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Visual cortical organization at the single axon level: a beginning
Neuroscience Research 42 (2002) 155 /166 www.elsevier.com/locate/neures
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Visual cortical organization at the single axon level: a beginning Kathleen S. Rockland * Laboratory for Cortical Organization and Systematics, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan Received 22 October 2001; accepted 12 December 2001
Abstract Single axon analysis of visual cortical connections is an important extension of previous anterograde studies using 3H-amino acids or wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). The higher resolution tracers */Phaseolus vulgaris leucoagglutinin (PHA-L), biocytin, biotinylated dextran amine (BDA) and dextran-conjugates */have already produced new results, simply by providing improved visualization, concerning laminar definition and possible subtypes of connections, as well as the beginning of a database of morphometrics and microstructure. The comparative approach, comparing geniculocortical terminations and cortical connections across several areas, has suggested both specific structural /functional correlations (for example, in extrastriate area MT/V5) and more subtle, possibly gradient-wise variations. Likely future directions for this line of research include more direct correlations of axon geometry with functional architectures, investigations of microcircuitry at the level of electron or confocal microscopy, anatomical and functional investigations of connectional convergence and interactions, and, not least, a more comprehensive database. # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Anterograde; Convergence; Divergence; Feedback; Feedforward; Pulvinar
1. Introduction A common approach to understanding visual cortical organization has been to examine successive transformations in the neural representations of the visual world, and to try to identify the underlying neural substrates. This has been extremely successful in the early part of the visual pathway. For example, combined physiological recording and intraaxonal filling of geniculocortical connections demonstrated the laminar segregation of the magno- and parvocellular pathways, promoted the idea of further segregation in the efferent pathways from V1 to extrastriate areas, and provided the baseline data necessary for interpreting developmental and plasticity paradigms (Blasdel and Lund, 1983; Freund et al., 1989; Florence and Casagrande, 1990). Comparable studies of long distance cortical connections beyond V1, however, have been more difficult, in part because of the sheer length and intricacy of these connections. Of the many anatomical studies addressing * Tel.: 81-48-467-6427; fax: 81-48-467-6420. E-mail address: [email protected] (K.S. Rockland).
cortical connectivity, most have been carried out using large injections of horseradish peroxidase (HRP) or fluorescent dyes as retrograde tracers, and 3H-amino acids or WGA-HRP as anterograde. These tracers have been excellent for demonstrating the general lay-out of interconnected structures, as well as providing data about topographic and laminar organization and, to some extent, relative density of connections. Especially the anterograde tracers, however, have left unanswered basic questions concerning the fine organization of connections, related to the morphometrics of terminal arbors and boutons. These data are necessary for further consideration of how microstructural features correlate with known functional specializations and receptive field properties in different areas, and how different areas might interact. Greatly improved visualization of morphological features became routinely possible with the introduction of higher resolution anterograde tracers: Phaseolus vulgaris -leucoagglutinin (PHA-L) in 1984, and over the next few years, biocytin, biotinylated dextran amine (BDA), and dextran conjugates such as fluoro-ruby (FR) and fluoro-emerald. These tracers could be easily delivered extracellularly, and still resulted in a Golgi-like image of individual labeled profiles. This was often
0168-0102/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 1 ) 0 0 3 2 1 - 2
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Fig. 1. The better visualization of high resolution tracers can itself provide important information. The photomicrograph in (A) shows a very dense field of terminations in area V4, labeled by a BDA injection in V2. (Some retrogradely labeled neurons are evident in layers 5/6 and 2/3.) Terminations are densest in layer 4, but extend toward layer 2. Higher magnification (B) indicates that the label above layer 4 in fact consists of terminations, but that the infragranular label (arrows in A and C) is mainly preterminal. Calibration bar in (A) is 200 mm and in (B) and (C), 20 mm.
comparable in detail to the quality of intracellular fills (Figs. 1 /4). In this article, I review the available data for several systems of cortical connections, with some discussion of implications for visual functions (Sections 3/5). I include a brief description (Section 5) of connections between visual cortical areas and the pulvinar nucleus, which are likely to have important contributions to cortical organization; and conclude by considering future directions for extending this line of research (Section 6). While there is a substantial body of single axon work in rodent neocortical areas (e.g. Pinault and Deschenes, 1998; Sugihara et al., 2001), there are still not many studies on extrinsic connections of monkey visual cortex at the single axon level. Thus, in this review I will be drawing heavily on results from my own laboratory. I begin with general background and a brief technical description of axon reconstruction. (For callosal connections and the systems of horizontal intrinsic collaterals most data are from bulk injections and will not be considered here.)
2. Background, technique, and shortcomings Injections of WGA-HRP or 3H-amino acids demonstrated that connections terminate with laminar specificity. Three generally recognized classes of connections were distinguished as feedforward, feedback and lateral connections (Maunsell and Van Essen, 1983). Feedforward connections terminated mainly in layer 4, and were at least implicitly assumed to be similar to geniculocortical connections in V1. Feedback connections terminated densely in layer 1, sometimes along with some combination of other layers but not layer 4; and lateral connections had a columnar configuration, with terminations in several layers. Parent neurons also had distinct laminar patterns (see Rockland, 1997 for further discussion). The first single axon studies were aimed to extend the previous experiments by establishing the configurations of individual axons: were there collateral terminations in other layers or areas, what was the distribution of arbors and the number of terminations? What were the
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Fig. 2. Photomicrographs of an isolated single arbor (asterisk in A and D). Portions are shown at higher magnification in (B). This field would be appropriate for carrying out serial section reconstruction. As examples, two sequential sections are shown in (C) and (D). The arrowheads indicate cut segments at the top of section (C), which continue at the bottom of section (D). These would be used to guide and verify the match of the profile of interest (asterisk). (A) and (D) are the same field, but photographed in different focal planes. Calibration bar is 100 mm in (A), (C) and (D) and 20 mm in (B).
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Fig. 3. FR and BDA can be used as double anterograde tracers. The low power photomicrograph in (A) shows two injections in area V2 (semitangential section at the tip of the lunate sulcus, LS). The arrow points to an injection of FR in layers 1 /3 (in brown); the asterisk indicates an injection of BDA in layers 4 /6 (in black). The injections, even though spatially separate, result in partially convergent terminations (shown in B) in area V4. The short arrow in (B) indicates BDA-labeled terminations extending into layers 1 and 2. These are shown at higher magnification in (C). (D) shows a field of black, BDA-labeled profiles (arrowheads) mixed with brown, FR-labeled profiles. Calibration bar in (A) is 500 mm, 200 mm in (B), 100 mm in (C) and 20 mm in (D).
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Fig. 4. High resolution tracers can demonstrate easily differences in size and shape of terminal arbors and boutons. These photomicrographs show terminations in MT/V5 labeled by BDA injections in areas V1 and V2. The connections from V2 end in small boutons but those from V1 (arrow) have large terminal boutons and a thick main axon. Calibration bar is 100 mm in (A), 20 mm in (B).
convergent/divergent architectures that had been hidden within the denser projection foci? It was thought that comparisons across different visual areas might contribute to understanding the anatomical basis for specializations, as well as suggest principles of sensory cortical organization. In order to assure completeness of label, what might be called the first generation of axon studies primarily used large extracellular injections (diameter ]/1.0 mm). Large injections label thousands of axons, and detailed analysis of individual axons requires a match and reconstruction through serial sections. This is not particularly difficult, but it is slow. Briefly, the first step is to select a ‘profile of interest’ (POI). Two criteria in the selection process are: (1) feasibility */some projection foci are simply too dense (Fig. 1); and (2) identifying at least one feature of interest (a branch point or a portion of terminal arbor (Fig. 2A). It is helpful to know at the outset which is the distal and proximal portions of a selected profile. It is usually best to select terminal portions in grey matter, so that useful
data can be collected even if the reconstruction is only partial. The actual procedure relies on a 3D match through adjacent sections. This is done by identifying the region of interest at low magnification (40 / or 100 /), and matching a set of 4 /6 landmarks (blood vessels and darkly labeled profiles) in the x /y planes. Next, at higher magnification (200 / or 400 /), the process is repeated, with the POI and surrounding landmarks. Profiles are matched in the z, as well as x /y planes (Fig. 2C, D). Reconstruction can be done either by a camera lucida microscope attachment, with data projected onto paper, or by a computer-linked microscope (such as Neurolucida, MicroBrightField Inc., Colchester, Vt), with data entered into computer files. The computerized technique is slower, but allows 3D rotation and automatic application of statistical analyses. There are several shortcomings associated with axon reconstructions. The most serious may be that there is typically a small sample of axons per study. Another related problem is that the technique requires some selection and more particularly, for practical purposes,
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is biased toward regions of sparser terminations. Both these pitfalls need to be kept in mind, but to some extent can be controlled for by complementary single arbor analyses, and even by qualitative observation of individual sections. These strategies are effective for questions of laminar distributions, arbor size, and number of boutons per arbor. They are less useful for investigations of branching or total axon configuration, for which the more extensive reconstructions are necessary. Another issue is that differences at the single axon level ideally should be corroborated by additional criteria. For geniculocortical connections, that is, strong subdivisions were established on the basis of correlated differences in parent neurons, axon caliber, size and laminar distributions of terminal arbors, size of terminal specializations, and functional properties (Blasdel and Lund, 1983; Freund et al., 1989). For corticocortical axons labeled by extracellular injections, the terminal portion of the axon usually cannot be traced back to the cell of origin. Parent neurons are too far away and in addition are obscured within the injection site. Intracellular or smaller injections, delivered by the juxtacellular technique (Pinault, 1996), are one answer to this problem; but so far, juxtacellular injections have been used most extensively in rodent brain (but see Murphy and Sillito, 1996). Axon analysis also needs to be combined with other techniques for more direct investigation of functional issues. In contrast with the superbly precise data contributed by single axon analysis concerning divergence, there is a relative lack of data on cortical convergence. This issue is, however, becoming more approachable. Additional high resolution anterograde tracers are now available, which can be colorimetrically distinguished. PHA-L and BDA, and BDA and FR have been successfully used together (for example, Ojima and Takayanagi, 2001; Ojima and Rockland, 2001; and Fig. 3).
3. Feedforward connections 3.1. Connections to areas V2 and V4: uniformity? Connections from area V1 to V2 and from V2 to V4 have been shown by many studies to terminate mainly in layer 4 (‘feedforward’). In confirmation of the importance of these connections, inactivation studies have shown that an intact V1 is necessary for the visual responsiveness of neurons in V2 and V4 (Bullier et al., 1994). There have so far been no electron microscopic and only two single axon studies in primate (Rockland, 1992; Rockland and Virga, 1990); but from these, several comparisons can be made between geniculocortical input and the cortical afferent input to V2 and V4. First, with single axon resolution it was possible to detect terminations in other layers than layer 4. In both
V2 and V4, some arbors targeted layer 3, and some axons had collaterals in other layers, usually layer 5. In V4, two of 20 axons from V2 terminated mainly in layer 1 (Fig. 15 in Rockland, 1992). Continuing work showed that within projection foci, BDA-labeled terminations can easily be seen in layers 1 and 2 (Fig. 3B, C). The different laminar patterns are important, although the exact significance is not clear. They may well be related to heterogeneity in the parent cell populations. A few neurons in the deeper layers of area V1, for example, are known to give rise to feedforward projections in monkeys (Kennedy and Bullier, 1985; Sincich and Horton, 2001). These may project, in an inverted depth gradient, to the supragranular layers. These laminar differences may be an indication of subcategories of feedforward connections, although additional criteria are necessary to establish this point. Currently, one can only speculate about whether or not these are functionally sharp distinctions (like the subpopulations of geniculocortical connections). Another implication of these complex laminar patterns is that there is not an exclusive interlaminar relay from layer 4 to supra- and infragranular layers. Rather, neurons outside later 4 may directly receive a small number of extrinsic inputs, in addition to indirect influences through layer 4. Moreover, since layer 4 is invaded by apical and basal dendrites of neurons in other layers, these neurons may be receiving input at several different locations of their dendritic tree. Second, axon reconstruction yielded specific data about arbor size and number. In terms of size, most of the arbors in layer 4 in both V2 and V4 were found to be small (diameter is about 200 mm), about the same size as parvocellular geniculocortical terminations in area V1. The similarity in arbor size between the two areas was somewhat surprising given the differences in magnification factor, topography, and functional properties that have been shown by other techniques, and the area specific morphometric differences reported for pyramidal neurons (e.g. Elston et al., 1999) and intrinsic horizontal connections (Lund et al., 1993; Malach et al., 1993). No obvious differences were found, either, in the terminal specializations in the two areas. It is possible, however, that more work and a larger sample size from different visual field locations would succeed in identifying distinctive features. Although there is a general tendency for arbor size constancy in V2 and V4, the issue is complicated by intra-areal variability in arbors. When a single axon has multiple arbors, these are typically not the same in either size or shape. Rather, one (‘principal’) arbor will be larger and have more boutons. While there was no strong indication for area-specific size of arbors, there was clear evidence of an areaspecific trend in the number of arbors per axon. Of 20
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V2 axons examined in V4, 19 had two to four spatially separate arbors (Rockland, 1992), whereas most V1 axons terminating in V2 had single arbors. How much does this trend continue in higher cortical areas? Two investigations of axons projecting from TE to perirhinal cortex report axons with five separate arbors (Saleem and Tanaka, 1996; and Figs. 12, 13 in Cheng et al., 1997). This divergence may be related to the emergence of more complex receptive field properties, or to a complex divergent/convergent network architecture, still to be elucidated. In summary, single axon analysis of feedforward connections to V2 and V4 has raised many intriguing questions, which need further investigation. Do laminar differences indicate functional specialization like the geniculocortical connections to area V1? Are microstructural differences between V2 and V4 negligible, or perhaps only subtle and more gradient-like? 3.2. Feedforward connections to area MT/V5: an instance of specialization Area MT is closely involved with motion processing, and receives connections from both V1 and V2 (Orban, 1997). Inactivation experiments have suggested that this area is less dependent than V2 and V4 on intact input from V1 (Bullier et al., 1994); but somewhat paradoxically, single axon analysis has demonstrated that the connections from V1 have several specializations consistent with fast and/or secure transmission (Rockland, 1989, 1995). The most obvious specializations are first, the large caliber of the afferent axons, many of which are up to 3.0 mm in diameter (this contrasts with an average of 1.0 mm for most corticocortical axons), and second, the large size and complex beaded shape of the terminations (Fig. 4). Of the corticocortical connections examined so far, these features seem to be unique to the V1-to-MT pathway. The reason may be because they originate from an unusual combination of neurons; namely, pyramidal neurons in layer 4B, stellate cells in layer 4B, and large Meynert cells in the deeper layers (Shipp and Zeki, 1989). Remarkably, it is still unknown whether there are further specializations associated with these distinct neuronal subpopulations, but this would seem likely. Electron microscopic studies (Anderson et al., 1998) have confirmed the large size of the terminations from V1, and further determined that individual boutons form on average 1.7 synapses. The boutons formed asymmetric synapses with spines (54%), dendrites (33%), and somata (13%). Thirteen percent is a rather high proportion of contacts onto what are probably the somatas of inhibitory interneurons. By comparison, geniculocortical axons contact 52 /68% spines, 33/47% shafts, and 0/3% somata (Freund et al., 1989).
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The distinctively large size of the V1 terminations is suggestive of a high degree of synaptic efficacy relative to other inputs. Only indirect evidence is available on this point; but it has been noted (Anderson et al., 1998), as one gauge of efficacy, that the postsynaptic densities in V5 are only slightly smaller (B/0.13 mm2) than those of the thalamocortical synapses in the cat (0.18 mm2). By analogy with the better investigated cat system, the size of the MT/V5 synapses suggest that the mean amplitudes of the unitary AMPA receptor EPSPs would be 1/ 2 mV. Additionally, the ultrastructural investigation found that single afferents can make multisynaptic contacts with single neurons in MT/V5. If this occurs commonly, this would be another mechanism for large EPSPs. Another distinctive specialization of the axons from V1 is that they have a bistratified distribution in both layers 4 and 6 of MT. The collaterals to layer 6 are unusual and suggest a particular importance for this layer in the microcircuitry. The most likely possibilities are: a tight, neuron-to-neuron feedback loop between the feedforward-projecting neurons in layers 4B and 5 of V1, and the feedback-projecting neurons in layer 6 of MT; interlaminar intrinsic connections from layer 6 to layers 3/4 within MT; or subcortical connections from the deeper layers of MT to the pulvinar, superior colliculus, or pons. It is worth noting that geniculocortical afferents frequently have collaterals in layer 6 of V1 (Blasdel and Lund, 1983; Freund et al., 1989). Axons terminating in MT commonly have two arbors in layer 4 and one in layer 6. The principal arbor in layer 4 is about the same size (200 mm in diameter) as the majority of V1 arbors in V2, and V2 arbors in V4. The arbors in layer 6 are smaller, and usually offset from the supragranular arbors. This contrasts with geniculocortical axons, where collaterals to layer 6 are shown in register with overlying arbors to layer 4 (Blasdel and Lund, 1983; Freund et al., 1989). The pattern of multiple arbors may relate to the functional architecture of MT, which consists of patches of neurons tuned for disparity or directionality (Albright et al., 1984; Born and Tootell, 1992; DeAngelis and Newsome, 1999). Further work is necessary, however, to determine the exact relationships. The terminations from V2 to MT do not have the conspicuous specializations of the terminations from V1 (Fig. 4), and are rather similar to the V2 cortical terminations in V4 (Rockland, 1995). Unlike the connections from V1, those from V2 had thinner axons (1.0 vs. 3.0 mm), delicate terminal boutons, and no collaterals in layer 6. Of 15 single arbors from V2, 12 were concentrated in layers 3/4 and were 200 /250 mm in diameter. Three arbors were larger (d/400 mm) and had a columnar shape, extending from layer 4 toward layer 1. Of seven more fully reconstructed axons, five had three arbors (d B/200 mm) in layers 3/4, separated by 200 /600 mm. Two axons terminated in what appeared
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to be a single focus in layers 3/4. How the V1 and V2 connections interact and whether they share postsynaptic targets is still unknown.
4. Feedback connections: divergence Feedback connections have attracted considerable interest in the context of ‘top down’ effects (cf. Bullier et al., 2001; Naya et al., 2001; von Stein et al., 2000). They have been considered as contributing to processes related to perceptual modulation, while feedforward connections may be more directly responsible for receptive field tuning properties (Lamme et al., 1998). Experiments to evaluate changes in receptive field properties in a target area after inactivation of feedback connections have led to the suggestion that feedback connections have a potentiating effect on the responses of neurons in areas V1, V2, and V3, and that these effects are fast, influencing the early phase of the response (reviewed in Bullier et al., 2001). The physiological properties of these connections have also been investigated in slice preparations in rats. Shao and Burkhalter (1996, 1999) have shown that IPSPs are smaller in feedback than in feedforward, thalamocortical or horizontal intrinsic axons. EPSPs are similar in all three pathways. Unfortunately, there have been no combined anatomical-physiological studies in primates; and there are as yet few direct points of contact between anatomical and physiological studies of feedback connections. Feedback connections have been investigated at the single axon level for V2 to V1 (Rockland and Virga, 1989), V4 and TEO to V2 (Rockland et al., 1994), from TE to TEO (Suzuki et al., 2000), and from MT/V5 to V1 and V2 (in squirrel monkeys: Rockland and Knutson, 2000). As there have so far been no reports of any obvious specializations, these systems will be considered together in one section. As for feedforward connections, the better visualization from high resolution tracers showed that the laminar termination pattern of feedback connections is complex. For connections from V2 to V1, and from V4, axons targeted layer 1, but frequently emitted collaterals in the deeper layers (Rockland and Virga, 1989; Rockland et al., 1994). From TE, four of nine reconstructed axons in TEO had terminations in layers 1 /3, one axon terminated in layer 1 alone, two axons in layers 5 and 6 only, and two axons in layers 1, 2, 3, 5 and 6 (Suzuki et al., 2000). From MT, two of nine axons terminated in V1 in layer 1 alone, three axons only in layer 4B, one axon in layers 1 and 4B, and 3 axons in layers 1, 4B and 6 (Rockland and Knutson, 2000). These fine laminar differentiations, as commented in the previous section, may be an indication of further functionally significant subtypes, in this case, subtypes
of feedback connections (see also the Discussion in Suzuki et al., 2000). This possibility is likely, given the heterogeneity of feedback efferent neurons. In most areas, these have a bistratified distribution in layers 6 and 3A, and the population within layer 6 is itself morphologically mixed. There is no direct evidence, however, that these two populations in layer 3A and 6 have different terminal configurations; and more detailed studies, such as depth-specific injections, are still needed. The more precise laminar data imply that feedback connections have a mixed postsynaptic population, and that the frequent emphasis on proximal/distal complementarity of feedforward and feedback connections may need to be qualified. Feedback terminations in the deeper layers are clearly not targeting distal apical dendrites. Moreover, as stated in Section 3.1, some feedforward terminations occur in layers 1 and 2, and potentially might directly converge with feedback connections. A second result from single axon analysis concerns the spatial extent of feedback systems. Terminal fields are typically divergent, extending over distances /1.0 mm. This confirms previous experiments using WGAHRP. That is, feedback terminations in layer 1 extended further than feedforward projecting neurons in layer 3, and feedback projecting neurons in layer 6 extended further than feedforward terminations in layer 4 (Section V.C in Salin and Bullier, 1995; Sections 2.2, 3.2 in Rockland, 1997; and the Discussion in Rockland and Knutson, 2000). Axon reconstruction demonstrated the divergence more directly, and further showed that terminations are often distributed continuously, somewhat like parallel fibers in the cerebellum. The activated postsynaptic population, therefore, will likely be a cohort of neighboring neurons. This contrasts with the geometry of feedforward connections. Even when there are multiple arbors, these are comparatively small, spherical, and dispersed, contacting spatially separate populations. Indirect evidence suggests that feedback connections probably extend across functional modules, although this is again an issue that requires more work. There is some indication from single axon analysis that the divergence factor increases with distance between source and target area (Rockland and Virga, 1989; Rockland et al., 1994; the Discussion in Rockland and Knutson, 2000). Connections from V2 to V1 (about 1.0 /4.0 mm long) are shorter than those from V4 to V2 (about 3.0 /5.0 mm long). In the case of axons bifurcating to two areas, terminal fields often span larger territories in the more distant area; for example, axons from TEO or V4 to V1 extend up to 6.0 and 5.0 mm, respectively, in V1, but B/5.0 mm in V2. Similarly, analysis of one MT axon with branches to V1 and V2 showed that the terminal field in V1 was larger (Fig. 6, Rockland and Knutson, 2000).
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5. Connections between pulvinar and cortex Connections between visual cortical areas and pulvinar subnuclei have been investigated extensively with retrograde and older anterograde tracers. They have frequently been discussed in general terms as reciprocal loops (but see Descheˆnes et al., 1998), and implicated in thalamic oscillations (Contreras et al., 1996; Jones, 2001). Their functional role and fine scale organization is largely mysterious, but it seems likely that they have an important role in visual cortical processing, and will need to be incorporated in the current framework of feedforward, feedback, and intrinsic connectivity (Robinson and Cowie, 1997; Grieve et al., 2000). As it turned out, the higher resolution tracers dramatically demonstrated that there are at least two types of corticopulvinar terminations, which had not been distinguishable with 3H-amino acids or WGAHRP (Rockland, 1996). One (‘elongate’ or type 1) is characterized by an elongated terminal field and a large number of terminal specializations that are distinctively spinous. The second (‘round’ or type 2) has a small spherical terminal arbor (d/100/125 mm), with a small number ( B/200 per arbor) of mainly large, beaded endings. The second type is numerically sparse from all areas examined so far except for V1 (Rockland, 1996). These two types of terminations have now been demonstrated in the corticothalamic pathways of cat auditory (Ojima, 1994) and cat visual cortices (Sherman and Guillery, 2001), and seem to be common across several species and areas (reviewed in Rouiller and Welker, 2000). Earlier electron miscroscopic studies had reported large and small corticopulvinar boutons (Ogren and Hendrickson, 1979), but the single axon approach has been significant in providing direct visualization, additional classification criteria, and a larger sample size. The functional significance of these different corticopulvinar connections requires further investigation. One hypothesis has been that the large, type 2 connections have a driving influence, whereas the type 1 connections are more modulatory (for example, Sherman and Guillery, 2001). This inference is in part from analogy with the retino- and corticogeniculate pathways, which differ in several structural and functional respects, possibly consistent with, respectively, ‘driving’ and ‘modulatory’ effects (Section IX, Sherman and Guillery, 2001). Direct evidence for modulatory roles, however, is difficult; and it should be kept in mind that the parameters of synaptic efficacy are complex and highly context-dependent. In the reverse, pulvinocortical (PC) direction, one recent single axon study reports on the connections from two injections at the lateral border of the lateral pulvinar (Rockland et al., 1999). Projection foci to
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extrastriate aeras (V2, V3, and V4) were densest in layer 3 and 5, as previously reported (Levitt et al., 1995), but individual axons were found to have terminations in different laminar combinations. In area V2, PC axons frequently had collaterals in layer 1 in addition to terminations in layers 3, 4 and 5 (see Figs. 6 /8, 14 of Rockland et al., 1999). Thus, at least in V2, PC terminations occur in layers targeted both by feedforward and feedback cortical connections. Like corticocortical connections, PC axons (n /25) were found to have multiple, spatially separate arbors. Curiously, the overall size of the PC arbors was not uniform across areas. In MT/V5, arbors were small (d B/ 200 mm), and in V4, they were large (d is about 600 mm). In V2, arbor size was more variable, but typically measured 250/500 mm. Since, as reviewed in Section 3, the arbor size of cortical connections (CC) is relatively constant in V2, V4, and MT, the conclusion is that there are area-specific differences in the size ratios of PC to CC terminal fields. How do corticopulvinar and pulvinocortical fields compare? Despite the earlier emphases on reciprocity, the arbor configuration is actually asymmetrical. Type 1 corticopulvinar fields sweep over large distances in the pulvinar, seemingly larger than pulvinocortical arbors in the cortex (at least in V2 and MT/V5); but type 2 corticopulvinar arbors are smaller than most pulvinocortical arbors (100 vs. 200 mm). A larger sample, especially from different pulvinar subdivisions, will be important in extending and interpreting these results.
6. Summary and future directions From these investigations of single axon connectivity, we are beginning to have a reasonable database of morphometrics and microstructure. This has already led to results in several spheres. First, the quantitative data are a source of realistic parameters for modeling studies, and can be used as one measure of connectional efficacy (e.g. Budd, 1998). Second, comparisons across different areas in some cases have pointed to definite structuralfunctional correlates (notably, in MT/V5). In general, correlates have been less obvious, but this in itself may be significant if, as suggested by other evidence (Kondo et al., 1999), features change in a gradient-wise fashion. Third, there have been significant clarifications simply by looking with the improved visualization of high resolution tracers. These include better laminar definition, clearer criteria of possible subtypes (on the basis of size and shape of boutons and arbors), and confirmation of axon branching. There are several major avenues for future extensions. One is direct correlation of axon geometry with functional architecture. This has been difficult because the need for unbroken serial sections in axon analysis is not
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easily compatible with cytochrome oxidase or other standard markers of functional architecture. Several studies, however, have now used optical imaging of the intrinsic cortical signal to pre-define functional modules. These are then targeted for tracer injections and aligned with the subsequent histological sections. This approach has been very successful in area V1, where ocular dominance and other modules have been compared with intrinsic excitatory (Malach et al., 1993; Yoshioka et al., 1996; Li et al., 2000), and intrinsic inhibitory connections (cat area 18: Buzas et al., 2001). It has recently been used in monkey inferotemporal cortex to compare the distribution of horizontal intrinsic connections with the distribution of activation maps elicited by the presentation of complex visual objects (Tanifuji et al., 2001). In areas V2 and MT/V5, despite the distinct modularity of these areas, it is still unknown whether the multiple arbors of a single axon terminate within the same or related modules. When terminations are in different layers, are these located in similar or opposite compartments? For that matter, direct evidence is still needed for whether feedback connections, in V1 and V2 especially, cross over functional domains, as is implied by their very extended configuration. A second important direction is microcircuitry, at the level of electron or confocal microscopy. Data about specific postsynaptic targets will be essential for progress in how receptive field properties are built up and for specific comparisons among the geniculo-, cortico- and pulvinocortical pathways. Ultrastructural studies in rodents, for example, report that feedback connections from area LM to V1 terminate with a distinctively high proportion of excitatory contacts mainly onto other pyramidal neurons (Johnson and Burkhalter, 1996); but this needs to be confirmed for primates. Within the feedback systems alone, there remain many basic questions: what are the differences between the terminations in layer 1 and those in the deeper layers? Between feedback from MT/V5 and from V2, in V1? Between feedback from MT/V5 in V1 and V2? Do feedback terminations from V2 and V4 converge on the same neurons in V1? A third direction is the issue of how connectional systems interact. Connectional influences have been addressed by techniques such as antidromic stimulation or selective inactivation (for example, Hupe´ et al., 2001a,b). There are still, however, very little data on how two or more afferent systems interact. Anatomically, precise information is necessary about convergence. This can now be acquired by injections of double anterograde tracers, especially if combined with electron or confocal microscopic preparations. Functionally, perhaps in vivo modifications can be devised of the elegant in vitro techniques now available, such as using
calcium indicators to detect neurons activated by stimulating a ‘trigger’ neuron (Peterlin et al., 2000). Finally, despite the interest in moving to functionally relevant experimental paradigms, there will need to be a much larger database of axon morphology */ different systems in different areas, species, and developmental ages. Ideally and maybe essentially, we need comparably detailed data for intrinsic, callosal, cortical, and subcortical connections. As one of many questions, for example, what are the rules governing the intrinsic collaterals and extrinsic terminations of different pyramidal neurons? Experiments in slice preparations, have clearly demonstrated differential signaling from the axon of a single neuron, as a function of postsynaptic targets (Markram et al., 1998). The same complexity may need to be considered for extrinsic connectivity, and will be important for understanding area specializations and interactions. Realistically, a more comprehensive database may depend on technical advances that can either circumvent or expedite the labor intensive process of serial section reconstruction. Smaller or juxtacellular injections may help in isolating labeled profiles; or this may be accomplished by alternative new techniques.
Acknowledgements I thank Dr Manabu Tanifuji for helpful discussion and Michiko Fujisawa for assistance with manuscript preparation.
References Albright, T.D., Desimone, R., Gross, C.G. 1984. Columnar organization of directionality selective cells in visual area MT of the macaque. J. Neurophysiol. 51, 16 /31. Anderson, J.C., Binzegger, T., Martin, K.A.C., Rockland, K.S. 1998. The connection from cortical area V1 /V5: a light and electron microscopic study. J. Neurosci. 18 (24), 10525 /10540. Blasdel, G.G., Lund, J.S. 1983. Termination of afferent axons in macaque striate cortex. J. Neurosci. 3 (7), 1389 /1413. Born, R.T., Tootell, R.B.H. 1992. Segregation of global and local motion processing in primate middle temporal visual area. Nature 357, 497 /499. Budd, J.M.L. 1998. Extrastriate feedback to primary visual cortex in primates: a quantitative analysis of connectivity. Proc. R. Soc. Lond. B 265, 1 /7. Bullier, J., Girard, P., Salin, P.-A. 1994. The role of area 17 in the transfer of information to extrastriate visual cortex. In: Peters, A., Rockland, K.S. (Eds.), Cerebral Cortex: Primary Visual Cortex in Primates, vol. 10. Plenum Press, New York, pp. 301 /330. Bullier, J., Hupe´, J.-M., James, A.C., Girard, P. 2001. The role of feedback connections in shaping the responses of visual cortical neurons. Prog. Brain Res. 134, 1 /12. Buzas, P., Eysel, U.T., Adorjan, P., Kisvarday, Z.F. 2001. Axonal topography of cortical basket cells in relation to orientation, direction, and ocular dominance maps. J. Comp. Neurol. 437, 259 /285.
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Maunsell, J.H.R., Van Essen, D.C. 1983. The connections of the middle temporal visual area (MT) and their relationship to a cortical hierarchy in the macaque monkey. J. Neurosci. 3, 2563 / 2586. Murphy, P.C., Sillito, M. 1996. Morphology of the feedback pathway from area 17 of the cat visual cortex to the lateral geniculate nucleus. J. Neurosci. 16, 1180 /1192. Naya, Y., Yoshida, M., Miyashita, Y. 2001. Backward spreading of memory-retrieval signal in the primate temporal cortex. Science 291, 661 /664. Ogren, M.P., Hendrickson, A.E. 1979. The morphology and distribution of striate cortex terminals in the interior and lateral subdivisions of the macaca monkey pulvinar. J. Comp. Neurol. 188, 179 / 199. Ojima, H. 1994. Terminal morphology and distribution of corticothalamic fibers originating from layers 5 and 6 of cat primary auditory cortex. Cereb. Cortex 4, 646 /663. Ojima, H., Rockland, K.S., 2001. Converging projections in prefrontal cortex (PFC) from auditory association cortex (AAC) in Japanese monkeys. 24th Annual Meeting of Japan Neurosci. Abstr., p. 225. Ojima, H., Takayanagi, M. 2001. Use of two anterograde axon tracers to label distinct cortical neuronal populations located in close proximity. J. Neurosci. Methods 104, 177 /182. Orban, G.A. 1997. Visual processing in macaque area MT/V5 and its satellites (MSTd and MSTv). In: Rockland, K.S., Kaas, J.H., Peters, A. (Eds.), Cerebral Cortex: Extrastriate Cortex in Primates, vol. 12. Plenum Press, New York, pp. 359 /434. Peterlin, Z.A., Kozloski, J., Mao, B.Q., Tsiola, A., Yuste, R. 2000. Optical probing of neuronal circuits with calcium indicators. Proc. Natl. Acad. Sci. USA 97, 3619 /3624. Pinault, D. 1996. A novel single-cell staining procedure performed in vivo under electrophysiological control: morpho-functional features of juxta-cellularly labeled thalamic cells and other central neurons with biocytin or neurobiotin. J. Neurosci. Methods 65, 113 /136. Pinault, D., Deschenes, M. 1998. Projection and innervation patterns of individual thalamic reticular axons in the thalamus of the adult rat: a three-dimensional, graphic, and morphometric analysis. J. Comp. Neurol. 391, 180 /203. Robinson, D.L., Cowie, R.J. 1997. The primate pulvinar: structural, functional, and behavioral components of visual salience. In: Steriade, M., Jones, E.G., McCormick, D.A. (Eds.), Thalamus: Organization and Function, vol. II. Elsevier Science, Amsterdam, pp. 53 /92. Rockland, K.S. 1989. Bistratified distribution of terminal arbors of individual axons projecting from area V1 to middle temporal area (MT) in the macaque monkey. Vis. Neurosci. 3, 155 /170. Rockland, K.S. 1992. Configuration, in serial reconstruction, of individual axons projecting from area V2 to V4 in the macaque monkey. Cereb. Cortex 2, 353 /374. Rockland, K.S. 1995. The morphology of individual axons projecting from area V2 to MT in the macaque. J. Comp. Neurol. 355, 15 /26. Rockland, K.S. 1996. Two types of corticopulvinar terminations: round (type 2) and elongate (type 1). J. Comp. Neurol. 368, 57 /87. Rockland, K.S. 1997. Hierarchy Revisited: Neuronal substrates of Interareal Connectivity. In: Rockland, K.S., Kaas, J.H., Peters, A. (Eds.), Cerebral Cortex: Extrastriate Cortex in Primates, vol. 12. Plenum Press, New York, pp. 243 /293. Rockland, K.S., Knutson, T. 2000. Feedback connections from area MT of the squirrel monkey to areas V1 and V2. J. Comp. Neurol. 425, 345 /368. Rockland, K.S., Virga, A. 1989. Terminal arbors of individual ‘feedback’ axons projecting from area V2 to V1 in the macaque monkey: a study using immunohistochemistry of anterogradely transported Phaseolus vulgaris leucoagglutinin. J. Comp. Neurol. 285, 54 /72.
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Rockland, K.S., Virga, A. 1990. Organization of individual cortical axons projecting from area V1 (area 17) to V2 (area 18) in the macaque monkey. Vis. Neurosci. 4, 11 /28. Rockland, K.S., Saleem, K.S., Tanaka, K. 1994. Divergent feedback connections from areas V4 and TEO in the macaque. Vis. Neurosci. 11, 579 /600. Rockland, K.S., Andresen, J., Cowie, R.J., Robinson, D.L. 1999. Single axon analysis of pulvinocortical connections to several visual areas in the macaque. J. Comp. Neurol. 406, 221 /250. Rouiller, E.M., Welker, E. 2000. A comparative analysis of the morphology of corticothalamic projections in mammals. Brain Res. Bull. 53 (6), 727 /741. Saleem, K.S., Tanaka, K. 1996. Divergent projections from the anterior inferotemporal area TE to the perirhinal and entorhinal cortices in the macaque monkey. J. Neurosci. 4, 4757 /4775. Salin, P.-A., Bullier, J. 1995. Corticocortical connections in the visual system: structure and function. Physiol. Rev. 75, 107 /154. Shao, Z., Burkhalter, A. 1996. Different balance of excitation and inhibition in forward and feedback circuits of rat visual cortex. J. Neurosci. 16 (22), 7353 /7365. Shao, Z., Burkhalter, A. 1999. Role of GABAB receptor-mediated inhibition in reciprocal interareal pathways of rat visual cortex. J. Neurophysiol. 81, 1014 /1024. Sherman, S.M., Guillery, R.W. 2001. Exploring the Thalamus. Academic Press, San Diego.
Shipp, S., Zeki, S. 1989. The organisation of connections between area V5 and V1 in macaque monkey visual cortex. Eur. J. Neurosci. 1, 309 /332. Sincich, L.C., Horton, J.C. 2001. A reappraisal of the projection pattern from macaque striate cortex to the pale, thin and thick cytochrome oxidase stripes in V2. Ann. Mtg. Soc. Neurosci. Abstr. 27, 11. Sugihara, I., Wu, H.S., Shinoda, Y. 2001. The entire trajectories of single olivocerebellar axons in the cerebellar cortex and their contribution to cerebellar compartmentalization. J. Neurosci. 21, 7715 /7723. Suzuki, W., Saleem, K.S., Tanaka, K. 2000. Divergent backward projections from area TE of the macaque inferotemporal cortex. J. Comp. Neurol. 422 (2), 206 /228. Tanifuji, M., Li, H., Yamane, Y., Rockland, K.S. 2001. Horizontal intrinsic connections as the anatomical basis for the functional columns in the macaque inferior temporal cortex. Ann. Meet. Soc. Neurosci. Abstr. 27, 1633. von Stein, A., Chiang, C., Ko¨nig, P. 2000. Top-down processing mediated by interareal synchronization. Proc. Natl. Acad. Sci. USA 97 (26), 14748 /14753. Yoshioka, T., Blasdel, G.G., Levitt, J.B., Lund, J.S. 1996. Relation between patterns of intrinsic lateral connectivity, ocular dominance and cytochrome oxidase-reactive regions in macaque monkey striate cortex. Cereb. Cortex 6, 297 /310.
Update Article
Visual cortical organization at the single axon level: a beginning Kathleen S. Rockland * Laboratory for Cortical Organization and Systematics, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan Received 22 October 2001; accepted 12 December 2001
Abstract Single axon analysis of visual cortical connections is an important extension of previous anterograde studies using 3H-amino acids or wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). The higher resolution tracers */Phaseolus vulgaris leucoagglutinin (PHA-L), biocytin, biotinylated dextran amine (BDA) and dextran-conjugates */have already produced new results, simply by providing improved visualization, concerning laminar definition and possible subtypes of connections, as well as the beginning of a database of morphometrics and microstructure. The comparative approach, comparing geniculocortical terminations and cortical connections across several areas, has suggested both specific structural /functional correlations (for example, in extrastriate area MT/V5) and more subtle, possibly gradient-wise variations. Likely future directions for this line of research include more direct correlations of axon geometry with functional architectures, investigations of microcircuitry at the level of electron or confocal microscopy, anatomical and functional investigations of connectional convergence and interactions, and, not least, a more comprehensive database. # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Anterograde; Convergence; Divergence; Feedback; Feedforward; Pulvinar
1. Introduction A common approach to understanding visual cortical organization has been to examine successive transformations in the neural representations of the visual world, and to try to identify the underlying neural substrates. This has been extremely successful in the early part of the visual pathway. For example, combined physiological recording and intraaxonal filling of geniculocortical connections demonstrated the laminar segregation of the magno- and parvocellular pathways, promoted the idea of further segregation in the efferent pathways from V1 to extrastriate areas, and provided the baseline data necessary for interpreting developmental and plasticity paradigms (Blasdel and Lund, 1983; Freund et al., 1989; Florence and Casagrande, 1990). Comparable studies of long distance cortical connections beyond V1, however, have been more difficult, in part because of the sheer length and intricacy of these connections. Of the many anatomical studies addressing * Tel.: 81-48-467-6427; fax: 81-48-467-6420. E-mail address: [email protected] (K.S. Rockland).
cortical connectivity, most have been carried out using large injections of horseradish peroxidase (HRP) or fluorescent dyes as retrograde tracers, and 3H-amino acids or WGA-HRP as anterograde. These tracers have been excellent for demonstrating the general lay-out of interconnected structures, as well as providing data about topographic and laminar organization and, to some extent, relative density of connections. Especially the anterograde tracers, however, have left unanswered basic questions concerning the fine organization of connections, related to the morphometrics of terminal arbors and boutons. These data are necessary for further consideration of how microstructural features correlate with known functional specializations and receptive field properties in different areas, and how different areas might interact. Greatly improved visualization of morphological features became routinely possible with the introduction of higher resolution anterograde tracers: Phaseolus vulgaris -leucoagglutinin (PHA-L) in 1984, and over the next few years, biocytin, biotinylated dextran amine (BDA), and dextran conjugates such as fluoro-ruby (FR) and fluoro-emerald. These tracers could be easily delivered extracellularly, and still resulted in a Golgi-like image of individual labeled profiles. This was often
0168-0102/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 1 ) 0 0 3 2 1 - 2
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Fig. 1. The better visualization of high resolution tracers can itself provide important information. The photomicrograph in (A) shows a very dense field of terminations in area V4, labeled by a BDA injection in V2. (Some retrogradely labeled neurons are evident in layers 5/6 and 2/3.) Terminations are densest in layer 4, but extend toward layer 2. Higher magnification (B) indicates that the label above layer 4 in fact consists of terminations, but that the infragranular label (arrows in A and C) is mainly preterminal. Calibration bar in (A) is 200 mm and in (B) and (C), 20 mm.
comparable in detail to the quality of intracellular fills (Figs. 1 /4). In this article, I review the available data for several systems of cortical connections, with some discussion of implications for visual functions (Sections 3/5). I include a brief description (Section 5) of connections between visual cortical areas and the pulvinar nucleus, which are likely to have important contributions to cortical organization; and conclude by considering future directions for extending this line of research (Section 6). While there is a substantial body of single axon work in rodent neocortical areas (e.g. Pinault and Deschenes, 1998; Sugihara et al., 2001), there are still not many studies on extrinsic connections of monkey visual cortex at the single axon level. Thus, in this review I will be drawing heavily on results from my own laboratory. I begin with general background and a brief technical description of axon reconstruction. (For callosal connections and the systems of horizontal intrinsic collaterals most data are from bulk injections and will not be considered here.)
2. Background, technique, and shortcomings Injections of WGA-HRP or 3H-amino acids demonstrated that connections terminate with laminar specificity. Three generally recognized classes of connections were distinguished as feedforward, feedback and lateral connections (Maunsell and Van Essen, 1983). Feedforward connections terminated mainly in layer 4, and were at least implicitly assumed to be similar to geniculocortical connections in V1. Feedback connections terminated densely in layer 1, sometimes along with some combination of other layers but not layer 4; and lateral connections had a columnar configuration, with terminations in several layers. Parent neurons also had distinct laminar patterns (see Rockland, 1997 for further discussion). The first single axon studies were aimed to extend the previous experiments by establishing the configurations of individual axons: were there collateral terminations in other layers or areas, what was the distribution of arbors and the number of terminations? What were the
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Fig. 2. Photomicrographs of an isolated single arbor (asterisk in A and D). Portions are shown at higher magnification in (B). This field would be appropriate for carrying out serial section reconstruction. As examples, two sequential sections are shown in (C) and (D). The arrowheads indicate cut segments at the top of section (C), which continue at the bottom of section (D). These would be used to guide and verify the match of the profile of interest (asterisk). (A) and (D) are the same field, but photographed in different focal planes. Calibration bar is 100 mm in (A), (C) and (D) and 20 mm in (B).
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Fig. 3. FR and BDA can be used as double anterograde tracers. The low power photomicrograph in (A) shows two injections in area V2 (semitangential section at the tip of the lunate sulcus, LS). The arrow points to an injection of FR in layers 1 /3 (in brown); the asterisk indicates an injection of BDA in layers 4 /6 (in black). The injections, even though spatially separate, result in partially convergent terminations (shown in B) in area V4. The short arrow in (B) indicates BDA-labeled terminations extending into layers 1 and 2. These are shown at higher magnification in (C). (D) shows a field of black, BDA-labeled profiles (arrowheads) mixed with brown, FR-labeled profiles. Calibration bar in (A) is 500 mm, 200 mm in (B), 100 mm in (C) and 20 mm in (D).
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Fig. 4. High resolution tracers can demonstrate easily differences in size and shape of terminal arbors and boutons. These photomicrographs show terminations in MT/V5 labeled by BDA injections in areas V1 and V2. The connections from V2 end in small boutons but those from V1 (arrow) have large terminal boutons and a thick main axon. Calibration bar is 100 mm in (A), 20 mm in (B).
convergent/divergent architectures that had been hidden within the denser projection foci? It was thought that comparisons across different visual areas might contribute to understanding the anatomical basis for specializations, as well as suggest principles of sensory cortical organization. In order to assure completeness of label, what might be called the first generation of axon studies primarily used large extracellular injections (diameter ]/1.0 mm). Large injections label thousands of axons, and detailed analysis of individual axons requires a match and reconstruction through serial sections. This is not particularly difficult, but it is slow. Briefly, the first step is to select a ‘profile of interest’ (POI). Two criteria in the selection process are: (1) feasibility */some projection foci are simply too dense (Fig. 1); and (2) identifying at least one feature of interest (a branch point or a portion of terminal arbor (Fig. 2A). It is helpful to know at the outset which is the distal and proximal portions of a selected profile. It is usually best to select terminal portions in grey matter, so that useful
data can be collected even if the reconstruction is only partial. The actual procedure relies on a 3D match through adjacent sections. This is done by identifying the region of interest at low magnification (40 / or 100 /), and matching a set of 4 /6 landmarks (blood vessels and darkly labeled profiles) in the x /y planes. Next, at higher magnification (200 / or 400 /), the process is repeated, with the POI and surrounding landmarks. Profiles are matched in the z, as well as x /y planes (Fig. 2C, D). Reconstruction can be done either by a camera lucida microscope attachment, with data projected onto paper, or by a computer-linked microscope (such as Neurolucida, MicroBrightField Inc., Colchester, Vt), with data entered into computer files. The computerized technique is slower, but allows 3D rotation and automatic application of statistical analyses. There are several shortcomings associated with axon reconstructions. The most serious may be that there is typically a small sample of axons per study. Another related problem is that the technique requires some selection and more particularly, for practical purposes,
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is biased toward regions of sparser terminations. Both these pitfalls need to be kept in mind, but to some extent can be controlled for by complementary single arbor analyses, and even by qualitative observation of individual sections. These strategies are effective for questions of laminar distributions, arbor size, and number of boutons per arbor. They are less useful for investigations of branching or total axon configuration, for which the more extensive reconstructions are necessary. Another issue is that differences at the single axon level ideally should be corroborated by additional criteria. For geniculocortical connections, that is, strong subdivisions were established on the basis of correlated differences in parent neurons, axon caliber, size and laminar distributions of terminal arbors, size of terminal specializations, and functional properties (Blasdel and Lund, 1983; Freund et al., 1989). For corticocortical axons labeled by extracellular injections, the terminal portion of the axon usually cannot be traced back to the cell of origin. Parent neurons are too far away and in addition are obscured within the injection site. Intracellular or smaller injections, delivered by the juxtacellular technique (Pinault, 1996), are one answer to this problem; but so far, juxtacellular injections have been used most extensively in rodent brain (but see Murphy and Sillito, 1996). Axon analysis also needs to be combined with other techniques for more direct investigation of functional issues. In contrast with the superbly precise data contributed by single axon analysis concerning divergence, there is a relative lack of data on cortical convergence. This issue is, however, becoming more approachable. Additional high resolution anterograde tracers are now available, which can be colorimetrically distinguished. PHA-L and BDA, and BDA and FR have been successfully used together (for example, Ojima and Takayanagi, 2001; Ojima and Rockland, 2001; and Fig. 3).
3. Feedforward connections 3.1. Connections to areas V2 and V4: uniformity? Connections from area V1 to V2 and from V2 to V4 have been shown by many studies to terminate mainly in layer 4 (‘feedforward’). In confirmation of the importance of these connections, inactivation studies have shown that an intact V1 is necessary for the visual responsiveness of neurons in V2 and V4 (Bullier et al., 1994). There have so far been no electron microscopic and only two single axon studies in primate (Rockland, 1992; Rockland and Virga, 1990); but from these, several comparisons can be made between geniculocortical input and the cortical afferent input to V2 and V4. First, with single axon resolution it was possible to detect terminations in other layers than layer 4. In both
V2 and V4, some arbors targeted layer 3, and some axons had collaterals in other layers, usually layer 5. In V4, two of 20 axons from V2 terminated mainly in layer 1 (Fig. 15 in Rockland, 1992). Continuing work showed that within projection foci, BDA-labeled terminations can easily be seen in layers 1 and 2 (Fig. 3B, C). The different laminar patterns are important, although the exact significance is not clear. They may well be related to heterogeneity in the parent cell populations. A few neurons in the deeper layers of area V1, for example, are known to give rise to feedforward projections in monkeys (Kennedy and Bullier, 1985; Sincich and Horton, 2001). These may project, in an inverted depth gradient, to the supragranular layers. These laminar differences may be an indication of subcategories of feedforward connections, although additional criteria are necessary to establish this point. Currently, one can only speculate about whether or not these are functionally sharp distinctions (like the subpopulations of geniculocortical connections). Another implication of these complex laminar patterns is that there is not an exclusive interlaminar relay from layer 4 to supra- and infragranular layers. Rather, neurons outside later 4 may directly receive a small number of extrinsic inputs, in addition to indirect influences through layer 4. Moreover, since layer 4 is invaded by apical and basal dendrites of neurons in other layers, these neurons may be receiving input at several different locations of their dendritic tree. Second, axon reconstruction yielded specific data about arbor size and number. In terms of size, most of the arbors in layer 4 in both V2 and V4 were found to be small (diameter is about 200 mm), about the same size as parvocellular geniculocortical terminations in area V1. The similarity in arbor size between the two areas was somewhat surprising given the differences in magnification factor, topography, and functional properties that have been shown by other techniques, and the area specific morphometric differences reported for pyramidal neurons (e.g. Elston et al., 1999) and intrinsic horizontal connections (Lund et al., 1993; Malach et al., 1993). No obvious differences were found, either, in the terminal specializations in the two areas. It is possible, however, that more work and a larger sample size from different visual field locations would succeed in identifying distinctive features. Although there is a general tendency for arbor size constancy in V2 and V4, the issue is complicated by intra-areal variability in arbors. When a single axon has multiple arbors, these are typically not the same in either size or shape. Rather, one (‘principal’) arbor will be larger and have more boutons. While there was no strong indication for area-specific size of arbors, there was clear evidence of an areaspecific trend in the number of arbors per axon. Of 20
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V2 axons examined in V4, 19 had two to four spatially separate arbors (Rockland, 1992), whereas most V1 axons terminating in V2 had single arbors. How much does this trend continue in higher cortical areas? Two investigations of axons projecting from TE to perirhinal cortex report axons with five separate arbors (Saleem and Tanaka, 1996; and Figs. 12, 13 in Cheng et al., 1997). This divergence may be related to the emergence of more complex receptive field properties, or to a complex divergent/convergent network architecture, still to be elucidated. In summary, single axon analysis of feedforward connections to V2 and V4 has raised many intriguing questions, which need further investigation. Do laminar differences indicate functional specialization like the geniculocortical connections to area V1? Are microstructural differences between V2 and V4 negligible, or perhaps only subtle and more gradient-like? 3.2. Feedforward connections to area MT/V5: an instance of specialization Area MT is closely involved with motion processing, and receives connections from both V1 and V2 (Orban, 1997). Inactivation experiments have suggested that this area is less dependent than V2 and V4 on intact input from V1 (Bullier et al., 1994); but somewhat paradoxically, single axon analysis has demonstrated that the connections from V1 have several specializations consistent with fast and/or secure transmission (Rockland, 1989, 1995). The most obvious specializations are first, the large caliber of the afferent axons, many of which are up to 3.0 mm in diameter (this contrasts with an average of 1.0 mm for most corticocortical axons), and second, the large size and complex beaded shape of the terminations (Fig. 4). Of the corticocortical connections examined so far, these features seem to be unique to the V1-to-MT pathway. The reason may be because they originate from an unusual combination of neurons; namely, pyramidal neurons in layer 4B, stellate cells in layer 4B, and large Meynert cells in the deeper layers (Shipp and Zeki, 1989). Remarkably, it is still unknown whether there are further specializations associated with these distinct neuronal subpopulations, but this would seem likely. Electron microscopic studies (Anderson et al., 1998) have confirmed the large size of the terminations from V1, and further determined that individual boutons form on average 1.7 synapses. The boutons formed asymmetric synapses with spines (54%), dendrites (33%), and somata (13%). Thirteen percent is a rather high proportion of contacts onto what are probably the somatas of inhibitory interneurons. By comparison, geniculocortical axons contact 52 /68% spines, 33/47% shafts, and 0/3% somata (Freund et al., 1989).
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The distinctively large size of the V1 terminations is suggestive of a high degree of synaptic efficacy relative to other inputs. Only indirect evidence is available on this point; but it has been noted (Anderson et al., 1998), as one gauge of efficacy, that the postsynaptic densities in V5 are only slightly smaller (B/0.13 mm2) than those of the thalamocortical synapses in the cat (0.18 mm2). By analogy with the better investigated cat system, the size of the MT/V5 synapses suggest that the mean amplitudes of the unitary AMPA receptor EPSPs would be 1/ 2 mV. Additionally, the ultrastructural investigation found that single afferents can make multisynaptic contacts with single neurons in MT/V5. If this occurs commonly, this would be another mechanism for large EPSPs. Another distinctive specialization of the axons from V1 is that they have a bistratified distribution in both layers 4 and 6 of MT. The collaterals to layer 6 are unusual and suggest a particular importance for this layer in the microcircuitry. The most likely possibilities are: a tight, neuron-to-neuron feedback loop between the feedforward-projecting neurons in layers 4B and 5 of V1, and the feedback-projecting neurons in layer 6 of MT; interlaminar intrinsic connections from layer 6 to layers 3/4 within MT; or subcortical connections from the deeper layers of MT to the pulvinar, superior colliculus, or pons. It is worth noting that geniculocortical afferents frequently have collaterals in layer 6 of V1 (Blasdel and Lund, 1983; Freund et al., 1989). Axons terminating in MT commonly have two arbors in layer 4 and one in layer 6. The principal arbor in layer 4 is about the same size (200 mm in diameter) as the majority of V1 arbors in V2, and V2 arbors in V4. The arbors in layer 6 are smaller, and usually offset from the supragranular arbors. This contrasts with geniculocortical axons, where collaterals to layer 6 are shown in register with overlying arbors to layer 4 (Blasdel and Lund, 1983; Freund et al., 1989). The pattern of multiple arbors may relate to the functional architecture of MT, which consists of patches of neurons tuned for disparity or directionality (Albright et al., 1984; Born and Tootell, 1992; DeAngelis and Newsome, 1999). Further work is necessary, however, to determine the exact relationships. The terminations from V2 to MT do not have the conspicuous specializations of the terminations from V1 (Fig. 4), and are rather similar to the V2 cortical terminations in V4 (Rockland, 1995). Unlike the connections from V1, those from V2 had thinner axons (1.0 vs. 3.0 mm), delicate terminal boutons, and no collaterals in layer 6. Of 15 single arbors from V2, 12 were concentrated in layers 3/4 and were 200 /250 mm in diameter. Three arbors were larger (d/400 mm) and had a columnar shape, extending from layer 4 toward layer 1. Of seven more fully reconstructed axons, five had three arbors (d B/200 mm) in layers 3/4, separated by 200 /600 mm. Two axons terminated in what appeared
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to be a single focus in layers 3/4. How the V1 and V2 connections interact and whether they share postsynaptic targets is still unknown.
4. Feedback connections: divergence Feedback connections have attracted considerable interest in the context of ‘top down’ effects (cf. Bullier et al., 2001; Naya et al., 2001; von Stein et al., 2000). They have been considered as contributing to processes related to perceptual modulation, while feedforward connections may be more directly responsible for receptive field tuning properties (Lamme et al., 1998). Experiments to evaluate changes in receptive field properties in a target area after inactivation of feedback connections have led to the suggestion that feedback connections have a potentiating effect on the responses of neurons in areas V1, V2, and V3, and that these effects are fast, influencing the early phase of the response (reviewed in Bullier et al., 2001). The physiological properties of these connections have also been investigated in slice preparations in rats. Shao and Burkhalter (1996, 1999) have shown that IPSPs are smaller in feedback than in feedforward, thalamocortical or horizontal intrinsic axons. EPSPs are similar in all three pathways. Unfortunately, there have been no combined anatomical-physiological studies in primates; and there are as yet few direct points of contact between anatomical and physiological studies of feedback connections. Feedback connections have been investigated at the single axon level for V2 to V1 (Rockland and Virga, 1989), V4 and TEO to V2 (Rockland et al., 1994), from TE to TEO (Suzuki et al., 2000), and from MT/V5 to V1 and V2 (in squirrel monkeys: Rockland and Knutson, 2000). As there have so far been no reports of any obvious specializations, these systems will be considered together in one section. As for feedforward connections, the better visualization from high resolution tracers showed that the laminar termination pattern of feedback connections is complex. For connections from V2 to V1, and from V4, axons targeted layer 1, but frequently emitted collaterals in the deeper layers (Rockland and Virga, 1989; Rockland et al., 1994). From TE, four of nine reconstructed axons in TEO had terminations in layers 1 /3, one axon terminated in layer 1 alone, two axons in layers 5 and 6 only, and two axons in layers 1, 2, 3, 5 and 6 (Suzuki et al., 2000). From MT, two of nine axons terminated in V1 in layer 1 alone, three axons only in layer 4B, one axon in layers 1 and 4B, and 3 axons in layers 1, 4B and 6 (Rockland and Knutson, 2000). These fine laminar differentiations, as commented in the previous section, may be an indication of further functionally significant subtypes, in this case, subtypes
of feedback connections (see also the Discussion in Suzuki et al., 2000). This possibility is likely, given the heterogeneity of feedback efferent neurons. In most areas, these have a bistratified distribution in layers 6 and 3A, and the population within layer 6 is itself morphologically mixed. There is no direct evidence, however, that these two populations in layer 3A and 6 have different terminal configurations; and more detailed studies, such as depth-specific injections, are still needed. The more precise laminar data imply that feedback connections have a mixed postsynaptic population, and that the frequent emphasis on proximal/distal complementarity of feedforward and feedback connections may need to be qualified. Feedback terminations in the deeper layers are clearly not targeting distal apical dendrites. Moreover, as stated in Section 3.1, some feedforward terminations occur in layers 1 and 2, and potentially might directly converge with feedback connections. A second result from single axon analysis concerns the spatial extent of feedback systems. Terminal fields are typically divergent, extending over distances /1.0 mm. This confirms previous experiments using WGAHRP. That is, feedback terminations in layer 1 extended further than feedforward projecting neurons in layer 3, and feedback projecting neurons in layer 6 extended further than feedforward terminations in layer 4 (Section V.C in Salin and Bullier, 1995; Sections 2.2, 3.2 in Rockland, 1997; and the Discussion in Rockland and Knutson, 2000). Axon reconstruction demonstrated the divergence more directly, and further showed that terminations are often distributed continuously, somewhat like parallel fibers in the cerebellum. The activated postsynaptic population, therefore, will likely be a cohort of neighboring neurons. This contrasts with the geometry of feedforward connections. Even when there are multiple arbors, these are comparatively small, spherical, and dispersed, contacting spatially separate populations. Indirect evidence suggests that feedback connections probably extend across functional modules, although this is again an issue that requires more work. There is some indication from single axon analysis that the divergence factor increases with distance between source and target area (Rockland and Virga, 1989; Rockland et al., 1994; the Discussion in Rockland and Knutson, 2000). Connections from V2 to V1 (about 1.0 /4.0 mm long) are shorter than those from V4 to V2 (about 3.0 /5.0 mm long). In the case of axons bifurcating to two areas, terminal fields often span larger territories in the more distant area; for example, axons from TEO or V4 to V1 extend up to 6.0 and 5.0 mm, respectively, in V1, but B/5.0 mm in V2. Similarly, analysis of one MT axon with branches to V1 and V2 showed that the terminal field in V1 was larger (Fig. 6, Rockland and Knutson, 2000).
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5. Connections between pulvinar and cortex Connections between visual cortical areas and pulvinar subnuclei have been investigated extensively with retrograde and older anterograde tracers. They have frequently been discussed in general terms as reciprocal loops (but see Descheˆnes et al., 1998), and implicated in thalamic oscillations (Contreras et al., 1996; Jones, 2001). Their functional role and fine scale organization is largely mysterious, but it seems likely that they have an important role in visual cortical processing, and will need to be incorporated in the current framework of feedforward, feedback, and intrinsic connectivity (Robinson and Cowie, 1997; Grieve et al., 2000). As it turned out, the higher resolution tracers dramatically demonstrated that there are at least two types of corticopulvinar terminations, which had not been distinguishable with 3H-amino acids or WGAHRP (Rockland, 1996). One (‘elongate’ or type 1) is characterized by an elongated terminal field and a large number of terminal specializations that are distinctively spinous. The second (‘round’ or type 2) has a small spherical terminal arbor (d/100/125 mm), with a small number ( B/200 per arbor) of mainly large, beaded endings. The second type is numerically sparse from all areas examined so far except for V1 (Rockland, 1996). These two types of terminations have now been demonstrated in the corticothalamic pathways of cat auditory (Ojima, 1994) and cat visual cortices (Sherman and Guillery, 2001), and seem to be common across several species and areas (reviewed in Rouiller and Welker, 2000). Earlier electron miscroscopic studies had reported large and small corticopulvinar boutons (Ogren and Hendrickson, 1979), but the single axon approach has been significant in providing direct visualization, additional classification criteria, and a larger sample size. The functional significance of these different corticopulvinar connections requires further investigation. One hypothesis has been that the large, type 2 connections have a driving influence, whereas the type 1 connections are more modulatory (for example, Sherman and Guillery, 2001). This inference is in part from analogy with the retino- and corticogeniculate pathways, which differ in several structural and functional respects, possibly consistent with, respectively, ‘driving’ and ‘modulatory’ effects (Section IX, Sherman and Guillery, 2001). Direct evidence for modulatory roles, however, is difficult; and it should be kept in mind that the parameters of synaptic efficacy are complex and highly context-dependent. In the reverse, pulvinocortical (PC) direction, one recent single axon study reports on the connections from two injections at the lateral border of the lateral pulvinar (Rockland et al., 1999). Projection foci to
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extrastriate aeras (V2, V3, and V4) were densest in layer 3 and 5, as previously reported (Levitt et al., 1995), but individual axons were found to have terminations in different laminar combinations. In area V2, PC axons frequently had collaterals in layer 1 in addition to terminations in layers 3, 4 and 5 (see Figs. 6 /8, 14 of Rockland et al., 1999). Thus, at least in V2, PC terminations occur in layers targeted both by feedforward and feedback cortical connections. Like corticocortical connections, PC axons (n /25) were found to have multiple, spatially separate arbors. Curiously, the overall size of the PC arbors was not uniform across areas. In MT/V5, arbors were small (d B/ 200 mm), and in V4, they were large (d is about 600 mm). In V2, arbor size was more variable, but typically measured 250/500 mm. Since, as reviewed in Section 3, the arbor size of cortical connections (CC) is relatively constant in V2, V4, and MT, the conclusion is that there are area-specific differences in the size ratios of PC to CC terminal fields. How do corticopulvinar and pulvinocortical fields compare? Despite the earlier emphases on reciprocity, the arbor configuration is actually asymmetrical. Type 1 corticopulvinar fields sweep over large distances in the pulvinar, seemingly larger than pulvinocortical arbors in the cortex (at least in V2 and MT/V5); but type 2 corticopulvinar arbors are smaller than most pulvinocortical arbors (100 vs. 200 mm). A larger sample, especially from different pulvinar subdivisions, will be important in extending and interpreting these results.
6. Summary and future directions From these investigations of single axon connectivity, we are beginning to have a reasonable database of morphometrics and microstructure. This has already led to results in several spheres. First, the quantitative data are a source of realistic parameters for modeling studies, and can be used as one measure of connectional efficacy (e.g. Budd, 1998). Second, comparisons across different areas in some cases have pointed to definite structuralfunctional correlates (notably, in MT/V5). In general, correlates have been less obvious, but this in itself may be significant if, as suggested by other evidence (Kondo et al., 1999), features change in a gradient-wise fashion. Third, there have been significant clarifications simply by looking with the improved visualization of high resolution tracers. These include better laminar definition, clearer criteria of possible subtypes (on the basis of size and shape of boutons and arbors), and confirmation of axon branching. There are several major avenues for future extensions. One is direct correlation of axon geometry with functional architecture. This has been difficult because the need for unbroken serial sections in axon analysis is not
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easily compatible with cytochrome oxidase or other standard markers of functional architecture. Several studies, however, have now used optical imaging of the intrinsic cortical signal to pre-define functional modules. These are then targeted for tracer injections and aligned with the subsequent histological sections. This approach has been very successful in area V1, where ocular dominance and other modules have been compared with intrinsic excitatory (Malach et al., 1993; Yoshioka et al., 1996; Li et al., 2000), and intrinsic inhibitory connections (cat area 18: Buzas et al., 2001). It has recently been used in monkey inferotemporal cortex to compare the distribution of horizontal intrinsic connections with the distribution of activation maps elicited by the presentation of complex visual objects (Tanifuji et al., 2001). In areas V2 and MT/V5, despite the distinct modularity of these areas, it is still unknown whether the multiple arbors of a single axon terminate within the same or related modules. When terminations are in different layers, are these located in similar or opposite compartments? For that matter, direct evidence is still needed for whether feedback connections, in V1 and V2 especially, cross over functional domains, as is implied by their very extended configuration. A second important direction is microcircuitry, at the level of electron or confocal microscopy. Data about specific postsynaptic targets will be essential for progress in how receptive field properties are built up and for specific comparisons among the geniculo-, cortico- and pulvinocortical pathways. Ultrastructural studies in rodents, for example, report that feedback connections from area LM to V1 terminate with a distinctively high proportion of excitatory contacts mainly onto other pyramidal neurons (Johnson and Burkhalter, 1996); but this needs to be confirmed for primates. Within the feedback systems alone, there remain many basic questions: what are the differences between the terminations in layer 1 and those in the deeper layers? Between feedback from MT/V5 and from V2, in V1? Between feedback from MT/V5 in V1 and V2? Do feedback terminations from V2 and V4 converge on the same neurons in V1? A third direction is the issue of how connectional systems interact. Connectional influences have been addressed by techniques such as antidromic stimulation or selective inactivation (for example, Hupe´ et al., 2001a,b). There are still, however, very little data on how two or more afferent systems interact. Anatomically, precise information is necessary about convergence. This can now be acquired by injections of double anterograde tracers, especially if combined with electron or confocal microscopic preparations. Functionally, perhaps in vivo modifications can be devised of the elegant in vitro techniques now available, such as using
calcium indicators to detect neurons activated by stimulating a ‘trigger’ neuron (Peterlin et al., 2000). Finally, despite the interest in moving to functionally relevant experimental paradigms, there will need to be a much larger database of axon morphology */ different systems in different areas, species, and developmental ages. Ideally and maybe essentially, we need comparably detailed data for intrinsic, callosal, cortical, and subcortical connections. As one of many questions, for example, what are the rules governing the intrinsic collaterals and extrinsic terminations of different pyramidal neurons? Experiments in slice preparations, have clearly demonstrated differential signaling from the axon of a single neuron, as a function of postsynaptic targets (Markram et al., 1998). The same complexity may need to be considered for extrinsic connectivity, and will be important for understanding area specializations and interactions. Realistically, a more comprehensive database may depend on technical advances that can either circumvent or expedite the labor intensive process of serial section reconstruction. Smaller or juxtacellular injections may help in isolating labeled profiles; or this may be accomplished by alternative new techniques.
Acknowledgements I thank Dr Manabu Tanifuji for helpful discussion and Michiko Fujisawa for assistance with manuscript preparation.
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