppenheim (1974) The development of synapses Tennyson, V. M. (1970) The fine structure of the axon and growth in the cerebellar cortex of the chick embryo. J. Neurocytol. 3: 277- cone of the dorsal root neuroblast of the rabbit embrvo. J. Cell Biol. 294. 44: 62-79. Fricke, R. (1975) Studies on the morphology and development of the Vaughn, J. E., and T. J. Sims (1978) Axonal growth cones and de- hippocampus and dentate gyrus. Unpublished doctoral dissertation, veloping axonal collaterals form synaptic junctions in embryonic mouse Washington University, St. Louis, MO. spinal cord. J. Neurocytol. 7: 337-363. Fricke, R., and W. M. Cowan (1977) An autoradiographic study of the development of the entorhinal and commissural afferents to the dentate gyrus of the rat. J. Comp. Neurol. 173: 231-250.