THE JQURNAL OF CQMPAFLUTVE NEUROLOGY 305:370-392 (1991)

Targets of Horizontal Connections in Macaque Primary Visual Cortex BARBARA A. McGUIRE, CHARLES D. GILBERT, PATRICIA K. RIVLIN, AND TORSTEN N. WIESEL The Rockefeller University, New York, New York 10021 (B.A.M., C.D.G., T.N.W.); Cornell University, Section of Neurobiology and Behavior, Ithaca, New York 14853 (P.K.R.)

ABSTRACT Pyramidal neurons within the cerebral cortex are known to make long-range horizontal connections via an extensive axonal collateral system. The synaptic characteristics and specificities of these connections were studied at the ultrastructural level. Two superficial layer pyramidal cells in the primate striate cortex were labeled by intracellular injections with horseradish peroxidase (HRP) and their axon terminals were subsequently examined with the technique of electron microscopic (EM) serial reconstruction. At the light microscopic level both cells showed the characteristic pattern of widespread, clustered axon collaterals. We examined collateral clusters located near the dendritic field (proximal) and approximately 0.5 mm away (distal).The synapses were of the asymmetric/round vesicle variety (type I), and were therefore presumably excitatory. Three-quarters of the postsynaptic targets were the dendritic spines of other pyramidal cells. A few of the axodendritic synapses were with the shafts of pyramidal cells, bringing the proportion of pyramidal cell targets to 80%. The remaining labeled endings were made with the dendritic shafts of smooth stellate cells, which are presumed to be (GABA)ergicinhibitory cells. On the basis of serial reconstruction of a few of these cells and their dendrites, a likely candidate for one target inhibitory cell is the small-medium basket cell. Taken together, this pattern of outputs suggests a mixture of postsynaptic effects mediated by monosynaptic excitation followed by combined disynaptic inhibition and excitation. As a consequence the horizontal connections may well be the substrate for the variety of influences observed between the receptive field center and its surround. Key words: synapses, circuitry, cerebral cortex, pyramidal cells, area 17

An intriguing recent discovery concerning the pattern of cortical circuitry is the existence of a rich plexus of horizontal connections running parallel to the cortical surface. The presence of such connections was first suggested by patterns of degeneration following focal cortical lesions (Fisken et al., '75). Subsequently, intracellular injections of HRP showed that the long-range connections (up to 6 mm in extent) originated from pyramidal cells within the same cortical area (Gilbert and Wiesel, '79, '83; Landry et al., '80; Martin and Whitteridge, '84). Horizontal connections have also been observed by using extracellular tracers (Rockland and Lund, '82 and '83). The finding of rich horizontal connections was a significant departure from the view of cortical connectivity revealed by Golgi impregnations, which led to the belief that cells projected primarily across the cortical layers, with relatively little lateral interaction parallel to the cortical surfzce (Cajal, '11; Lorente de No, '49; Lund, '73; Lund and Boothe, '75). An important feature of the horizontal connections is their clustered appearance. This clustering was shown by physiological and anatomical techniques to be in register with the columnar O

1991 WILEY-LISS, INC.

functional architecture, such that cells having similar orientation preference are interconnected (Ts'o et al., '86; Ts'o and Gilbert, '88; Gilbert and Wiesel, '89; though cross-orientation interactions have been observed in cat area V2, Matsubara et al., '87). In the present study we examine the cell-to-cell connections made by the horizontal connections of superficial layer pyramidal cells in striate cortex of the monkey. Single cells were injected intracellularly with HRP and their dendritic and axonal arbors reconstructed at the light microscopic level with 3-dimensional computer graphics. We attempted to determine whether these intrinsic connections are excitatory or inhibitory by characterizing their synaptic contacts. This was done by using the common ultrastructural criteria of associating synaptic morphology with excitation (represented by type I, asymmetric/round vesicle synapses) or inhibition (represented by type 11, symmetric/ffat vesicle synapses; Uchizono, '65; Bodian, '66; Ribak, '78). In addition, we identified the postsynaptic ~

Accepted November 21,1990.

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Fig. 1. tight microscope reconstruction of HRP-labeled cell 1 seen in parasagittal plane (anterior to left, posterior to right). This cell had a receptive field that belonged to ocular dominance group 5 and was Xo,x y2 ), centered approximately 4" below the fovea near the vertical midline, bi-directional, and not end-inhibited. The pyramidal cell body was 15 K r n in diameter, located in lower layer 3, and its apical dendrites

extended to the pial surface. Several axon collaterals (thinner lines) branch off the descending axon and then ramify near the dendritic tree and in three other clusters within layers 2 and 3. Outlines indicate the locations of blocks trimmed to mesas for electron microscopic analysis. Scale, 100 I L ~ .

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targets by serial EM reconstruction to establish whether the second stage of this intrinsic cortical circuit is inhibitory, via contacts with smooth stellate cells, or excitatory, via contacts with pyramidal cells. Some of these results were presented in a preliminary report (McGuire et al., '85).

MATERIALS AND METHODS Intracellular injections

The HRP-labeled axon terminals and their postsynaptic profiles were followed through the sections a t a print , then at a higher magnification first of about 8 , 7 0 0 ~and magnification of 26,000 x and synapses were characterized as axospinous (made directly on spine heads) or axodendritic (made directly on the shafts of dend.rites) by using standard criteria (see McGuire et al., '84; see also Fig. 5). When necessary, additional micrographs were taken to aid in the following of spine necks or postsynaptic profiles that

Layer 3 cells in Area 17 of two Macaca fascicularis monkeys were injected intracellularly with HRP after their receptive fields were determined, by using a procedure described previously (Gilbert and Wiesel, '79). After completing the injections each monkey was perfused with a saline rinse followed by a mixture of aldehydes (about 1,000 ml of 1% paraformaldehyde/2% glutaraldehyde) dissolved in 0.1 M phosphate buffer. The brain was left overnight in fixative before blocking and sectioning.

Histological preparation Both tissue blocks were sectioned at 50 pm on a Vibratome, the first cut transversely in a parasagittal plane (cell 1) and the second in a tangential plane (cell 2). All sections were reacted with diaminobenzidine with cobalt intensification (Adams, '77). At this point, every other section from each block was processed for electron microscopy and embedded flat on slides, as described in detail in McGuire et al. ('84). The alternate sections from the parasagittal block were mounted directly for light microscopy (see Gilbert and Wiesel, '79). The alternate sections from the tangential block were first processed for cytochrome oxidase histochemistry (Wong-Riley, '79) before mounting.

Light microscopy The dendritic tree and axonal collateral arborization were reconstructed at the light microscope level for each cell, including a detailed two-dimensional drawing and a three-dimensional computer graphic reconstruction (Gilbert and Wiesel, '79). For cell 1 sectioned transversely (Figs. 1-3) we determined the laminar position of the axon collaterals. For tangentially sectioned cell 2 (Fig. 4), the pattern of cytochrome oxidase staining in the superficial layers was traced with a camera lucida at low magnification (1OOx) and then matched to the higher magnification drawing of the cell reconstruction by using the pattern of blood vessels. The cell body position was determined by finding how many 50 pm sections were interspersed between the soma and the honeycomb pattern of cytochrome oxidase staining visible in layer 4A.

Electron microscopy For each cell, two regions of axonal collaterals were selected for examination electron microscopically: one close to the neuron within the arborization of the apical dendritic tree, and a second area further away within a synaptic terminal-rich zone. (Although cell 1 did have an extremely distant cluster that would have been interesting to examine, there were too few synaptic boutons to merit the time-consuming serial sectioning and analysis there.) The appropriate blocks containing these selected regions were trimmed to mesas (see outlines, Figs. 1 and 4), thin sectioned onto Formvar-coated slot grids and stained with heavy metals (see McGuire et al.,'84).

Fig. 2. Light micrograph of cell 1 in 50 pm section. The sections were incubated with DAB to reveal the dense HRP reaction product, and then osmicated and embedded flat in plastic, revealing surrounding pale somata of unlabeled neurons and glia. The labeled pyramidal cell has basal dendrites extending slightly into layer 4A and an apical dendritic arbor that reaches the cell-sparse layer 1,above. Thinner and well-labeled axon collaterals are also visible (open arrows). Regions of the apical dendrites shown in Figure 7 are indicated by small arrowheads. Scale, 50 pm.

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Fig. 4. Light microscope reconstruction of HRP-labeled cell 2 that was sectioned tangentially (antero-posterior and medio-lateral axes indicated). The receptive field of this cell was 1” x lo, centered at an azimuth of -4” and near the vertical midline, strongly end-inhibited and belonged to ocular dominance group 5. Spiny dendrites shown in thicker lines, and axon collaterals in thinner lines. Shaded circles

represent tne locations of cytochrome oxidase-labeled blobs. The cell body and dendritic arbor were located in an interblob space, and most of the axonal collaterals also avoided the blobs. Outlines indicate the shapes of blocks trimmed to mesas for ultrastructural analysis of horizontal connections. Scale, 100 +m.

Fig. 5. a*: Electron micrographs of HRP-labeled synapses. a: Labeled synapse contacting dendritic spine “s” with asymmetric postsynaptic thickening. In adjacent sections, spine was connected to horizontally-oriented dendrite “d” and not to the large pyramidal apical dendrite “P.” b: Two HRP-labeled synapses contact dendritic spines. One of the spines contains spine apparatus and is attached to its parent dendrite “d” via a thin spine neck (arrowheads). c: Three HRP-labeled en passant synapses contact dendritic spines “s.” The

dendrite “d” was found by serial reconstruction (see next panel) to be connected to the centermost spine. d: Serial reconstruction of a portion of the HRP-labeled axon collateral (in black) that contacts 5 spines, each from a swelling. “5,” same spines shown in (c). One spine was followed to its parent dendrite (in gray) that received symmetric (-) and asymmetric synapses ( 0 )along its length. Scales: a-c, 1 km; d, 2 wm.

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extended off the region being followed for the labeled axon. Some of the postsynaptic processes were reconstructed on acetate sheets, and all synaptic inputs to these dendrites were identified as to type, either asymmetric/round vesicle (Type I), or symmetric/flat vesicle (Type 11) according to vesicle shape and size and also the thickness of the postsynaptic density (Colonnier, '68). To compare the relative number of inputs to each dendrite, the density of synaptic inputs was calculated by counting the number of axodendritic contacts along those portions of dendrites that were fully reconstructed, and dividing this number by the dendritic length, adjusted according to the obliquity of each dendrite and the number of sections cut through it. To get an idea of whether the labeled axon collaterals were randomly contacting all processes or preferentially contacting some, an analysis of synapses within the same region was performed. We chose an area of neuropil near cell 2 that was about 25 x 27 pm2in area and contained few cell bodies so as to maximize the density of synapses. Thirteen sections were photographed at a magnification of 6,100X and final print magnification of 1 8 , 3 0 0 ~Then . every synapse with a postsynaptic thickening was identified in the middle 5 sections, scored as asymmetric/round vesicle or symmetric/flat vesicle, and their postsynaptic profiles followed through other sections as necessary to identify them as spines, dendritic shafts, somas, or axons. To learn more about the relative proportions of inputs that spiny cells received on their shafts and spines, we counted the number of spines on several portions of the dendritic tree of the first HRP-filled cell. Using a camera lucida at l , O O O X , stretches of apical and basal dendrites were drawn and every visible spine was counted. Then spine density was determined as a function of length for several different regions of dendrite. Finally, to provide an idea of scale for the postsynaptic dendrites, and to see whether any regions of a pyramidal cell of the same type as the HRP-filled cells might be the appropriate caliber for being Postsynaptic dendrites, the apical dendrite was photographed at 1 , 0 0 0 ~and printed at a scale matching the reconstructions, Then regions of apical dendrite of appropriate caliber were chosen and used to illustrate those stretches of dendritic tree of a similar cell that could possibly be contacted by horizontal connections.

RESULTS Two HRP-injected pyramidal cells were selected for ultrastructural examination. Both neurons had oriented, complex receptive fields and were non-selective for color. The fields were parafoveal and located near the vertical meridian. The soma of cell 1was located in layer 3B (Figs. 1and 2). The cell body seen in the parasagittal plane was obviously pyramidal (Fig. 2), having a rich basal dendritic tree drooping slightly into layer 4A and an apical dendritic tree that extended up to layer 1.The dendrites were spiny. The axon emerged from the base of the soma and eventually entered the white matter and could not be followed further. Several axon collaterals branched at right angles off the main axon primarily within layer 3, and then extended laterally or upwards to arborize within both layers 2 and 3 (Fig. 1). There were four clusters of axon collaterals aligned along the parasagittal plane, separated by boutonsparse gaps (Figs. 1 and 3). The entire axonal arbor extended for 2 mm, with the 3 densest clusters having a 400

TABLE 1. Synaptic Targets of Labeled Terminals Axospinous (pi)

Cell 1 Distal Proximal Subtotal

Cell 2 Distal Proximal Subtotal Totals

Axodendritic (76) 7(18J 7 (26)

31 (82) 20 (74) 51 ( 7 8 )

14 (22)

19 (76) 19 (70) 38 (73) 89 (76)

14 (27) 28 (24)

6 (241 8 (30)

Total (pi)

38 (100) 27 (100) 65 (100) 2.5 (100) 2 1 (1001

52 (100) 117 (100)

km center-to-center spacing. Two clusters were selected for ultrastructural analysis (outlined in Fig. l),one within the cell's dendritic field and the other displaced 400 pm away. Cell 2 was also pyramidal. It was located in layer 3B, just above the cytochrome oxidase stained web characteristic of layer 4A. Its tangential position was in an interblob region (Fig. 4), as would be expected from the fact that its receptive field was oriented and non-color selective (Livingstone and Hubel, '84). Interestingly, most of the axonal arbor was situated outside of blobs as well, including those regions that were chosen for ultrastructural analysis (Fig. 4).The HRP-filled axon collaterals extended in all directions but not as far as the first cell, reaching about 760 km end-toend at its widest extent. The clustered nature of the axon terminals was less prominent than for the first cell. As with the first cell, there were two clusters of terminals that were chosen for analysis, one within the dendritic field, and a more distal one, centered about 300 km away.

Electron microscopy The ultrastructural analysis was done in two stages, first locating and identifying the labeled synapses and then reconstructing some of the postsynaptic dendrites. The labeled synapses were easily identified from their electrondense HRP reaction product throughout the axoplasm (Fig. 5 ) . When the section was thin enough or the reaction product light enough, the synaptic vesicles could be distinguished as round. When the plane of section was appropriate for seeing the postsynaptic densities, the synapses were always characterized as asymmetric (Fig. 5 ) . The synapses tended to be enpassant (32 out of a sample of 49 boutons, or 65%),the remainder arising from spine-like side twigs (14) or terminal boutons (3).Out of a total of 116 boutons, only one formed more than a single output. The overall synaptic pattern for the two cells was the same, with the majority of contacts made on dendritic spines. Of 65 recovered synapses from cell 1,78% were with spines and 22% were with dendritic shafts; of 52 recovered synapses from cell 2, 73% were with spines and 27% were with dendritic shafts (Table 1).The Table also shows little difference in the relative proportions of axospinous and axodendritic synapses between clusters close to the cell body (proximal) and clusters farther away (distal). Out of the total sample of 117 labeled synapses, there were no synapses with either cell bodies or axon initial segments. Furthermore, there were no synapses back onto the parent cell (autapses). Spinypostsynaptic dendrites. Eleven out of 89 postsynaptic spines were reconstructed back to their dendrite of origin, 8 for cell 1 and 3 for cell 2. Seven of these postsynaptic dendrites were reconstructed. Each dendrite had many spines or spine necks along its length, and ranged from 0.2 to 1.2 pm in thickness, most being between 0.4 and

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Fig. 6. Electron microscopic serial section reconstructions of spiny dendrites receiving HRP-labeledcontacts (asterisks). Unlabeled synaptic contacts are shown in black (a)round vesicle/asymmetricsynapses; (-) flat vesicleisymmetric synapses; (0)unclassified synapses. Dendrites a-f from transversely sectioned cell 1; g-i from tangentially

sectioned cell 2. Four dendrites (b,c,d,and g) were vertically oriented in the tissue. These 9 dendrites ranged in diameter from 0.2 pm (h) to 1.1 pm (d), and the density of synapses made along their dendritic shafts varied from 0.5 (i) to 0.1 synapsesikm (c,e).Scale, 10 pm.

B.A. McGUIRE ET AL.

Fig. 7. a-c: Outlines of electron microscopic serially reconstructed apical dendrites. Asterisks mark sites of HRP-labeled inputs. a: Portion of postsynaptic dendrite 6b. b: Portion of postsynaptic dendrite 6d. c: Thick apical dendrite that was adjacent to HRP-labeled collateral but did not receive a labeled input from it. d-f: Photographs of portions of

HRP-labeled apical dendrites from cell 1 (locations indicated by arrowheads in Fig. 2 ) . d Primary and secondary branch, 10-30 pm from soma. e: 7th order branch, 130-150 pm from soma. f: 8th order branch, 160-180 Fm from soma. Scale, 10 pm.

0.8 pm (Fig. 6). The large majority of spines postsynaptic to a labeled input received only a single synapse, but a few received an additional symmetric/flat vesicle input. Five percent of the labeled synapses were located on the dendritic shafts of spiny neurons, bringing the total proportion of spiny cell targets to 81%. From the shape and orientation of the postsynaptic spiny dendrites, several appeared to be apical dendrites (e.g., Figs. Gb-d,g). Since the evidence suggested that a large fraction of the postsynaptic targets were apical dendrites, we wished to assess the likely position of the synapse within a pyramidal cell’s apical dendritic arbor. To this end, assuming the target cells could be similar to the ones injected, we compared the diameters of the postsynaptic apical dendrites with the HRP-labeled apical dendritic tree of cell 1 (see Fig. 7 and arrowheads, Fig. 2). The thinner postsynaptic spiny dendrites (Figs. 6b, 7a) were the same size as the HRP-labeled 8th order apical dendrites, about 175 pm above the cell body. The thicker postsynaptic spiny dendrites (Figs. 6d, 7b) matched the size of the HRP-stained 7th order branches, 140 pm from the soma. Finally, an even thicker apical dendrite (that did not receive a labeled input even though the labeled axon passed right by) was also reconstructed (Fig. 7c), and it was the same size as the primary and secondary apical dendrites of cell 1,just 20 km from the cell body. The match between the calibers of dendrites that are postsynaptic to labeled collaterals and the dendrites of the injected cells suggests that other layer 3 pyramids may be targets, though the possibility exists that peripheral dendrites of deeper lying pyramids may also be targets of horizontal collaterals.

Smooth postsynaptic dendrites. The majority of the dendrites contacted directly on their shaft by the HRPlabeled cells turned out to be smooth and as a group represented 19% of the total sample of postsynaptic sites. There were a total of 28 HRP-labeled axodendritic contacts, 27 of which were partially reconstructed. Five of these were spiny (see above). The remainder (81% of the axodendritic synapses) were smooth and received many synapses, sometimes even evident from a single electron microscopic cross-section (Fig. 8). Twelve of these partially reconstructed smooth dendrites are illustrated in Figures 9-12. Based simply on shape, there were 2 obvious dendritic types among the 22 reconstructed postsynaptic smooth dendrites: varicose and straight. Ten out of the 22 were beaded in shape (Fig. 9d,ij). The constricted portions were 0.1-0.4 krn in diameter and contained many microtubules and an electron dense matrix. Each swelling typically contained several mitochondrial cross-sections and measured about 1 pm at its widest extent. Synaptic inputs were located both on the swellings and along the interbead constrictions (see Fig. 9d). Nine were straight (Figs. 9a-c,g,h, 10, 12, and 15b), ranging in diameter from 0.3 to 1.2 pm. There was some indication that there were two types, one more slender (Figs. 9a,c) and the other more stout (e.g., Figs. 9b, 10, 12), but it is conceivable that these might represent more peripheral and proximal branches of the same cell. The remaining 3 dendrites were intermediate in type (see Figs. 9e,D. In two instances we managed to follow the postsynaptic dendrite to its parent cell body. Both cells were large smooth stellate cells, one in layer 2 and the other in layer 3.

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provided more than 1input over the length reconstructed, and one of these is shown in Fig. 9e. Both dendrites that received a pair of labeled inputs were smooth. We cannot say whether or not different dendrites from the same neuron each receive a labeled input, but it appears at the very least that the inputs must be very sparsely distributed on a neuron, suggesting very little convergence, especially when a composite reconstruction is examined (Fig. 13). Synaptic density. Our ability to distinguish among different types of postsynaptic dendrites was based on more than just qualitative differencesof shape. A useful quantitative tool for differentiating spiny from smooth postsynaptic dendrites was the density of contacts along their shafts. (This was fortunate, since the complete synaptic pattern would have been difficult to characterize, given that spines are often impossibleto reconstruct from unlabeled cells and associate with their parent dendrites in serial sections.) To give an example, consider the two vertically oriented dendrites at the left and right of Figure 13. Each received a single input from the same HRP-labeled axon collateral, and they were the same caliber. In individual electron micrographs, each one looked like it could be a pyramidal cell apical dendrite (e.g., Fig. 14). For the rightmost dendrite, serial reconstruction confirmed this likelihood,revealing many spines and spine necks and a preponderance of symmetriciflat vesicle inputs on the shaft (69% of the 16 identified axodendritic inputs; Fig. 15a). The shaft inputs were also distributed sparsely, about 1every 2 or 3 pm (0.39 synapses/pm). In contrast, the leftmost dendrite in Figure 13 turned out to be smooth (Fig. 15b), and most of the inputs (87% of 70 that were classified) contained round vesicles. The synaptic inputs were also more than 5 times as Fig. 8. Electron micrograph of HRP-labeled synapse with dendritic frequent as those on spiny dendrite 15a, with a synaptic shaft. Note several mitochondria within postsynaptic dendrite “d,” and density of 2.08 synapses every pm. 3 asymmetrichound vesicle unlabeled synapses (thick arrows) on same On average, the spiny dendrites received 1 synapse every cross section. Scale, 1 km. 2-10 pm (mean = 0.3 synapses/pm, N = 121, whereas the smooth dendrites were almost an order of magnitude more Because the reconstruction of the cell bodies was incom- densely innervated, receiving more than one synapse per plete, our figures on soma diameter are underestimates (11 pm (ranging from 0.8 to 3.9 synapses/pm; mean = 1.9; and 13 pm, Figs. 10-12). The labeled inputs were located N = 21, see Fig. 16). On the average, the shafts of spiny proximally on secondary dendrites of the straightlstout dendrites received 52% symmetridflat vesicle inputs (out of variety: the synapse on one cell was about 20 pm from the a total of 77 classifiable inputs), while those of smooth soma (Fig. 101, and the contact on the other cell was only dendrites had a much higher proportion of asymmetric inputs, 81% (out of a total of 514 classifiable inputs). The about 8 pm from the soma (Fig. 12). Both postsynaptic neurons were similar in their cytology, difference in innervation density of spiny and smooth having a rich complement of organelles within their cyto- dendrites confirmed that all of the dendrites classified as plasm. There were numerous broad mitochondria, dense smooth are in fact just that, and not spiny dendrites with lysosomes, clumps of Golgi apparatus and stacks of rough spines that were not recovered by reconstruction. For endoplasmic reticulum scattered around the soma, with example, smooth dendrite 9a had 6 times the synaptic many attached and free ribosomes (Fig. 11).Compared with density as a spiny dendrite of similar thickness, 6b, and a the mitochondria in neighboring dendrites, these cells’ much higher proportion of round vesicle inputs (72% vs. mitochondrial profiles were shorter and fatter with more 22%). Over comparable lengths, we were able to find 8 open cristae. The nucleus of each cell had several indenta- spines on dendrite 6b but none on 9a. The only borderline tions in the membrane. Each neuron had several stout case was perhaps smooth dendrite 9e, which had the lowest dendrites (slightly larger than 1 pm) that emerged directly synaptic density of all the dendrites classified as smooth, from the soma surface with little tapering there. The but a high proportion (71%)of asymmetric inputs. The synaptic density and pattern was the same for the primary dendrites were straight and uncurving, and for one cell, 3 of the 4 dendrites recovered were oriented parallel to postsynaptic smooth stellate cell bodies as it was for the apical dendrites (Fig. 10). The dendritic cytoplasm was partially reconstructed dendrites. A typical cross section moderately electron dense and relatively packed with or- through the soma had about 10 synaptic inputs present, ganelles, including numerous mitochondria, microtubules, and the 2 cells received 57 and 87 inputs over the sections reconstructed (Figs. 10, 12), suggesting that they each rosettes of ribosomes, and smooth endoplasmicreticulum. Number of labeled inputs per dendrite. Out of the received on the order of 200-300 inputs over their entire sample of 33 postsynaptic dendrites described here, there cell body surface. About three-quarters of the axosomatic were only 2 instances in which the HRP-labeled axon synapses were round vesicle ones, and these also predomi-

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Fig. 9. Electron microscopic serial reconstructions of smooth dendrites receiving HRP-labeled inputs (asterisks). Unlabeled synaptic contacts are shown in black: ( 0 )round vesicleiasymmetric synapses; (-1 flat vesicle/symmetric synapses; (D) unclassified synapses. Dendrites a-f from transversely-sectioned cell 1; g-j h m tang.entiaIly~sectionprtcell 2. Synaptic densities range from 3.9 ( g )to 0.8 synapseslpm (el. Scale, 10 pm.

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Fig. 10. Electron microscopic serial reconstruction of smooth stellate cell postsynaptic to cell 1. The HRP-labeled input (asterisk) was on a secondary dendrite, approximately 20 wm from the soma. The straight/stout dendrites of this cell are thickly studded with inputs from unlabeled synaptic terminals, shown in black: ( 0 ) round vesicle/ asymmetric synapses; (- flat vesicleisymmetricsynapses; (V)unclassi-

fied synapses. Three of the four dendrites shown here are oriented vertically within the tissue (parallel to pyramidal cell apical dendrites). The soma also received a high density of synapses, with a majority being asymmetric ones, features typical of the synaptology of smooth stellate interneurons. Since the mid-nuclear plane was not yet reached, the apparent soma diameter of 11.3 bm is an underestimate. Scale, 10 bm.

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Figure 11

CIRCUITRY OF CORTICAL HORIZONTAL CONNECTIONS nated on the dendrites, amounting to 70%-80% from the primary out to the secondary branches. The synaptic density of the dendrites was in the mid-range of all the smooth dendrites (2.53 and 2.08 inputs/pm, see Fig. 16). Postsynaptic dendrite orientation. The nature of the postsynaptic targets suggests a specificity for vertically oriented dendrites. Postsynaptic dendrites were classified as “vertical” (perpendicular to the pia and parallel to the most obvious apical dendrites in the vicinity) if they were longitudinally cut in the parasagittally cut sections, or exactly circular in cross section in the tangentially cut series. A few were called “nearly vertical” because they were estimated as being within 10” of vertical. Similar reasoning allowed us to identify branches as clearly “horizontal,” and all remaining dendrites were called “oblique.” For the 12 reconstructed spiny dendrites, 4 were vertical and 1 nearly so, 4 were oblique and 2 were horizontal, and 1 was a horizontal branch off a thick vertical dendrite. For the smooth dendrite targets, there was also a tendency for the horizontal connections to be made on vertical branches. Considering the 21 reconstructed smooth dendrites, 9 were vertical and 3 nearly so, 5 were oblique and 4 were horizontal. Target selectivity of projection. To determine whether the labeled synapses were specific in their selection of postsynaptic targets we evaluated the overall proportions of the various synaptic types in the vicinity of the labeled axons. A region containing primarily neuropil (rather than neuronal and glial cell bodies) was chosen near the soma of cell 1 for the purpose of identifying the proportions of synapses made within the same area of cortex. In an area about 25 pm x 27 pm (almost 700 pm’)),388 synapses with a synaptic cleft were identified over several serial sections. Of these, 318could clearly be identified as either asymmetric/ round vesicle or symmetric/flat vesicle: 287 were round (90%) and 31 were flat (10%). As shown in Table 2, two-thirds of the round vesicle synapses were axospinous; one-third was axo-dendritic; and very few were axosomatic. Of the flat vesicle synapses, the proportions were the opposite, with two-thirds of them contacting dendrites, and the remaining third split between spines and cell bodies. Within this area, presumably due to a small sample, there were no axo-axonic synapses or round vesicle axosomatic contacts even though both are known to exist. To the extent that the labeled collaterals contact spiny neurons, is there any tendency for the contacts to be made out of proportion to the overall distribution of round vesicle inputs onto spines and dendritic shafts? Selecting the dendrites of HRP-labeled cell #1 as representative for superficial layer pyramidal cells, we estimated the rough proportions of axo-dendritic and axo-spinous synapses. First we needed to get an estimate of the spine density, which ranged from 0 to 1.2 spines/pm and averaged about 0.8 spines/pm, similar to findings in cat sensorimotor cortex (Peters and Jones, ’84).Taking our figures of the proportions and densities of round and flat inputs onto the shafts of spiny dendrites, and assuming one asymmetric

Fig. 11. Electron micrograph of soma and proximal dendrites of postsynaptic neuron, reconstructed in Figure 12. Note slightly indented nucleus (n) and extensive cytoplasm packed with organelles. The mitochondria (open arrows) are thicker and paler than those in most neighboring dendrites. Frequent axosomatic synapses are present in this typical cross section, both asymmetric (thick arrows) and symmet-

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input per spine plus a small proportion of symmetric inputs (roughly 7% as reported in cat striate cortex, Colonnier, ’81; Feldman, ’841, we come to the figure that pyramidal cell dendrites have total synaptic densities of approximately 1 synapse/ym, of which 80-90% would be asymmetric. The dendritic distribution of these asymmetric inputs would range from 2:l to 20:l between spines and shafts. The labeled axon terminals in this study had a synaptic target ratio of 18:1, spines versus dendritic shafts.

DISCUSSION Pyramidal cells as postsynaptic targets Our results show that pyramidal cell spines are the most common target of the short- and long-range axon collaterals of layer 2-3 pyramidal cells. This is clear from several pieces of evidence. First, three-quarters of the HRP-labeled synapses were made with dendritic spines. Since spiny stellate cells and their dendrites are for the most part restricted to layer 4, these spines almost certainly belong to pyramidal cells, the most prevalent cell type in the superficial cortical layers (Cajal, ’11; Lund, ’73; Lund and Boothe, ’75; Valverde, ’85). In fact, an additional 6% of the postsynaptic dendrites received the labeled input directly on the dendritic shaft but were actually spiny, too, making the percentage of synaptic outputs onto pyramidal cells just over 80%. The morphology and synaptic pattern of the postsynaptic spiny dendrites are also consistent with the hypothesis that they belong to pyramidal cells: generally straight branches with pale cytoplasm, few mitochondria, sparse synapses along their dendritic stalks, many spines or spine necks, and usually a preponderance of symmetric/flat vesicle axodendritic inputs. Connectivity of other types of pyramidal cells. These findings are in close agreement with two other types of pyramidal cells, layer 3 and layer 5 cells in the cat. Using similar techniques, Kisvarday et al. (’86) showed that 84-87% of the elements postsynaptic to layer 3 pyramids were dendritic spines, and some of the inputs to dendritic shafts were also with pyramidal cell-like branches. Just as we show here for similar cells in the monkey, the pattern of synaptic contacts was the same in distal as it was in proximal clumps of axon terminals, suggesting that in both mammals, superficial pyramidal cells probably perform the same functions close to the soma that they do farther away. Large layer 5 pyramids in the cat had a similar synaptic circuitry, with 78-81% of the output onto spines, and no differences between the proximal and distal clumps (Gabbott et al., ’87). Undoubtedly, this prevalence of pyramidal cell synapses with dendritic spines that has been seen now with several cell types is the basis for the findings of Fisken et al. (’75) who saw mostly degenerating asymmetric axospinous synapses located laterally from a lesion in primate striate cortex. A few types of pyramidal cell, though, have been shown to primarily contact the shafts of dendrites rather than the

ric (arrowheads) ones. Primary and secondary dendrites (d) can be seen above, containing moderately electron-dense cytoplasm. The synaptic input along the dendrites is also very dense and primarily from asymmetrichound vesicle endings. The HRP-labeled input cannot be seen in this section. Scale, 10 pm.

Figure 12

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spines. In a study of a pyramidal cell in layer 213 of monkey SI cortex (Winfield et al., 'Ma), 60% of the labeled axon terminals contacted dendritic shafts. The reason for a difference of this magnitude between pyramidal cells in two different areas of primate sensory cortex is unclear, but it suggests that cells can be quite specific and distinct in their projection target. This difference in specificity can apply to particular elements in the cortical circuit, such as the projection by layer 6 pyramidal cells to layer 4.The axon collaterals of cat layer 6 pyramids in layer 4 make 72% of their synapses with dendritic shafts, many of which have the morphology of smooth or sparsely spiny dendrites (McGuire et al., '84). Another pyramid, a layer 4 cell in rat visual cortex, made only half of its 16 recovered synapses with spines, the remainder being shafts (Somogyi, '78). Finally, corticothalamic cells in mouse SmI cortex, as a group, have been shown to have 90% of their synapses on dendritic shafts (White and Keller, 1987). It is tempting to speculate that these differences in synaptic target reflect not simply species differences but also unique target specificities required for the receptive field properties that are generated by a given cortical connection (e.g., Gilbert and Wiesel, '81; Bolz and Gilbert, '86). From the present study, taken together with earlier work, it seems possible to predict the nature of the postsynaptic targets of thalamic a e r e n t s and pyramidal cells simply from the morphology of their presynaptic endings. Afferent axons from the lateral geniculate nucleus with boutons en passant contact dendritic spines (see, e.g., Ferster and LeVay, '78; Winfield et al., '81b), while axons with boutons at the end of spine-like side twigs contact dendritic shafts (e.g., layer 6 pyramidal cells projecting to layer 4, McGuire et al., '84; layer 2/3 pyramidal cell, Winfieldet al., '81a). This appears not to be true for smooth stellate cells, such as chandelier and basket cells, which form en passant contacts but do not contact spines (Somogyi, '79; Fairen and Valverde, '80; Somogyi et al., '82; Kisvarday et al., '87). It seems as if side twigs and spines allow their parent axons and dendrites to come within sufficientlyclose range to another process to form a contact, thereby allowing the smooth axonal collateral or dendritic shaft itself to avoid an otherwise tortuous course. It is interesting to note that we have many examples of spiny dendrites receiving frequent asymmetric synapses on their dendritic shafts. This was unexpected in view of the classic view of spiny cells having primarily symmetric contacts on their dendritic shafts (LeVay, '73; Parnavelas et al., '77; White et al., '80; Hornungand Garey, '81; Feldman, '84; Saint Marie and Peters, '85). The dendrites that received frequent asymmetric input tended to have the densest overall input for the spiny group, but though they approached the characteristic of smooth stellate cells in this regard, there was no indication that they represented intermediate forms ("sparsely spiny"). It remains to be seen whether the spiny branches with many round vesicle inputs belong to specific cell types or to certain locations within the dendritic field, such as the most distal branches.

In any event, it is clear that the proportion of asymmetric inputs on the shaft is not a sufficient criterion for classifying a dendritic profile as belonging to a smooth stellate cell. Sparse interconnectiuity. There are remarkably few contacts between the labeled axon and individual postsynaptic dendrites. In just 2 cases out of 117 were there two inputs to a single dendrite. It is possible that different branches of the same cell may each receive a single contact, but the geometry of the axon and postsynaptic dendrites as seen in Figure 13 suggests to us that this could only happen rarely, and then on widely separated dendrites of the same cell. Therefore, we do not believe that there is significant convergence of synaptic contacts onto a single postsynaptic cell. These data agree well with the results of Kisvarday et al. ('86),Gabbott et al. ('871, and McGuire et al. ('84), who all found that most of the synapses from pyramidal cells were made sparsely on their targets, usually only one at any location. Perhaps this helps to explain the physiological results of Ts'o et al. ('86) and Ts'o and Gilbert ('88), who only found weak excitatory interactions between neurons in cat and monkey visual cortex. Individual horizontal connections, then, are weak relative to interlaminar or afferent connections, which show strong cross-correlations (Toyama et al., '81a,b; Tanaka, '83), though anatomical studies do not show the expected richness of innervation for afferent inputs onto identified postsynaptic cells (Freund et al., '85). This does not mean, however, that the horizontal connections represent a weak influence, but rather that their influence is exerted by a mass-action effect, involving the activation and convergence of input from hundreds of cells in many columns, as opposed to the vertical connections, which exert their influence by a relatively small number of cells in a single column.

Fig. 12. Electron microscopic serial reconstructionof neuron shown in Figure 11 and viewed in the tangential plane. The HRP-labeled synapse (asterisk) was made on a secondary dendrite, about 8 km lateral from the soma. Three straightistout dendrites emerged directly from the soma that was partially reconstructed, not yet to the mid-

nuclear plane. The entire neuron received very dense synaptic input, predominantly from asymmetric/roundvesicle synapses (01,with the remainder being from symmetric/flat vesicle (-1, and unclassified (0) synapses. The morphology and cytology and synapticpattern of this cell match those for the cell illustrated in Figure 10.Scale, 10 km.

Smooth stellate cells as postsynaptic targets One-fifth of the projection from layer 213 pyramidal cells is onto dendrites having the morphology and synaptic patterns of smooth stellate cells. The evidence supporting their identity is substantial: their dendrites lacked spines and spine necks entirely; they received a preponderance of asymmetric inputs (a well-established characteristic of smooth stellate cells, Colonnier, '81); and they received a higher density of synaptic inputs than did the spiny dendrites (Fig. 16). These results agree qualitatively with the analysis of layer 3 pyramids in the cat (Kisvarday et al., '86), though these authors found that no more than 5%of their postsynaptic targets belonged to smooth stellate cells, compared to our figure of 20%in the monkey. We do yet not know whether this fourfold difference in the estimate of postsynaptic inhibitory interneurons is due to species differences or to differences in technique. Variation in shape and size suggest that the postsynaptic smooth dendrites belong to several different cell types. The one about which we have the most information is the smooth dendrite that in two examples we were able to follow back to parent cell bodies. The soma diameters

B.A. McGUIRE ET AL.

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,

I

Fig. 13. Composite electron microscopic serial reconstruction of a portion of the HRP labeled axon collateral from the distal patch of cell 1, mostly within lower layer 2. Also shown (thinner lines) are outlines of 7 of its postsynaptic dendrites. Asterisks, sites of synaptic output, usually at en passant swellings. It is clear from the trajectory of the axon that it

1

often has only a single opportunity to contact many dendrites, so that the innervation of individual dendrites and neurons must be extremely sparse. Many of the postsynaptic processes are vertically oriented within the tissue. Two of these (at the far left and right) are shown in more detail in Figure 14. Scale, 10 pm.

CIRCUITRY OF CORTICAL HORIZONTAL CONNECTIONS

Fig. 14. Low power electron micrograph of HRP-labeled synapse (asterisk and arrow) with the shaft of a pyramidal cell apical dendrite (“Ap”). To right, another apical dendrite and a pyramidal cell soma (“P”) with its primary apical dendrite extending upward toward the pia. The postsynaptic apical dendrite shaft receives only 3 unlabeled

387

inputs in this section: one asymmetric (large arrow), one symmetric (arrowhead) and one unclassified (open arrow). Two asymmetric inputs (indicated by arrows) a r e seen on spines (“s”) that were connected in serial sections. See serial reconstruction of this dendrite in Figure 15a. Scale, 10 Fm.

B.A. McGUIRE ET AL.

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Synaptic Density Fig. 16. Histogram of synaptic density for the 33 postsynaptic dendrites described in this paper. There was no overlap between the synaptic densities of the spiny and smooth postsynaptic dendrites. Most smooth dendrites received more than 1synapse per pm along the shaft. Every postsynaptic spiny dendrite received 1synapse or less every 2 pm along its shaft ( 5 0 . 5 synapsesipm). Though there were 4 times as many postsynaptic spiny as smooth dendrites in the total sample, fewer spiny branches were reconstructed because of the difficulty in following spine heads back to their dendrites of origin.

(13-15 km) place them among the largest probable GABAcontaining neurons that have been seen in the upper layers of monkey somatosensory cortex (Hendry and Jones, '81). Other smooth stellate cell classes are either too big (giant cells and Type I large basket cells) or too small (neurogliaform, chandelier, and double bouquet cells) to be suitable candidates (Jones, '75). The best candidate is the small or medium basket cell, reported to be 12-15 km in size, with thick, straight dendrites that branch infrequently and are primarily vertically oriented within the cortex (Type I1 cells, Jones '75; Tombol, '78; Jones and Hendry, '84).The cytoplasm of our reconstructed cells, moreover, had cytoplasm similar to large basket cells of the monkey sensorimotor cortex and cat striate cortex, being relatively electrondense and having many mitochondria and other organelles as well as extremely dense synaptic input (Sloper et al., '79; Somogyi et al., '83). There is good evidence that this cell type, as well as other smooth stellate cells, is inhibitory (Ribak, '78; Freund et al., '83; Somogyi et al., '84; DeFelipe et al., '86; Somogyi, '86; Kisvarday et al., '87). Each basket cell (in the cat) makes contacts on pyramidal cell somata, dendrites, and spines, with as many as 8 inputs seen with a

Fig. 15. Electron microscopic serial reconstructions of 2 dendrites postsynaptic to cell 1 that each receive an HRP-labeled contact on their shaft (asterisk). Other synaptic inputs shown in black: ( 0 )asymmetric/ round vesicle synapse; (- symmetric/flat vesicle synapse; (V)unclassified synapse. a: Pyramidal cell apical dendrite (see profile, far right, Fig. 13). Note frequent spine necks and spines. A majority (65%) of the identified inputs along the shaft contained flat vesicles, and the overall synaptic density along the shaft was 0.4 synapsesum. b: Smooth stellate dendrite (see profile, far left, Fig. 14). The orientation and caliber are similar to apical dendrite. However, this dendrite lacks spines, has primarily round vesicle inputs, and a synaptic density of 2.1 synapsesipm along the completely reconstructed portion. Scale, 10 %m.

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389

TABLE 2. Synaptic Targets Within Layer 3 Neuropil

Asymmetric Symmetric Totals

Axospinous (70)

Axodendritic (70)

Axosomatic

181 (68) 11 (33) 192 (64)

83 (31) 17 (52) 100 (33)

3 (1) 5 (151 8 (3)

(%I

Axoaxonic (96)

Total

0 (0) 0 (0) 0(0)

267 (90) 33 (10) 300 (100)

(%I

~

single soma and up to 8 or more contacts on a single apical dendrite (Somogyi et al., '83). The second class of postsynaptic smooth dendrite was varicose. The ones seen in our study appear similar to those seen as targets of layer 2/3 pyramidal cells in monkey somatosensory cortex (Winfield et al., '81a). They may belong to the types of cells known as neurogliaform cells (Tombol, '78; Jones, '841, "local plexus" interneurons (Peters and Saint Marie, '84, Fig. 9)' and/or horsetail neurons (Tombol, '78).

cells may be amplified by being located closer to their cell bodies, in comparison to the seemingly peripheral placement of synapses on the pyramidal targets, and this could counter the smaller numbers of synapses involved. Further, the effect of the subsequent connections between the inhibitory interneurons and their targets can be amplified by several factors. One is that the synapses of the postsynaptic interneurons, particularly those of the basket cells that appear to be involved, are on the somata of their target cells, so they are likely to be very effective. Secondly, the axonal arbors of smooth stellate cells tend to be more Target selectivity of horizontal connections focused, limited in their tangential extent, compared to Our results do not lead one to conclude that the horizon- those of pyramidal cells. This would tend to make the tal connections preferentially contact particular cell types. spread of excitation sparser and covering a larger territory The proportion of synapses onto smooth stellate cells (20%) (thus weaker at any given site), whereas the inhibitory matches the fraction of GAEiAergic neurons in striate influences would cover a smaller territory but be relatively cortex found by Hendry et al. ('87). The percentage of more influential within the area covered. We lack the contacts by the horizontal connections and spines (75%)is necessary information to assess the relative importance of quite similar to the overall proportion of axospinous Type I the inhibitory and excitatory influences. For example, one synapses in the superficial layers (68%,this report; 80% in would need to establish the effect of activating a distal the cat, Beaulieu and Colonnier, '85). This might lead one synapse on a pyramidal cell-is it propagated with little to believe that the projection ends randomly on all neurons within a terminal cluster, and does not select for spines decrement to the cell body, as suggested in the hippocamversus dendritic shafts, in contrast to the specificity men- pus (Andersen et al., '80, '86), or is it less effective than the tioned above for the projection from layer 6 pyramidal cells proximal inhibitory contacts? One also needs more compreto layer 4 (80%smooth and sparsely spiny stellate contacts, hensive information on the targets of the postsynaptic McGuire et al., '84). The possibility does remain, however, smooth stellate cells. Physiological evidence for long-range excitation has been that the target cells could be a subpopulation of superficial layer cells, specific for a characteristic other than smooth seen using cross-correlation analysis (Ts'o et al., '86; Ts'o versus spiny dendrites. Our analysis does not rule this out. and Gilbert, '88), though this technique tends to be better Our results give an indication that horizontal connec- for seeing monosynaptic and common input excitation than tions prefer vertical dendrites of both spiny and smooth disynaptic inhibition. Experiments in tissue slices show cells. This is subject to the caveat that we do not know the both excitatory and inhibitory influences resulting from total dendritic length of vertical versus non-vertical den- activation of the horizontal connections, and the balance drites within the tissue. Nonetheless, for pyramidal cells we between excitation and inhibition is dependent on the state would like to suggest that there may be some preference for of activation of the recipient cell (Hirsch and Gilbert, '89). the horizontal connections to contact apical dendrites. For Clearly, the results presented here provide the anatomical smooth stellate cells, this tendency may either reflect a substrate for such combined influences, and other inputs to similar partitioning onto vertical dendrites or may indicate the cells receiving horizontal connections can determine the a preference for smooth stellate cell types with vertically net effect of the excitatory and disynaptic inhibitory connecelongated dendritic trees, such as the small-medium basket tions arising from the horizontal inputs. The consequences of activating the horizontal conneccell. This is reminiscent of the pattern of connectivity in the cerebellum and hippocampus, where the horizontal parallel tions on receptive field structure can therefore be thought fibers contact the vertical branches of Purkinje cells, and of in terms of both excitatory and inhibitory features. perforant path fibers contact the apical dendrites of pyrami- End-inhibition appears to be generated by layer 6 cells (Bolz dal cells. and Gilbert, '86), whose large receptive fields require integration made possible by the horizontal connections Functional consequences of the pattern of (Bolz and Gilbert, '89). Receptive fields also have sidesynaptic innervation by horizontal bands, where a stimulus cannot by itself activate a cell, but connections can modulate a cell's response in an excitatory or inhibitory At the outset, the predominance of horizontal input via direction to a stimulus presented within the receptive field asymmetric synapses with other excitatory (pyramidal) core (Bishop et al., '73; Nelson and Frost, '78, '85; Allman neurons would lead one to expect an excitatory role for et al., '85; Tanaka et al., '86; Orban et al., '87; Wiesel and these connections (see summary diagram, Fig. 17). How- Gilbert, '89; Van Essen et al., '89; Grinvald et al., '89; ever, there could still be a substantial role of inhibition even Gilbert and Wiesel, '90). These influences require input if a minority of the synapses are with inhibitory interneu- from a larger area of cortex than would be necessary to rons. For example, the effect of the input to smooth stellate produce the receptive field core alone, and it is tempting to

Fig. 17. Schematic diagram of intrinsic horizontal connections described in this paper. Superficial layer pyramidal cell (in black) has discrete clusters of axon terminals. Within each cluster, the axon terminals contact other pyramidal cells (usually on spines, not shown), and some smooth stellate cells. There are several morphological types of smooth stellates contacted, suggested by different shapes here. Actual laminar position of pyramidal cells is not known.

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CIRCUITRY OF CORTICAL HORIZONTAL CONNECT10INS speculate that the horizontal connections are the basis for this class of interactions. One cannot help but realize from the work presented here that the characterization of cortical microcircuitry is still just beginning. Our lack of knowledge about the cortex is particularly striking in view of what is known about the retina, where considerably more has been described about cell types, their connectivity and pharmacology. Part of the challenge making the cortex more difficult for ultrastructural analysis is the considerable extent of the axonal and dendritic fields of cortical neurons. In the future, however, this analysis will be greatly facilitated by the increasing availability of markers specific for particular cell types. The use of double label techniques will then minimize the necessity for extensive serial electron microscopic reconstruction, and allow one to characterize more easily and completely the targets of all cortical connections.

ACKNOWLEDGMENTS This work was supported by NEI grants EY06792 (B.A.M.), EY05253 (T.N.W.), NSF grants BNS 8351738, and BNS 8615935 (C.D.G.) and an award from the Rita Allen Foundation (C.D.G.). We also acknowledge our excellent support staff: Peter Peirce for photographic assistance, Joyce Powzyk for cell reconstructions, and Julie Dollinger and Eric Velazquez for technical support.

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