visual cortical organization at the single axon le el: a...

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 transfor-

mations 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 geni-

culocortical connections demonstrated the laminar seg-

regation 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 develop-

mental and plasticity paradigms (Blasdel and Lund,

1983; Freund et al., 1989; Florence and Casagrande,

1990).

Comparable studies of long distance cortical connec-

tions 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

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 organi-

zation of connections, related to the morphometrics of

terminal arbors and boutons. These data are necessary

for further consideration of how microstructural fea-

tures 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* Tel.: �81-48-467-6427; fax: �81-48-467-6420.

E-mail address: [email protected] (K.S. Rockland).

Neuroscience Research 42 (2002) 155�/166

www.elsevier.com/locate/neures

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

mailto:[email protected]

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 demon-

strated that connections terminate with laminar specifi-

city. Three generally recognized classes of connections

were distinguished as feedforward, feedback and lateral

connections (Maunsell and Van Essen, 1983). Feedfor-

ward connections terminated mainly in layer 4, and were

at least implicitly assumed to be similar to geniculocor-

tical connections in V1. Feedback connections termi-

nated densely in layer 1, sometimes along with some

combination of other layers but not layer 4; and lateral

connections had a columnar configuration, with termi-

nations 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

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.

K.S. Rockland / Neuroscience Research 42 (2002) 155�/166156

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).

K.S. Rockland / Neuroscience Research 42 (2002) 155�/166 157

Fig. 3. FR and BDA can be used as double anterograde tracers. The low power photomicrograph in (A) shows two injections in area V2 (semi-

tangential 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).


convergent/divergent architectures that had been hidden

within the denser projection foci? It was thought that

comparisons across different visual areas might con-

tribute 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 auto-

matic 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,

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).


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 indivi-dual 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, strongsubdivisions 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 inaddition are obscured within the injection site. Intracel-

lular 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 inves-

tigation 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 inlayer 4 (‘feedforward’). In confirmation of the impor-

tance 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 geniculocor-tical 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 showedthat 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 andBullier, 1985; Sincich and Horton, 2001). These may

project, in an inverted depth gradient, to the supragra-

nular 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 connec-tions).

Another implication of these complex laminar pat-

terns 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 inother 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 wassomewhat surprising given the differences in magnifica-

tion factor, topography, and functional properties that

have been shown by other techniques, and the area

specific morphometric differences reported for pyrami-

dal 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 ispossible, 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 belarger and have more boutons.

While there was no strong indication for area-specific

size of arbors, there was clear evidence of an area-

specific trend in the number of arbors per axon. Of 20


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? Twoinvestigations 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 feedforwardconnections 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 micro-

structural 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 paradoxi-

cally, single axon analysis has demonstrated that the

connections from V1 have several specializations con-sistent 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 sofar, 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 thiswould 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 thesomatas of inhibitory interneurons. By comparison,

geniculocortical axons contact 52�/68% spines, 33�/47%

shafts, and 0�/3% somata (Freund et al., 1989).

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 ampli-

tudes of the unitary AMPA receptor EPSPs would be 1�/

2 mV. Additionally, the ultrastructural investigation

found that single afferents can make multisynapticcontacts 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 possibilitiesare: 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 geniculocor-

tical 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 geniculocor-

tical axons, where collaterals to layer 6 are shown inregister 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 theconspicuous specializations of the terminations from V1

(Fig. 4), and are rather similar to the V2 cortical

terminations in V4 (Rockland, 1995). Unlike the con-

nections 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) andhad 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


to be a single focus in layers 3/4. How the V1 and V2

connections interact and whether they share postsynap-

tic 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 forreceptive 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 anato-

mical-physiological studies in primates; and there are asyet 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 obviousspecializations, these systems will be considered together

in one section.

As for feedforward connections, the better visualiza-

tion 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; Rock-land 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 6and 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 de-

tailed studies, such as depth-specific injections, are still

needed.

The more precise laminar data imply that feedback

connections have a mixed postsynaptic population, andthat the frequent emphasis on proximal/distal comple-

mentarity 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 con-

nections.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 WGA-

HRP. 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 (Sec-tion 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, some-

what like parallel fibers in the cerebellum. The activated

postsynaptic population, therefore, will likely be a

cohort of neighboring neurons. This contrasts with thegeometry 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 distancebetween 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 bifurcat-

ing 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).


5. Connections between pulvinar and cortex

Connections between visual cortical areas and pulvi-

nar subnuclei have been investigated extensively withretrograde and older anterograde tracers. They have

frequently been discussed in general terms as reciprocal

loops (but see Deschenes 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 offeedforward, feedback, and intrinsic connectivity (Ro-

binson 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 WGA-

HRP (Rockland, 1996). One (‘elongate’ or type 1) is

characterized by an elongated terminal field and a largenumber 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 beendemonstrated 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 directvisualization, additional classification criteria, and a

larger sample size.

The functional significance of these different cortico-

pulvinar 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 analogywith 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

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 axonsfrequently 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 feedfor-

ward 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 notuniform 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 pulvino-

cortical 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 ledto 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 structural-

functional 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 (Kondoet 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 defini-

tion, 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 func-

tional architecture. This has been difficult because the

need for unbroken serial sections in axon analysis is not


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 mod-

ularity 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 ques-

tions: 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. Anatomi-

cally, precise information is necessary about conver-

gence. 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 amuch 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 sub-

cortical connections. As one of many questions, for

example, what are the rules governing the intrinsic

collaterals and extrinsic terminations of different pyr-

amidal neurons? Experiments in slice preparations, haveclearly 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 specializa-

tions and interactions. Realistically, a more comprehen-

sive database may depend on technical advances that

can either circumvent or expedite the labor intensiveprocess 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 organiza-

tion 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.


Cheng, K., Saleem, K.S., Tanaka, K. 1997. Organization of corticos-

triatal and corticoamygdalar projections arising from the anterior

inferotemporal area TE of the macaque monkey: a Phaseolus

vulgaris leucoagglutinin study. J. Neurosci. 15, 7902�/7925.

Contreras, D., Destexhe, A., Sejnowski, T.J., Steriade, M. 1996.

Control of spatiotemporal coherence of a thalamic oscillation by

corticothalamic feedback. Science 274, 771�/774.

DeAngelis, G.C., Newsome, W.T. 1999. Organization of disparity-

selective neurons in Macaque area MT. J. Neurosci. 19, 1398�/

1415.

Deschenes, M., Veinante, P., Zhang, Z.-W. 1998. The organization of

corticothalamic projections: reciprocity versus parity. Brain Res.

Rev. 28, 286�/308.

Elston, G.N., Tweedale, R., Rosa, M.G.P. 1999. Cortical integration

in the visual system of the macaque monkey: large scale morpho-

logical differences of pyramidal neurons in the occipital, parietal

and temporal lobes. Proc. R. Soc. Lond. Ser. B 266, 1367�/1374.

Florence, S.L., Casagrande, V.A. 1990. The development of geniculo-

cortical axon arbors in a primate. Vis. Neurosci. 5, 291�/311.

Freund, T.F., Martin, K.A.C., Soltesz, I., Somogyi, P., Whitteridge,

D. 1989. Arborisation pattern and postsynaptic targets of physio-

logically identified thalamocortical afferents in striate cortex of the

macaque monkey. J. Comp. Neurol. 289, 315�/336.

Grieve, K.L., Acuna, C., Cudeiro, J. 2000. The primate pulvinar

nuclei: vision and action. TINS 23, 35�/39.

Hupe, J.-M., James, A.C., Girard, P., Bullier, J. 2001. Response

modulations by static texture surround in area V1 of the macaque

monkey do not depend on feedback connections from V2. J.

Neurophysiol. 85, 146�/163.

Hupe, J.-M., James, A.C., Girard, P., Lomber, S.T., Payne, B.R.,

Bullier, J. 2001. Feedback connections act on the early part of the

responses in monkey visual cortex. Neurophysiology 85, 134�/145.

Johnson, R.R., Burkhalter, A. 1996. Microcircuitry of forward and

feedback connections within rat visual cortex. J. Comp. Neurol.

368, 383�/398.

Jones, E.G. 2001. The thalamic matrix and thalamocortical synchrony.

TINS 24 (10), 595�/601.

Kennedy, H., Bullier, J. 1985. A double-labeling investigation of the

afferent connectivity to cortical areas V1 and V2 of the macaque

monkey. J. Neurosci. 5 (10), 2815�/2830.

Kondo, H.T., Tanaka, K., Hashikawa, T., Jones, E.G. 1999.

Neurochemical gradients along monkey sensory cortical pathways:

calbindin-immunoreactive pyramidal neurons in layers II and III.

Eur. J. Neurosci. 11, 4197�/4203.

Lamme, V.A.F., Super, H., Spekreijse, H. 1998. Feedforward,

horizontal, and feedback processing in the visual cortex. Curr.

Opin. Neurobiol. 8, 529�/535.

Levitt, J.B., Yoshida, T., Lund, J.S. 1995. Connections between the

pulvinar complex and cytochrome oxidase-defined compartments

in visual area V2 of macaque monkey. Exp. Brain Res. 104, 419�/

430.

Li, H., Fukuda, M., Tanifuji, M., Rockland, K.S. 2000. Correlation of

Meynert cell intrinsic collaterals with ocular dominance columns in

monkey primary visual cortex. Ann. Mtg. Soc. Neurosci. Abstr. 30,

143.

Lund, J.S., Yoshioka, T., Levitt, J.B. 1993. Comparison of intrinsic

connectivity in different areas of macaque monkey cerebral cortex.

Cereb. Cortex 3, 148�/162.

Malach, R., Amir, Y., Harel, M., Grinvald, A. 1993. Relationship

between intrinsic connections and functional architecture revealed

by optical imaging and in vitro targeted biocytin injections in

primate striate cortex. Proc. Natl. Acad. Sci. USA 90, 10469�/

10473.

Markram, H., Wang, Y., Tsodyks, M. 1998. Differential signaling via

the same axon of neocortical pyramidal neurons. Proc. Natl. Acad.

Sci. 95, 5323�/5328.

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 distribu-

tion of striate cortex terminals in the interior and lateral subdivi-

sions of the macaca monkey pulvinar. J. Comp. Neurol. 188, 179�/

199.

Ojima, H. 1994. Terminal morphology and distribution of corticotha-

lamic 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 fea-

tures 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.


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., Konig, 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.


visual cortical organization at the single axon le el: a...

Documents