THE JOURNAL OF COMPARATIVENEUROLOGY 303:233-244 (1991)

Synapses Made bykons of CaIlosal ProjectionNeurons inMouse Somatosenso~Cortex:Emphasison Intrinsic Connections EDWARD L.WHlTE AND DAVID CZEIGER Department of Morphology, Corob Center for Health Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel

ABSTRACT This is one of a series of papers aimed at identifying the synaptic output patterns of the local and distant projections of subgroups of pyramidal neurons. The subgroups are defined by the target site to which their main axon projects. Pyramidal neurons in areas 1and 40 of mouse cerebral cortex were labeled by the retrograde transport of horseradish peroxidase (HRP) transported from severed callosal axons in the contralateral hemisphere. Terminals of the local axon collaterals of these neurons (“intrinsic” terminals) were identified in somatosensory areas 1 and 40, and their distribution and synaptic connectivity were examined. Also examined were the synaptic connections of “extrinsic” callosal axon terminals labeled by lesion induced degeneration consequent to the severing of callosal fibers. A post-lesion survival time of 3 days was chosen because by this time the extrinsic terminals were all degenerating, whereas the intrinsic terminals were labeled by HRP. Both intrinsic and extrinsic callosal axon terminals occurred in all layers of the cortex where they formed only asymmetrical synapses. Layers I1 and I11 contained the highest concentrations of both types of callosal axon terminal. Analyses of serial thin sections through layers I1 and I11 in both areas 1 and 40 yielded similar results: 97% of the extrinsic (277 total sample) and of the intrinsic (1215 total sample) callosal axon terminals synapsed onto dendritic spines, likely those of pyramidal neurons; the remainder synapsed onto dendritic shafts of both spiny and nonspiny neurons. Thus the synaptic output patterns of intrinsic vs. extrinsic callosal axon terminals are strikingly similar. Moreover, the high proportion of axospinous synapses formed by both types of terminal contrasts with the proportion of asymmetrical, axospinous synapses that occur in the surrounding neuropil where only about 80% of the asymmetrical synapses are onto spines. This result is in accord with previous quantitative studies of the synaptic connectivities of both extrinsic and intrinsic axonal pathways in the cortex (White and Keller, 1989: Cortical Circuits; Boston: Birkhauser): in all instances, axonal pathways are highly selective for the types of elements with which they synapse. Key words: cerebral cortex, callosum, horseradish peroxidase

A consistent feature of pyramidal neurons in the cerebral 1984) to mostly spines (e.g., Kisvarday et al., 1986; and see cortex is the presence of an extensive set of axon collaterals White, 1989c),or to roughly equal proportions of spines and that ramify within the general area of the parent cell body dendrites (e.g., Haberly and Presto, 1986; and see White, and dendritic tree (see Feldman, 1984; White, 1989b). 1989~).Thus, considered as a group, the local axons of Terminals belonging to these “local” or “intrinsic” axon pyramidal neurons exhibit a variety of synaptic output collaterals make asymmetrical synapses onto spines and patterns. However, it may be that subgroups of pyramidal dendritic shafts (e.g., LeVay, 1973; Parnavelas et al., 1977, neurons exist whose local axons display consistently a and see White, 1989~1, and rarely onto neuronal cell bodies preference for one or another type of postsynaptic element. (Kisvarday et al., 1986). Reports on the proportions of This possibility has begun to be explored in a series of pyramidal cell intrinsic synapses formed with different studies aimed at identifying the local synaptic output types of postsynaptic elements have been inconsistent, Accepted September 25, 1990. ranging from mainly dendritic shafts (e.g., McGuire et al., C)

1991 WILEY-LISS, INC.

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with area 3 (Caviness, 1975; Yorke and Caviness, 1975). Then the sections were placed for 30 minutes in aqueous 1%osmium containing 1.5%potassium ferrocyanide, dehydrated and embedded for electron microscopy. During dehydration the sections were placed for 2 hours in 1% uranyl acetate dissolved in 70% ethanol. Large, single thin sections spanning the entire width of the cortex in parts of areas 1 and 40 that were heavily labeled with HRP were examined for each survival time to determine the laminar distribution and relative concentration of axon terminals labeled by HRP or by lesion induced degeneration. The results of this analysis indicated that extrinsic callosal axon terminals, labeled by lesion induced degeneration, and intrinsic ones, labeled by the intraaxonal transport of HRP, could be identified and differentiated from each other at a post-lesion survival time of 3 days. The efficacy and reliability of the combined lesion/HRP approach for identifying extrinsic and intrinsic axon terminals in a single preparation will be discussed subsequently. At all survival times, both extrinsic and intrinsic callosal axon terminals occurred most frequently in layers I1 and 111. In view of this, analyses of the numbers and types of synapses made by labeled callosal axon terminals were performed on serial thin sections through these layers. In all, six series, one for area 1and one for area 40 in each of three mice, at 3 to 3%day survival times, were mounted on slotted grids and examined with the electron microscope. Each series contained from 200 to 350 thin sections representing a volume of about 900 x mm3. All elements postsynaptic to HRP labeled or to degenerating axon terminals were followed in serial thin sections to identify them as dendritic spines or shafts, and, where possible, to determine the cell type of origin of the postsynMATEXIALSANDMETHODS aptic element. One dendrite, postsynaptic to an HRPYoung adult male CD/1 mice were injected intravenously labeled axon terminal, was graphically reconstructed by the with 1g/kg of mannitol45 minutes prior to being anaesthe- procedure outlined in White and Keller (1987). Labeled tized with sodium pentobarbitol. Craniotomies were per- terminals made only asymmetrical synapses. In order to formed to expose an area of parietal and frontal cortex, compare the proportion of synapses onto spines vs. denapproximately 1by 5 mm in size, parallel and just lateral to dritic shafts made by labeled axon terminals with that of all the superior sagittal sinus. Exposed cortex was aspirated asymmetrically synapsing terminals in the neuropil, counts down to the corpus callosum, and the cavity packed with were made of other asymmetrical, axospinous, and axodencotton to stem bleeding. The cotton was removed about 10 dritic synapses in samples of neuropil from each series. minutes later, whereupon the needle of a 10 pl syringe was Typically, synapse counts of the neuropil were made from inserted under the corpus callosum as close as possible to 100 serial electron micrographs of fields roughly 8 by 8 pm the midline and lifted up to sever the callosal fibers. in size; these counts included all asymmetrical synapses Simultaneously, the cavity was filled with a 40% aqueous made by both labeled and unlabeled terminals. solution of horseradish peroxidase (HRP, Seravac, Ltd.) A determination of the proportion of synapses made en ejected from the syringe. The bone flap was replaced about passant vs. by boutons terminaux was made for HRP15 minutes later, and the animals allowed to recover for labeled axon terminals. Because of the time consuming various periods. nature of tracing neuronal processes, even when labeled, From 2 to 7, and 14 days later, the animals were from one grid to the next, efforts to differentiate synapses anaesthetized with chloral hydrate and perfused intracaren passant from those made by boutons terminaux made dially with a solution containing 2.0% glutaraldehyde and 0.5% paraformaldehyde buffered to pH 7.3 with 0.1 M SCrensen’s phosphate buffer. Hemispheres contralateral to the lesion/injection site were tissue chopped at 125 pm and reacted for HRP as described previously (Elhanany and Figs. 1 and 2. Light micrographs of 125 p n thick coronal sections White, 1990), with the exception that 0.01 M imidazol through the mouse brain from preparations in which horseradish (Straus, 1982) and 0.5 mM nickel ammonium sulfate peroxidase (HRP) was placed onto the cut ends of callosal fibers in the (Adams, 1981) were added to the second diaminobenzidine contralateral hemisphere. Parts of cortical areas 1 (Fig. 1)and 40 (Fig. solution. Drawings were made of each section using a light 21, that border on cortical area 3, contain high concentrations of HRP microscope equipped with a drawing tube noting, in partic- (double arrowheads). Other areas of the cortical grey matter also ular, regions of somatosensory parietal cortex containing contain high concentrations of HRP (single arrowheads). Because of the refraction of light, in these thick sections, by high concentrations of high concentrations of HRP reaction product. By compari- myelinated fibers, certain areas, especially in subcortical structures, son with a set of similar sections stained for Nissl, these appear dark in thesephotographs; except forthe corpus callosum, this is areas were identified as the border regions of areas 1 and 40 not due to thepresence ofHRP. x 14.

patterns of pyramidal neurons defined as belonging to specific subgroups according to the distant site to which their “main” or “principal” axons project. In the first of these studies, White and Keller (1987) examined synapses in mouse SmI cortex made by the local axon collaterals of corticothalamic projection cells. More than 90% of these synapses were onto dendritic shafts. In contrast, the local axon collaterals of corticocortical cells in the same region synapse primarily with dendritic spines (Elhanany and White, 1990). In both studies, the proportion of axospinous vs. axodendritic synapses formed by the local output pathways differed significantly from that within the surrounding neuropil. Thus in both instances, the local output pathways are shown to be highly selective for the type of postsynaptic element with which they synapse. The present study is an effort to extend these results by examining the local synaptic output of an additional type of pyramidal neuron, i.e., the callosal projection cell. Callosal projection cells are distinguished from other types of projection neurons by the fact that the distant branches of their principal axons typically synapse at very high frequencies with dendritic spines (see Colonnier, 1981). Two related purposes of the present study are 1)to see if the local synaptic output of callosal cells is characterized by the same high degree of selectivity for postsynaptic elements that is exhibited by the local axon collaterals of other types of cortical projection cell, and 2) to see if the local axon collaterals of callosal neurons display the overwhelming preference for spines shown by the branches of their long, projecting axons.

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Figures 1 and 2

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focused on grids containing large numbers of thin sections (60-120 sections per grid).

RESULTS Light microscopy The approach used in this study involved cutting the corpus callosum and immediately bathing the cut ends of the callosal axons with HRP. From 2 to 6 days postoperative, dense concentrations of HRP reaction product were observed in all areas of the contralateral hemisphere known to receive or to provide callosal connections (e.g., Yorke and Caviness, 1975). Reaction product was particularly concentrated in the regions of cortical areas 1 and 40 that border on the area 3 representation of the large mystacial vibrissae (Figs. 1and 2) (Caviness, 1975; Yorke and Caviness, 1975; Woolsey and Van der Loos, 1970). Pyramidal cell bodies labeled by the retrograde transport of HRP were especially numerous in layers I1 and I11 of these border regions but occurred also in layers V and VI. Many labeled pyramidal cell bodies contained a sparse, granular deposit of HRP, but some were more densely and homogeneously labeled such that their cell bodies, dendrites, spines, and axons were clearly visible with the light microscope. Small dots of HRP reaction product, presumably axon terminals that at times were connected by thin, labeled fibers, occurred in all layers but were most numerous in layers I1 and 111. HRP reaction product was relatively light at 6 and 7 days postoperative and not at all visible at 14 days. The identification of areas 1 and 40 in 14 day animals was made by comparison with drawings of sections from animals of shorter survival times. The laminar distribution of axon terminals labeled either by HRP or by degeneration was assessed at each survival time in single thin sections approximately 1 mm wide stretching from the pia to the white matter.

by glia (Fig. 5). At all postoperative times, degenerating terminals occurred most frequently in layers I1 and 111. HRP reaction product was not observed within degenerating axon terminals; presumably the enzyme was degraded, or in some other way inactivated, early on in the degeneration process. Prior to the onset of anterograde degeneration, it is likely that a proportion of HRP-labeled axon terminals belonged to extrinsic afferents labeled by the anterograde transport of HRP. Because no early stages of degeneration were observed at, or after, 3 days postoperative, we concluded that all callosal axon terminals of extrinsic origin were clearly degenerating by this time. It follows then that all terminals labeled with HRP at day 3 and later were of intrinsic or local origin; that is, they belonged to the local axon collaterals of retrogradely labeled callosal projection neurons. The appearance of HRP-labeled neuronal elements in thin sections was consistent with previous reports (e.g., Hersch and White, 1981, 1982; White et al., 1980; White and Hersch, 1981, 1982). Labeled profiles contained a high concentration of electron-dense material that was easlly visualized in thin sections (e.g., Figs. 6-10). The range of labeling was such that profiles were either very densely or somewhat more lightly labeled, but no profiles were so lightly labeled as to cause difficulties with identifying them as labeled or not. Cell body profiles containing a few electron dense granules were observed, but no similar deposit was observed in dendrites or axons. Axon terminals labeled with HRP occurred in cortical layers I1 through VI, but most frequently in layers I1 and 111. HRP-labeled, intrinsic axon terminals were differentiated from the degenerating terminals of extrinsic callosal afferents by several distinct characteristics. Degenerating terminals occasionally contained vacuolous debris (Fig. 4), but organelles such as mitochondria and synaptic vesicles were difficult to identify within them. Because of this the

Electronmicmpy The combined lesionlinjection approach was designed to differentiate between the terminals of extrinsic callosal afferents, labeled by lesion induced, anterograde degeneration, and terminals of intrinsic or local origin labeled by the retrograde transport of HRP from the operated hemisphere. This approach has been applied previously to identify other intrinsic and extrinsic projections (Elhanany and White, 1990; White and Keller, 1987). No degenerative changes were observed in cell bodies or dendrites, even at 14 days postoperative, and so the possibility that callosal projection cells were affected by retrograde degeneration was excluded (cf. Lund and Lund, 1970). For this reason, all degenerating terminals were considered to belong to cell bodies located in the contralateral hemisphere; that is, they were the terminals of extrinsic callosal afferents. At 2 days post-lesion, degenerating terminals in the unoperated hemisphere showed early to middle stages of degeneration (cf. Jones and Powell, 1970); their cytoplasm was very electron dense and they were filled with many synaptic vesicles and swollen mitochondria. By 3 days postoperative, all degenerating terminals were in middle to late stages of degeneration; some terminals contained disrupted mitochondria, but typically the terminals were very electron dense and no organelles were discernable within them (Fig. 3). By postoperative day 4 and afterward, degenerating terminals were very electron dense (Fig. 4); many were markedly shrunken and had become engulfed

Figs. 3 and 4. Electron micrographs showing asymmetrical synapses made by degenerating, extrinsic callosal axon terminals (A) onto spines (S). Organelles are difficult to discern within the cytoplasm of these terminals, which thus appears more homogeneous than that of the vesicle-filled terminals labeled by HRP (see text). Figure 3 is from area 1 at 3 days post-lesion; Figure 4 is from area 40 at 6 days post-lesion. g, glia. X50,OOO. Fig. 5. Electron micrograph showing a markedly shrunken degenerating, extrinsic callosal axon terminal from area 40 at 7 days post-lesion. This terminal, which is enveloped by glia (g), may synapse onto an unlabeled element a t the bottom of the micrograph. X50,OOO. Figs. 6-9. Electron micrographs showing asymmetrical synapses between HRP labeled intrinsic, callosal axon terminals (A) and spines (S) in area 40 at 3 days postoperative. Synaptic vesicles, which at times appear as light spheres against the very dark reaction product in the background axoplasm (e.g., Fig. B), and mitochondria are visible within HRP-labeled terminals. Shown at the top of Figure 6 is an asymmetrical synapse from an unlabeled axon terminal (An) onto a spine (S). x50.000.

Fig. 10. Electron micrograph showing three boutons of an HRP labeled intrinsic callosal axon (A) joined by preterminal axons of typically small diameter. In this section, spines (S) are postsynaptic to two of the labeled boutons. ~ 3 2 ,0 0 0 .Inset: Electron micrograph of a n adjacent section showing, at higher magnification, the synapse indilcated by the arrow. X50,OOO.

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238 axoplasm of degenerating axon terminals was much more homogenous than that of HRP-labeled,intrinsic axon terminals which contained these organelles (cf. Figs. 3 and 4 with Figs. 6-10]. In addition, the axoplasm of degenerating axon terminals tended towards dark gray as distinct from the blacker tone of the HRP reaction product within the axoplasm of HRP-labeledterminals. An impression gained from examination of both single and serial thin sections was that degenerating axon terminals occurred in small groups or patches situated at varying distances away from the main concentrations of HRPlabeled axon terminals. These patches, only a few pm across and separated by only a few pm, may have reflected the terminal distribution of single afferent fibers, but the absence by postoperative day 3 of the preterminal axon segments of degenerating fibers precluded an assessment of this possibility. In all preparations, the intrinsic, HRPlabeled axon terminals were more numerous than the extrinsic, degenerating ones. The larger numbers of HRP-labeled terminals observed may derive in part from this segregation of terminal types: Except for 14 day preparations (see above), the choice of material for electron microscopy was guided by the lightmicroscopic examination of thick sections in which only the HRP label was visible; concentrations of degenerating terminals that may have occurred nearby, but outside the main concentrations of HRP labeled axon terminals, would not have been included in our samples. Long series of thin sections oriented obliquely to the pial surface so as to include only layers I1 and 111were examined to determine the numbers and types of synapses formed by degenerating and HRP labeled axon terminals in areas 1 and 40. HRP-labeled, intrinsic callosal axon terminals were usually just under 1 pm in diameter, but occasionally somewhat larger terminals were encountered. Preterminal axonal segments of intrinsic axons were among the processes of smallest diameter in the neuropil; diameters of approximately 0.2 km were typical for these elements (Fig. 10). Intrinsic callosal terminals contained mitochondria and large numbers of densely packed, round synaptic vesicles (see Figs. 6-10,13, and 14), the latter sometimes appearing as relatively light spheres against the very dark deposit of reaction product in the background axoplasm (Fig. 8). Terminals with relatively lightly stained axoplasm were also observed; in these the density of the background axoplasm was similar, and in places lighter than that of the stained material within the synaptic vesicles (Fig. 9). When followed in serial thin sections, HRP-labeled afferents were observed to have boutons as frequently as every 2 pm, although it was more common to encounter several pm of preterminal axon between successive boutons. In a sample of 593 synapses made by HRP-labeled terminals, 59% were en passant and 4% were made by bouton terminaux that were connected by narrow stalks to the parent axon. The remaining terminals (37%)were not followed for distances sufficiently long to determine whether the terminal formed a discrete bouton or continued on as part of a longer axon. Because the preterminal axons of extrinsic callosal afferents could not be discerned in our preparations, an assessment of synapses made en passant vs. by boutons terminaux was not made for extrinsic terminals. Callosal terminals of both extrinsic and intrinsic origin made only asymmetrical synapses (Colonnier, 1968), usually with one postsynaptic element; but about 10% of time

two (Fig. 9), and rarely three postsynaptic elements were contacted by a single axon terminal. In both areas 1and 40, approximately 97% of all the elements postsynaptic to both types of callosal axon terminal were dendritic spines (Table 1) (see Figs. 6-10, and 14), the remaining synapses were onto dendritic shafts (see Figs. 13 and 15). By comparison, 83%of the asymmetrical synapses in the neuropil of area 1, and 80% of those in the neuropil of area 40 were presynaptic to spines, the remainder in each instance synapsed onto dendritic shafts (Table 2). Dendritic shafts postsynaptic to labeled axon terminals were followed in serial thin sections in about equal numbers TABLE 1. Synaptic Connectivity of Callosal Axon Terminals' Postsynaptic element Shafts

SDines Intrinsic terminals' Extrinsic terminals'

1,174 (96.6%) 269 (97.1%)

41 (3.4%) 8 12.9%)

Totals 1,215 277

'Table showing the numbers of synapses formed by callosal axon terminals of intrinsic and extrinsic origin with spines and dendritic shafts. 'As assessed by x2 tests, no difference was observed in results from t h e two regions of cortex examined and so these have been combined. Values ofP a. 0.0001 were obtained in 'x tests comparing t h e numbers of asymmetrical, axospinous vs. axodendritic synapses formed by intrinsic or extrinsic axon terminals with samples of all the asymmetrical, axospinous and axodendritic synapses in the neuropil of layers I1 and 111in each area.

TABLE 2. SynapticComposition of the Neuropil I Spines

Postsynapticelement Shafts

Totals

_ _ _ _ _ _ _ _ _ ~

Area 1 40

331 (832%) 309 179 6%)

67 I16 8%) 79 120 4%)

398 368

'Table showing the numbers of asymmetrical axospinous and axodendritic synapses formed in t h e neuropil of layers I1 and 111.

Fig. 11. Graphic reconstruction from serial thin sections of a varicose, nonspiny dendrite from layer I11 of area 40. The dendrite receives many asymmetrical synapses from unlabeled presynaptic elements (approximate locations of these synapses are indicated by dots), and a few symmetrical synapses (indicated by bars). Arrowhead indicates location of synapse made by the HRP-labeled intrinsic, callosal axon terminal shown in Figure 13. Black lines crossing to Figure 12 indicate the location of the thin section shown in this figure. Vertical bar = 10 +m. Fig. 12. Electron micrograph showing one of the serial thin sections that was used to make the reconstruction shown at the left in Figure 11. In this section, the dendrite (D) is postsynaptic at asymmetrical synapses to five axon terminals (A);one of these (A*) is shown at higher magnification in Figure 14.~35,000. Fig. 13. Electron micrograph showing an asymmetrical synapse from an HRP-labeled, intrinsic callosal axon terminal (A) onto the dendrite (D) reconstructed in Figure 11.The terminal is packed with synaptic vesicles that appear as relatively light spheres (v, arrows) against the darkly stained background axoplasm. Pre- and postsynaptic membranes, difficult to discern due to the presence of reaction product within the synaptic cleft, are indicated by fine, unlabeled arrows. ~80,000. Fig. 14. Terminal (A*) shown at higher magnification in order to illustrate better the components of the synapse shown at lower magnification in Figure 12.~80,000. Fig. 15. Electron micrograph showing asymmetrical synapses from HRP-labeled, intrinsic callosal axon terminals (A),in area 1, onto a dendrite (D) and onto a spine (S). ~40,000.

Figures 11-15

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in areas 1and 40, in order to examine their shapes, and the numbers and kinds of synapses they received. Nine of thirty-three dendrites postsynaptic to intrinsic axon terminals (i.e., terminals of local origin labeled with HRP), and four of the five dendrites postsynaptic to extrinsic callosal axon terminals (i.e., terminals from the contralateral hemisphere labeled by lesion induced degeneration) were followed for distances too short to enable them to be identified reliably as spiny or nonspiny. Five dendrites postsynaptic to intrinsic axon terminals were traced for 3-5 pm. These dendrites had spines arising from their shafts at a frequency greater than one spine per p-m and received no additional synapses. On this basis, we conclude that these dendrites belonged to spiny neurons. The remaining 19 dendrites were followed for distances of about 1.5 pm (three dendritss, including one dendrite postsynaptic to an extrinsic axon terminal), 3-5 km (eight dendrites), 8-11 pm (four dendrites), 13-20 p-m (three dendrites), and 70 pm (one dendrite, shown in Fig. 11).These dendrites received large numbers of asymmetrical synapses (Figs. 11 and 12) and rarely, if at all, had spines; about half of them were varicose (Fig. 11). The lack or paucity of spines coupled with the presence of large numbers of asymmetrical synapses onto dendritic shafts are features typical of nonspiny or sparsely spiny, nonpyramidal neurons (White, 1989b).

DISCUSSION Pyramidal cells in cortical areas 1 and 40 of mouse somatosensory cortex were labeled by the retrograde transport of HRP placed onto severed callosal fibers in the contralateral hemisphere. Terminals belonging to the local axon collaterals of callosal projection neurons form only asymmetrical synapses, within layers I1 and 111 these are primarily onto dendritic spines. Dendritic shafts of both spiny and nonspiny neurons also receive intrinsic callosal synapses, but infrequently.

Areasexamined The identification, according to the system of Caviness (1975), of cortical areas 1and 40 and of other regions in the mouse that were well labeled by callosally transported HRP was assisted by comparing material prepared for the present study with Nissl stained sections through similar regions of the cortex. Our observations on the general pattern of callosal connectivity in the mouse were in accord with previous results (e.g.,Yorke and Caviness, 1975). A consistent feature of the primary somatosensory area of mammalian cerebral cortex is that those regions involved in axial or midline functions are characterized by dense interhemispheric connections (Conti et al., 1986; Killackey et al., 1983; Manzoni et al.,1989; Shanks et al., 1985; Yorke and Caviness, 1975). Accordingly, physiological recordings in the mouse indicate that the region of area 1examined in this study corresponds to the representation in SmI cortex of the trunk and proximal limbs (Welker, 1971; Woolsey, 1978). A somewhat different situation obtains for the secondary somatosensory areas where interhemispheric integration occurs also in areas representing highly lateralized functions (Manzoni et al., 1989). The region of area 40 examined in this study contains the SmII representation of the trunk and proximal hindlimbs, but the evoked potential (Woolsey, 1978) and microelectrode recording (Welker and Sinha, 1972) maps suggest that a part of SmII representing

the distal hindlimbs may have been included in our preparations. The approaches taken in this study precluded any precise identification of the areas of the body represented in the regions of cortex examined.

Experimental approach Horseradish peroxidase injected into the brain, or placed onto cut axons, is transported in both the retrograde and anterograde directions (e.g., Adams and Warr, 1976; LaVaii and LaVail, 1972; White et al., 1980). Under certain conditions, HRP transported in the retrograde direction fills neuronal processes emanating from the cell body, including dendrites and their spines and local axon collaterals (White and Hersch, 1981, 1982; White et al., 1980). The bidirectional transport of HRP means that “target” areas of the brain reciprocally connected with an injection site are liable to contain HRP labeled axon terminals from two different sources. Such an area would contain 1) a set of extrinsic axon terminals labeled by the anterograde transport of HRP from cell bodies and cut axons at the injection site, and 2) a set of intrinsic terminals belonging to the local axon collaterals of retrogradely filled cell bodies situated within the target area. In order to differentiate extrinsic from intrinsic callosal axon terminals, we lesioned the corpus callosum and allowed the animals to survive for 3 days. By this time the extrinsic terminals are labeled by lesion induced degeneration, whereas the intrinsic terminals are labeled by HRP. This approach has been used successfully to identify the intrinsic axon terminals of corticothalamic (White and Hersch, 1982) and of corticocortical (Elhanany and White, 1990) projection cells and to differentiate them from labeled, extrinsic axon terminals. The background axoplasm of HRP-labeled axon terminals was either very electron dense or somewhat less so. The absence of very lightly labeled terminals containing HRP suggests that no intrinsic callosal axon terminals went undetected, i.e., none were so lightly labeled as to appear unlabeled. However, it is not possible to determine if every callosal projection neuron was labeled by the retrograde transport of HRP, nor could we be certain that all the local axon collaterals of the labeled neurons were themselves labeled.

Distributionofc d d terminals The axon terminals of degenerating, extrinsic callosal afferents were more highly concentrated in layers I1 and I11 than in the deeper layers of the cortex. This observation is in agreement with the distribution of extrinsic callosal axon terminals as determined in studies of areas 1 and 40 in the mouse (Yorke and Caviness, 19751, of visual cortex in the mouse (Olavarria and van Sluyters, 1984; Yorke and Caviness, 1975), and in studies of auditory (Cipolloni and Peters, 1983; Cipolloni and Peters, 1979) and somatosensory cortex in the rat (Wise and Jones, 1976) and monkey (Sloper and Powell, 1979). Various cortical areas of the cat and monkey exhibit an additional concentration of extrinsic callosal terminals in layer IV (Jacobson and Marcus, 1970; Sloper and Powell, 1979; Innocenti, 1986). The laminar distribution of extrinsic callosal axon terminals, and their organization into columns or bands has been well documented in a review by Innocenti (1986; and see Malach, 1989). In our preparations, extrinsic callosal axon terminals were not visible with the light microscope, and so we could not discern any columnar or banding pattern (cf.

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INTRINSIC CALLOSAL SYNAPSES Cipolloni and Peters, 1979) in the gross distribution of these terminals. Light microscopic banding patterns should not be confused with the patches of extrinsic terminals we observed with the electron microscope; the latter being orders of magnitude smaller than the bands observed with the light microscope. Intrinsic callosal axon terminals labeled by HRP occurred more frequently in layers I1 and 111 than in other layers of the cortex; to our knowledge there are no other reports on the laminar distribution of intrinsic callosal axon terminals.

other layers or even in adjacent cytoarchitectonic areas of the cortex.

synapsesofmledterminals

Thalamocortical axon terminals to area 3 of mouse somatosensory cortex usually form 2-3 synapses per bouton (White and Keller, 1987). In contrast, both extrinsic and intrinsic callosal axon terminals in areas 1 and 40 typically form only one synapse per terminal, as do terminals belonging to the local axon collaterals of corticothalamic (White and Keller, 1987) and corticocortical (Elhanany and White, 1990) cells in mouse barrel cortex. The In all species examined, callosal connections are typically intrinsic axon terminals of superficial and deep pyramidal most dense between homotopic areas of the two hemi- neurons in the visual cortex of the cat (Kisvarday et al., spheres, but heterotopic projections also have been ob- 1986; McGuire et al., 1984) and monkey (Winfield et al., served in the mouse (Porter and White, 1983; White and 1981) have also been observed to form predominately one DeAmicis, 1977) and in other species (Barbaresi et al., 1989; synapse per terminal. Thus, on the basis of light microCipolloni and Pandya, 1989; Killackey et al., 1983; Shanks scopic observations, it might be possible to predict the et al., 1985).Thus it is possible that extrinsic callosal axon numbers of synapses formed by the local axon collaterals of terminals examined in the present study included terminals pyramidal cells. To be accurate, such predictions require of axons originating in both homotopic and heterotopic that each bouton forms only one synapse, and that the synapses are formed only by boutons large enough to be areas of the contralateral hemisphere. The corpus callosum is considered to be composed only of detected with the light microscope. Depending on the axons emitted by pyramidal neurons (Innocenti, 1986), pathway examined, these conditions may be difficult to except in visual cortex where the axons of nonpyramidal obtain: Some axonal swellings do not form synapses (Peters neurons are also involved (Buhl and Singer, 1989; and see and Proskauer, 1980; Winfield et al., 1981), and some Innocenti, 1986). Thus, the intrinsic axon terminals exam- boutons of pyramidal cell axon collaterals form more than ined in this study are presumed to arise from pyramidal one synapse (in the present study, about 10% of the neurons. Cortical pyramids typically have local axon collat- collateral boutons formed more than one synapse). In erals that ramify extensively within close proximity to the addition, synapses may be made en passant, sometimes by parent cell body and its dendritic tree (see White, 1989b),a portions of the axon that are not sufficiently swollen to be feature reported for area VI in the rat (Hedlich and recognized as boutons with the light microscope. In the Winkelman, 1982; Parnavelas et al., 1983; Peters and Kara, present study, the majority of intrinsic axon terminals 19851, cat (Gilbert and Wiesel, 1981; O’Leary, 19411, and formed synapses en passant, as did about 15% of the local monkey (Lund and Boothe, 1973, VII in the monkey (Lund axon terminals of pyramidal neurons examined in a previet al., 1981; Valverde, 19781, AI of the cat (Winer, 19841, ous study (Elhanany and White, 1990). and for SmI in the mouse (Lorente de N6, 1922, 1938). In Both extrinsic and intrinsic callosal axon terminals formed addition to having an axonal ramification near the cell body, only asymmetrical synapses, a finding consistent with pyramidal cells may have collateral axonal branches that numerous previous reports of the synaptic connectivities of ascend or descend to other layers of the cortex, or that cortical pyramids, in general, and of callosal projection cells, travel for long distances horizontally within it (see the in particular (Cipolloni and Peters, 1983; Fi’;ken et al., comprehensive review by Feldman, 1984). In most in- 1975; Jones and Powell, 1970; Lund and Lund, 1970; stances, the horizontal collaterals of pyramidal cells remain Porter and White, 1984, 1986; Sloper and Powell, 1979; within the cytoarchitectonic area of the parent cell body Vaughan and Peters, 1985; and see White, 1 9 8 9 ~ ) . Nearly all elements (97%) postsynaptic to intrinsic and (Gilbert and Wiesel, 1979; Rockland et al., 19821, but a collateral may cross from one cytoarchitectonic area to extrinsic callosal axon terminals are dendritic spines, alanother (DeFelipe et al., 1986), rendering the “local” in though some dendritic shafts are also contacted. These local axon collateral somewhat of a misnomer. The variety findings agree with data showing that 95 to 100% of the of arrangements that characterize the distribution of pyra- synapses made by extrinsic callosal afferents to the auditory midal cell axon collaterals makes it impossible, in the cortex of the rat (Sloper and Powell, 1979),to the somatosenabsence of direct evidence, to determine their cell bodies of sory cortex of the cat (Jones and Powell, 1970) and monkey (Cipolloni and Peters, 19831, and to the motor cortex of the origin. The intrinsic callosal axon terminals examined in this monkey (Cipolloni and Peters, 1983) are onto spines. In study were not traced back to their parent cell bodies, and contrast Fisken et al., (1975) report that extrinsic callosal so their origin could not be determined. However, in view of afferents in monkey visual cortex form only 76% of their the prominence of collateral ramifications within the immed- synapses onto dendritic spines. icate vicinity of a pyramidal cell body and its dendrites, The results reported in this study are the first that enable often far more extensive than more distant collateralisa- a comparison between the kinds of postsynaptic elements tions (e.g., Kisvarday et al., 1986; Gilbert and Wiesel, 1981; contacted by intrinsic vs. extrinsic terminals belonging to a Parnavelas, 1984; Cipolloni and Pandya, 1989), it seems single type of projection cell. The similarity between the likely that the intrinsic callosal terminals examined in this output patterns of intrinsic and extrinsic callosal axon study originated mainly from pyramidal cells in layers I1 terminals - both synapse nearly exclusively onto spines - is and 111. It cannot be excluded that some HRP labeled axon striking. It would be interesting to determine if similar terminals belonged to pyramidal cell bodies situated in arrangements characterize the types of synaptic connec-

242

E.L. WHITE AND D. CZEIGER

tions formed by the local and distant projections of other types of projection neurons.

Origin of postsynapticelements The origin of the spines postsynaptic to callosal axon terminals is uncertain. One possibility is that the spines belong to any or to all of the variety of spiny and sparsely spiny nonpyramidal neurons that occur in the cortex (e.g., Feldman and Peters, 1978; Fairen et al., 1984; Peters and Jones, 1984). Likely candidates for the origin of these spines are superficial and deep pyramidal cells whose spine-laden dendrites are a particularly frequent component of the neuropil in layers I1 and I11 of all cortical areas (Feldman, 1984; Cajal, 1909-11; Lorente de N6, 1922; 1938).Moreover, the general impression obtained from the examination of Golgi preparations is that, except within layer IV, where dendrites of spiny stellate cells abound, there are few spines which belong to nonpyramidal cell types (cf. White, 1989~). These considerations imply that superficial and deep pyramidal cells are the main targets for synapses made in layers I1 and 111, of areas 1 and 40, by callosal axon terminals from both extrinsic and intrinsic sources. Recent evidence has linked callosal neurons, which form only asymmetrical, presumed excitatory synapses (Innocenti, 1986; White, 1989a,c; Toyama et al., 1969) with the excitatory neurotransmitters glutamate and aspartate (Streit, 1984; Dinopoulos et al., 1989; Giuffrida and Rustioni, 1989). The likelihood that other pyramids are the primary targets of both extrinsic and intrinsic callosal axon terminals suggests that callosal neurons play a significant role in augmenting cortical activity both ipsilaterally and contralaterally, a feature which may account, in part, for the efficacy of cutting the corpus callosum in cases of intractable epilepsy (Walton, 1985). A wealth of data indicates that axonal pathways are presynaptic to every type of neuron within their target region (see White, 1989~). Likewise, in the present study, both intrinsic and extrinsic callosal axon terminals synapse not only onto spines and dendritic shafts belonging to spiny neurons but also onto the dendritic shafts of nonspiny and sparsely spiny neurons.

Specificityof d o s a l synapticrelationships As outlined above, extrinsic callosal axon terminals consistently form most of their synapses onto dendritic spines, a pattern displayed in different regions of the cortex and in different species. Similarly, the intrinsic axon terminals of corticocortical (Elhanany and White, 1990) and corticothalamic (White and Keller, 1987) projection cells consistently show the same patterns of output, although different for each pathway, within the separate layers of the mouse barrel cortex in which these pathways terminate. In the present study, intrinsic callosal axon terminals display identical patterns of output in two different regions of the mouse cortex, and as discussed above, these patterns are identical to those of extrinsic callosal axon terminals in the same regions. However, these synaptic relationships should not be taken to mean that identical output patterns necessarily will characterize separate axonal projections that originate from a single source, or from similar sources. For instance, claustral afferents to layers I, IV, and VI of the primary visual cortex of the cat make, respectively, 96%, 50%, and 88% of their synapses with dendritic spines (LeVay, 1986).

Quantitative data on the synaptic output patterns of cortical pyramids have been adduced to support the contention that both extrinsic and intrinsic axonal projections are highly selective with regard to the kinds of targets with which they synapse (Elhanany and White, 1990; White, 1989c; White and Keller, 1989). When considering the degree of selectivity exhibited by populations of axon terminals for their postsynaptic elements, it is critical to take into account the composition of the neuropil within which the terminals occur. Only in this way can it be determined whether the proportions of the different postsynaptic elements contacted by an axonal pathway are merely a reflection of the relative concentration of the different postsynaptic elements in the neuropil or, alternatively, whether the axons have sought out and formed synapses only with specific postsynaptic elements. For example, the ratio of asymmetrical, axospinous to axodendritic synapses in the neuropil of layer I11 in rat auditory cortex is 84:16, whereas the ratio for synapses made by extrinsic callosal afferents to this region is significantlydifferent at 93:7 (x2test P < 0.01) (Vaughan and Peters, 1985). These findings, indicate that axonal pathways are highly selective for their postsynaptic elements, a proposition consistent with additional results from the same region of cortex: At survival times of 3 months after a callosal lesion, thalamic afferents that have grown into the deafferented region (Vaughan and Foundas, 1982) form synapses at essentially the same spine to shaft ratio of 80:20 as reported for thalamic afferents in normal animals (Vaughan and Peters, 1985). Previous results from layers 111, IV, and V of mouse primary somatosensory cortex, where the ratio of asymmetrical, axospinous to axodendritic synapses in the neuropil approximates 60:40, exhibit ratios of 87:13 for intrinsic synapses made by corticocortical projection neurons (Elhanany and White, 1990), and 8:92 for those made by corticothalamic projection neurons (White and Keller, 1987). In both instances, the proportions of spines and dendritic shafts contacted by the intrinsic pathways differs significantly (x2 tests, P < < 0.0001) from the proportions of these elements present in the neuropil (Elhanany and White, 1990; White and Keller, 1989). In the present study, the ratio of asymmetrical, axospinous to axodendritic synapses in the neuropil of layers I1 and I11 is approximately 80:20, significantly different from the 97:3 ratio that characterizes the synaptic output relationships of both extrinsic and intrinsic callosal axon terminals in these layers. These findings are taken as additional evidence that axonal pathways are highly selective for the types of postsynaptic elements with which they synapse.

ACKNOWLEDGMENTS This study supported by NIH grant number 20149 and BSF grant number 89000-52 to ELW and Professor A. Peters, Department of Anatomy, Boston University School of Medicine. We are particularly indebted to Elizabeth and Harry Corob for their recent donation to us of an ultramicrotome.

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