The axon collaterals of dentate granule cells have been analyzed with the aid of a computerized microscope, following intracellular injections of horseradish peroxidase in hippocampal slice preparations. The axon of each granule cell gives rise to approximately seven primary collaterals; these collaterals usually divide into secondary and tertiary branches, which form an extensive plexus within the hilar region of the dentate gyrus. Individual axon collaterals vary greatly in length, but most have been found to be between 100 and 300 microns long. On average, the summed lengths of the collaterals (exclusive of the parent mossy fiber) are approximately 2,300 microns. Except for an occasional collateral that is given off by a mossy fiber in the proximal part of field CA3 of the hippocampus, the collaterals of the granule cell axons are confined to the hilar region; they are rarely seen in the granule cell layer itself and have never been observed in the molecular layer. In the longitudinal dimension of the dentate gyrus, most of the collaterals are contained within a zone about 400 microns wide. The distribution of the collaterals within the hilar region is correlated with the location of the granule cell body. Those that arise from cells near the tip of the suprapyramidal blade tend to be confined to the region above field CA3; those from cells nearer the crest and from the infrapyramidal blade ramify widely throughout the hilus. Two types of varicosities are present on the collaterals. Numerous small (approximately 2 microns), round varicosities are distributed unevenly along the collaterals; in electron micrographs these varicosities can be seen to make asymmetric synaptic contacts with dendritic shafts. On average, each granule cell collateral plexus has about 160 of these varicosities. The second type of varicosity is irregular in shape and ranges from 2 to 4 microns in diameter; there is usually only one such varicosity per collateral. In all respects except size, these varicosities resemble the expansions found on the parent mossy fibers. Mossy fiber trajectories in the proximal part of field CA3 were studied after extracellular injections of HRP into localized regions of the granule cell layer. Granule cells at different locations around the blade send their mossy fibers to different depths within the pyramidal cell layer in the proximal part of field CA3. However, further distally, mossy fibers from all parts of the granule cell layer contribute to the suprapyramidal bundle that occupies the stratum lucidum.

Figure 1

Light micrographs of HRP-filled granule cells. A. The animal used for this experiment was perfused with Oxypherol to eliminate red blood cells (curved arrows). The axon (arrowhead) can be seen exiting from the granule cell body that is located in the suprapyramidal blade. B. The cell shown here was from a nonperfused animal; numerous red blood cells (curved arrows) are stained because of their endogenous peroxidase activity. This cell was located in the infrapyramidal blade; note the ventricular space (asterisk) above the dendritic layer. Scale bar = 100mm

Figure 2

Figure 3

A camera lucida drawing of an HRP-filled dentate granule cell located in the middle of the suprapyamidal blade (see insert). The primary or parent mossy fiber extends through the hilar region (H) to the pyramidal cell layer (PL) of the hippocampus. The dashed lines show the approxitmate border between the hilar region of the dentate gyrus and field CA₃ of the hippocampus; the broken lines in the inset drawing marks the location of field CA₃. The collaterals that are given off by the mossy fiber within the hilus have small, round varicostities (some are indicated by arrowheads) and large, irregular varicosities (enclosed by open squares) in CA₃. Cut collaterals are indicated by solid circles. Note that some collaterals extend completely across the hilus and approach the infrapyramidal blade (IPB). Note also the absence of large mossy expansions on the parent axon until after it enters field CA₃. Scale bar = 100mm

Figure 4

A camera lucida drawing of another HRP-filled granule cell, located in the infrapyramidal blade near the crest (see insert). Note that the collaterals of this cell extend throughout much of the hilar region. The computer plot of this cell is shown in Figure 5D. Symbols indicating collateral varicosities and expansions are the same as in Figure 3. Scale Bar = 100mm.

Figure 5

Computer reconstructions of the collateral plexuses of dentate granule cells from four different animals. The cells in A and B were located in the suprapyramidal blade3 (SPB), whereas those in C and D were in the infrapyamidal blade (IPB). In each plot, the dendrites extent to the top of the molecular layer (ML), the cell bodies are in the granule cell layer (GL), and the axons travel through the hilus (H) to reach the pyramidal cell layer (PL) of the hippocampus (CA₃). The exact locations of both types of collateral varicosities were entered into the computer; in the plots, the smaller varicosities are represented by a fixed-size dot, whereas the larger varicosities (open circles) are plotted to scale. Mossy fiber expansions (open squares) occur on the parent axon, usually after it has entered the pyramidal cell layer, but occasionally within the hilus as in A and B. A. The collaterals of this cell are confined to the region of the hilus (the approximate limits of which are indicated by the broken line) directly beneath the granule cell layer. Note that the mossy fiber passes directly from the hilus to CA₃, avoiding the proximal portion of the pyramidal cell layer; in this case the parent mossy fibers and two of the collaterals were cut (as indication by the filled circle at its termination) when the slice was made. B. The granule cell shown here had the greatest total collateral length of the cells we have studied. Note that the collaterals extend to the IPB and that almost all of them exhibit a large varicosity (open circles). The parent mossy fiber was not cut in this case, but the HRP staining faded until it was no longer visible beyond the proximal part of the field CA₃. C. This is the same cell shown in the camera lucida drawing of Figure 4. Note the long primary collateral extending to the SPB. D. This cell was in the middle of the IPB. Note that its collaterals do not quite reach the GL at the crest, but do approach the SPB, and that there are two large expansions (open circles just to the right of the "H") on one collateral. Scale bar = 100mm

Figure 6

A computer reconstuction of the collateral plexus of a granule cell axon on which the lengths of the individual collaterals (excluding the three that were cut-filled circles) are indicated. The small varicosities are shown as dots but the large collateral expansions are not marked. The dendrites and most of the parent mossy fiber (arrowhead) are not illustrated. Scale bar = 100mm.

Figure 7

Composite diagram of the territories of the five representative collateral plexuses shown in Figures 3 - 5. The different symbols mark a line drawn around the distal tips of the collaterals in each case. Note that the collaterals of the cell shown in Figure 5A (marked here by asterisks) do not overlap with those of the cells located near the crest, whereas the collateral cells around the crest region have extensive overlap within the hilus. Scale bar = 100mm

Figure 8

Computer reconstructions of the collateral plexuses of two granule cells in the infrapyamidal blade. The computer plots have been rotated 90° about the Y axis, so that the cut surfaces of the slices (indicated by the dotted lines) are visible. Note that the majority of the collaterals remain within the thickness of the slices though three branches in each plexus had been amputated (filled circles) when the slices were made. The ends of the parent mossy fibers are indicated by arrowheads. Scale bar = 100mm.

Figure 9

Computer reconstructions of two adjacent granule cells that had been simultaneously filled with HRP. The collaterals of cell 1 (drawn with broken lines) have a slightly greater extent in the transverse plane than do the collaterals of cell 2 (continuous line). The total collateral length (excluding the parent axon) of cell 1 was 2,334mm and that of cell 2 was 1,554mm. Cell 1 had five primary branches exiting from the parent mossy fiber, whereas cell 2 had six; both cells had collaterals cut (filled circles) at the same edge of the slice, although the cut ends on cell 2 were closer to the cell body, which perhaps accounts for the smaller collateral length of this cell. Scale bar = 100mm.

Figure 10

Electron micrographs of sections through one HRP-filled small, collateral varicosity that forms an asymmetric synapse upon a dendritic shaft. Note the vesicles in the presynaptic process and the marked postsynaptic density (arrowheads). A second presynaptic process (asterisks), not filled with HRP, contacts the same dendrite. Scale bar = 0.5mm

Figure 11

An electron micrograph of a small collateral varicosity. The insert shows a micrograph (of the same varicosity) taken several sections away. Note the presynaptic vesicles and postsynaptic density (arrowheads). Scale bar = 0.5mm.

Figure 12

A photomicrograph of a coronal section through the rostral part of the dentate gyrus stained by the Timm sulphide silver method for heavy metals. The mossy fibers are stained darkly and extent from the hilus (H) into field CA₃ of the hippocampus (white arrowheads). In the proximal part of CA₃ stained mossy fibers are also located within the pyramidal cell layer (white curved arrow) although it is difficult to distinguish distinct intra- and infrapyramidal bundles. The hilar region is darkly and uniformly stained indicating the presence of the innumerable collateral varicosities. GL = granule cell layer; ML = molecular layer. Scale bar = 500mm.

Figure 13

Light micrographs of mossy fibers labeled by an extracellular HRP injection. A. This shows the injection in the crest region (open arrow), labeled mossy fibers and collaterals in the hilar region (H), and labeled mossy fibers (arrowhead) in the pryamidal cell layer (PL) of field CA₃. Scale bar = 200 mm. B. This is a higher-power micrograph of the proximal part of the pyramidal cell layer (PL). Note the mossy fibers (arrowhead) in the deep portion of the pyramidal cell layer. Some of these pass through the pyramidal layer to enter the stratum lucidum. Scale bar = 50mm. C. A section cut from the same 400-mm slice shown in A and B. Note the mossy fiber expansions (arrowheads) in the proximal part of the pyramidal cell layer (PL) and near the edge of the hilus (H). Scale bar = 50 mm.

Figure 14

A schematic diagram to summarize the major features of the mossy fiber trajectories in the proximal part of the hippocampus. Axons from granule cells in the in suprapyramdal blade (SPB) are represented by solid lines and those from granule cells in the crest and infrapyamidal blade (IPB) are indicated by dashed lines. Note that mossy fibers from cells at the tip of the SPB cross directly through the stratum radiatum (asterisk) to reach the stratum lucidum (open arrow), and in so doing avoid the proximal part of the pyramidal cell layer, whereas fibers that arise from other parts of the granule cell layer through the hilus (H) and then contact pyramidal cells in the proximal part of field CA₃. Mossy fibers from all parts of the granule cell layer (GL) are found above the distal portion of the pyramidal cell layer (PL, indicated by heavy dotted line) in the strtum lucidum. Scale bar = 100mm.

Figure 15

A histogram of the distances between expansions on mossy fibers in field CA₃ of the hippocampus.

Figure 16

A diagram of the dentate gyrus and proximal portion of the hippocampus to summarize the major synaptic relationships of the mossy fibers. Granule cells (marked 1 and 2) give rise to extensive collateral plexuses that are distributed throughout much of the hilus. These collaterals have two types of synaptic varicosities (several of the larger varicosities are enclosed within circles) that terminate on cells of the hilar region. Some of these varicosities contact the basal dendrites of pyramidal basket cells (3) which give rise to the GABAergic basket plexus that terminates on the somata and proximal dendrites of the granule cells (black arrows); this serves as an inhibitory feedback circuit. Other collateral varicosities terminate on the mossy cells (4) of the hilar region that give rise to the associational and commissural afferents to the dentate gyrus that terminate in the inner third of the molecuar layer (arrowheads); these connections serve as an excitatory feedback circuit. While it is likely that the mossy fiber collaterals terminate on many other types of hilar neurons, these relationships have not yet been experimentally demonstrated. As the parent mossy fiber approaches the pyramidal cell layer, the large presynaptic expansions begin to appear (some are enclosed in squares) and are found approximately 140mm apart for the entire length of the axon. These expansions form complex synapses with the specialized spines (thorny excresences) on the apical and basal dendrites of the pyramidal cells (5). A few collaterals usually arise from the mossy fibers in the proximal part of the pyramidal cell layer (open arrows) and terminate in the hilar region of the infra-, or more commonly, the suprapyramidal blade; these collaterals often have a large expansion in the hilar region. Asterisks mark the origin of the axon for each of the cells illustrated. Scale bar = 100mm.