Download The occipitoparietal pathway of the macaque monkey: comparison

The Occipitoparietal Pathway of the
Macaque Monkey: Comparison of
Pyramidal Cell Morphology in Layer III of
Functionally Related Cortical Visual Areas
Guy N. Elston and Marcello G. P. Rosa
The dendritic morphology of pyramidal cells located at the base of
layer III in the primary visual area (V1), the second visual area (V2),
the middle temporal area (MT), the ventral portion of the lateral
intraparietal area (LIPv) and in the portion of cytoarchitectonic area
7a within the anterior bank of the superior temporal sulcus was
revealed by injecting neurons with Lucifer Yellow in fixed, flattened
slices of macaque monkey visual cortex. These areas correspond to
different levels of the occipitoparietal cortical ‘stream’, which
processes information related to motion and spatial relationships in
the visual field. The tissue was immunocytochemically processed to
obtain a light-stable diaminobenzidine reaction product, revealing
the dendritic morphology in fine detail. Retrogradely labelled
MT-projecting neurons in supragranular V1 (layer IIIc of Hassler’s
nomenclature, corresponding to Brodmann’s layer IVb) were
predominantly pyramidal, although many spiny multipolar (stellate)
cells were also found. The average basal dendritic field area of
pyramidal neurons in sublamina IIIc of V1 was significantly smaller
than that in the homologous layer of V2, within the cytochrome
oxidase-rich thick stripes. Furthermore, the average basal dendritic
field areas of V1 and V2 pyramidal neurons were significantly
smaller than those of neurons in MT, LIPv and area 7a. There was no
difference in basal dendritic field area between layer III pyramidal
neurons in areas MT, LIPv and 7a. While the shape of most basal
dendritic fields was circularly symmetrical in the dimension
tangential to the cortical layers, there were significant biases in
complexity, with dendritic branches tending to cluster along
particular axes. Sholl analysis revealed that the dendritic fields of
neurons in areas MT, LIPv and 7a were significantly more complex
(i.e. had a larger number of branches) than those of V1 or V2
neurons. Analysis of basal dendritic spine densities revealed
regional variations along the dendrites, with peak densities being
observed 40–130 µm from the cell body, depending on the visual
area. The peak spine density of layer III pyramidal neurons in V1 was
lower than that observed in V2, MT or LIPv, which were all similar.
Pyramidal neurons in area 7a had the greatest peak spine density,
which was on average 1.7 times that found in V1. Calculations based
on the average spine density and number of dendritic branches at
different distances from the cell body demonstrated a serial increase
in the total number of basal dendritic spines per neuron at
successive stations of the occipitoparietal pathway. Our observations, comparing dendritic fields of neurons in the homologous
cortical layer at different levels of a physiologically defined ‘stream’,
indicate changes in pyramidal cell morphology between functionally
related areas. The relatively large, complex, spine-dense dendritic
fields of layer III pyramidal cells in rostral areas of the
occipitoparietal pathway allow these cells to sample a greater
number of more diverse inputs in comparison with cells in ‘lower’
areas of the proposed hierarchy.
visual areas located in the parietal and temporal lobes.
Connections between areas in these two pathways are
lamina-specific, and contrasting patterns of laminar origin and
termination of pathways have been used to arrange visual areas
into anatomical hierarchies (Rockland and Pandya, 1979;
Wong-Riley, 1979a; Tigges et al., 1981; Maunsell and Van Essen,
1983a; Colby et al., 1988; Andersen et al., 1990; Boussaoud et
al., 1990; Sousa et al., 1991; Rosa et al., 1993). Projections that
originate mainly in the supragranular layers and terminate
around layer IV are referred to as ‘feedforward’, and projections
that originate mainly from infragranular layers and terminate
outside layer IV are referred to as ‘feedback’ (e.g. Rockland and
Pandya, 1979; Maunsell and van Essen, 1983a). Thus, V1 is at the
‘low’ end of the proposed hierarchy, and the visual areas of the
temporal and parietal lobes are at the ‘high’ end (for review see
Felleman and Van Essen, 1991).
Basal dendritic territories of pyramidal neurons at an
‘intermediate’ level of the occipitotemporal pathway (the fourth
visual area, V4) are larger than those in ‘lower’ stations such as
V1 and the second visual area (V2; Lund et al., 1993). In addition,
recent studies in macaque have shown an increase in the
cross-sectional area of axonal clusters of intrinsic connections
along the proposed hierarchy of visual areas, from V1 to parietal
area 7a and the inferior temporal cortex (Yoshioka et al., 1992;
Amir et al., 1993; Lund et al., 1993; Fujita and Fujita, 1996). This
trend has been interpreted as a correlate of the anatomical
hierarchy of processing, whereby the more widespread intrinsic
connections, coupled with the larger centre to centre spacing,
would ref lect the ‘more global computational roles proposed for
higher-order visual areas’ (Amir et al., 1993). In parallel with
these obser vations, Lund and co-workers (1993) also
demonstrated that there is a proportional relationship between
the cross-sectional area of intrinsic axonal clusters and the
dendritic spread of supragranular pyramidal neurons in cortical
areas: the larger the intrinsic axonal clusters in a given area, the
larger the dendritic fields of pyramidal neurons. In view of these
results, one might expect the dendritic field areas of pyramidal
neurons to increase progressively along the pathways that link
V1 to the parietal and temporal lobes. Indeed, in a recent study
of marmoset monkey visual cortex (Elston et al., 1996), we found
a progressive increase in the dendritic field area of layer III
pyramidal cells between V1, V2, the dorsolateral area (DL) and
the fundus of the superior temporal area (FST). Although these
observations could be interpreted as supporting the anatomicalhierarchical model, the evidence is still inconclusive, mainly
because comparisons have been made between areas belonging
to the occipitoparietal and occipitotemporal pathways, which
process different modalities of vision. For example, both DL in
the marmoset and its macaque homologue (V4) are linked to the
processing of object shape and colour, whereas FST and area 7a
process motion and spatial relationships (e.g. Boussaoud et al.,
The visual system of primates comprises multiple cortical areas,
located in the occipital, temporal and parietal lobes. Two main
cortical pathways have been proposed on the basis of functional
and anatomical criteria, streaming from striate cortex (V1) into
Vision, Touch and Hearing Research Centre, Department of
Physiology and Pharmacology, The University of Queensland,
Queensland 4072, Australia
Cerebral Cortex Jul/Aug 1997;7:432–452; 1047–3211/97/$4.00
Table 1
Animals and visual areas in which neurons were injected
Animal
Age (months)
Sex
Weight (kg)
Areas studied
Plane of sectioning
AM 1
RM 13
RM 14
RM 16
14
20
20
96
male
male
female
female
1.3
1.5
1.5
2.2
V2
V1, 7a
MT, LIPv
V1
tangential
tangential
tangential
coronal
1990; Distler et al., 1993). Furthermore, previous studies did not
establish whether the neurons sampled in V1 and V2 were
part of the occipitoparietal or occipitotemporal pathways. We
addressed this problem by investigating the dendritic fields of
pyramidal neurons in striate cortex and four extrastriate areas
that correspond to different levels of the proposed motionprocessing pathway of the macaque visual system. Pyramidal
cells located at the base of layer III in V1 (layer IVb of
Brodmann), the origin of the occipitoparietal pathway, were
compared with basal layer III pyramidal neurons located within
the thick cytochrome oxidase-rich stripes of the second visual
area (V2), in the middle temporal area (MT), in the ventral
portion of the lateral intraparietal area (LIPv; Blatt et al., 1990)
and in the portion of cytoarchitectonic area 7a located in the
dorsal superior temporal sulcus (Hikosaka et al., 1988; Andersen
et al., 1990). This was achieved by intracellular injection of the
naphthalamide tracer Lucifer Yellow in fixed cortical slices.
These data also allowed the study of other aspects of basal
dendritic field morphology in extrastriate cortex that have not
been addressed by previous studies, such as the relative
complexity of dendritic fields, dendritic biases and the spine
density of neurons of different visual areas.
Materials and Methods
Animals and Tracer Injection
Four macaque monkeys (Macaca fascicularis) were used in this study
(Table 1). The monkeys were used in non-related acute experiments
involving electrophysiological recording or optical imaging, under
pentobarbitone sodium anaesthesia (Calford et al., 1995). Three of these
cases (AM 1, RM 13 and RM 14), which yielded the bulk of the data, were
juveniles (14–20 months old). In a fourth, 8-year-old animal (RM 16), a
1 µl injection of the f luorescent tracer Fluoroemerald (7% in H2O;
Molecular Probes D 1820) was placed in area MT at the beginning of a
non-related 3 day electrophysiological recording experiment, in order to
label neurons retrogradely in V1. In this case, after initial anaesthesia
(ketamine, 50 mg/kg and xylazine, 6 mg/kg; pentobarbitone sodium, 7
mg/kg), the animal was placed in a stereotaxic frame and the skull
overlying the superior temporal and lateral sulci was removed. The
injection was made with the aid of a 1 µl microsyringe (Scientific Glass
Engineering, Australia) mounted in a stereotaxic manipulator. The
microsyringe was lowered into the cortex from the surface of the anterior
bank of the superior temporal sulcus to a depth of 5 mm (Fig. 1A). This
anterior–posterior parasagittal approach resulted in penetration of the
posterior bank, including area MT. A series of 200 nl injections of
Fluoroemerald was made at 3–5 min intervals at slightly different points
along the track, to make a total of 1 µl. The microsyringe was left in place
for 10 min after the final injection, then retracted. The resulting injection
site was centred on area MT (Fig. 1B), but also leaked into the underlying
white matter and anterior bank of the superior temporal sulcus, probably
involving the medial superior temporal area (MST; Maunsell and Van
Essen, 1983a). As no projection from V1 to MST has been demonstrated,
this did not affect our results.
Histological Processing and Identification of Visual Areas
Upon completion of the electrophysiological experiments, the animals
were administered an i.v. injection of 200 mg/kg of sodium
Figure 1. Schematic showing (A) the approach used for the injection of Fluoroemerald
in animal RM16. The injection syringe penetrated via the anterior bank of the superior
temporal sulcus, but deposited the tracer mainly in the posterior bank of this sulcus.
Serial reconstruction of coronal sections revealed the core of the injection was in the
middle temporal area (MT). Two coronal sections (B) in which the injection site was
clearly visible are illustrated. Scale bar = 1 cm.
pentobarbitone. They were then perfused intracardially with normal
saline, followed by 4% paraformaldehyde (in 0.1 M phosphate buffer, pH
7.2). Blocks of tissue (Fig. 2) were excised from the occipital operculum
(V1), the posterior bank of the inferior occipital sulcus (V2), the posterior
and anterior banks of the dorsal superior temporal sulcus (including areas
MT and 7a) and the lateral bank of the intraparietal sulcus (including area
LIPv). Only the hemispheres contralateral to those involved in
electrophysiological recording experiments were used for intracellular
injection. Identification of areas MT, LIPv and 7a within the slices (Fig. 2)
was based on the sulcal morphology, using maps derived from previously
published studies (Gattass and Gross, 1981; Van Essen et al., 1981;
Desimone and Ungerleider, 1986; Andersen et al., 1990; Blatt et al., 1990;
Boussaoud et al., 1990; Shipp and Zeki, 1995). We were particularly
careful to avoid the regions around the caudal lip of the superior temporal
sulcus, which include parts of areas V4 and V4t, and around the lips and
fundus of the intraparietal sulcus, which include the dorsal subdivision of
LIP and the ventral intraparietal area respectively (Schein et al., 1982;
Maunsell and Van Essen 1983a; Gattass et al., 1988; Blatt et al., 1990;
Colby et al., 1993). The ventral portion of LIP, which receives input from
MT (Desimone and Ungerleider, 1986; Blatt et al., 1990; Shipp and Zeki,
1995), was selected for injection, as opposed to the dorsal portion which
receives input from both TEO and TE (Webster et al., 1994; Shipp and
Zeki, 1995). The blocks were f lattened between glass slides (e.g. Rosa et
al., 1988), left overnight in fixative (4% paraformaldehyde in 0.1 M
phosphate buffer) and then sectioned on a Vibratome. The exception was
animal RM 16, whose cortex was sectioned coronally in order to allow
reconstruction of the Fluoroemerald injection site in area MT (Fig. 1B)
and the morphological classification of retrogradely labelled cells in a
more traditional plane of sectioning. Alternate 50 and 250 µm slices were
obtained from the f lat-mounted blocks of tissue including V1 and V2, the
Cerebral Cortex Jul/Aug 1997, V 7 N 5 433
Figure 2. Schematics showing the parts of the brain from which blocks of tissue were excised (grey, in the inserts) and the relative location of individually injected neurons (dots) in
flat-mounted slices obtained from areas V1, V2, MT, LIPv and 7a. In the V2 diagram the occipital operculum was everted, revealing the posterior banks of the lunate and inferior
occipital sulci. The magnified view of the posterior bank of the inferior occipital sulcus indicates the location of cytochrome oxidase-rich thick stripes (dark grey) where MT-projecting
neurons are located, thin stripes (light grey) and cytochrome oxidase-poor interstripes (white). In the MT diagram the approximate borders of this area are indicated, based on the
heavier myelin content, which is visible even in unstained tissue. The scale bar (under the V2 diagram) corresponds to 5 mm.
50 µm slices being reacted for cytochrome oxidase (Wong-Riley, 1979b)
to reveal laminar features, the ‘blobs’ of V1 and the ‘stripes’ of V2 (Horton
and Hubel, 1981; Tootell et al., 1983). Only 250 µm slices were obtained
from the other areas, and the identification of layers was based on
cytoarchitecture (see below).
Nomenclature of Cortical Layers
Throughout this paper the nomenclature of cortical layers follows the
scheme proposed by Hassler (1966) rather than that proposed for
macaque striate cortex by Brodmann (1909). The main differences
between Hassler’s and Brodmann’s nomenclatures are summarized in
Table 2. We have decided to use the Hassler scheme because, based on
cytology and efferent connections, there is now ample evidence to
support the view that some of the subdivisions of Brodmann’s layer IV
(layers IVa and IVb) are more accurately described as subdivisions of layer
III (for a review see Casagrande and Kaas, 1994). Unlike the true granular
layer of primary sensory cortex, Brodmann’s sublamina IVb contains
many pyramidal neurons which provide direct ‘feedforward’ input to
other cortical areas in both the ipsi- and contralateral hemispheres (Lund
434 Supragranular Pyramidal Cells in Visual Cortex • Elston et al.
Table 2
Comparison between the nomenclature for the cortical layers of primate V1 used in the present
study (Hassler, 1966) and the one proposed by Brodmann
Hassler
Brodmann (1909, tr. Garey 1994)
I
II
IIIa
IIIb
IIIc
IVα
IVβ
V
VI
I
II
III
IVa
IVb
IVcα
IVcβ
V
VI
et al., 1975; Rockland and Pandya, 1981; Maunsell and Van Essen, 1983a;
Kennedy et al., 1986; Shipp and Zeki, 1989a; present results). Thus,
Brodmann’s sublamina IVb is more properly regarded as a subdivision of
layer III, as made explicit by Hassler’s nomenclature (IIIc). All of our
sample, in different areas, comes from supragranular cortex just above
the upper limit of Hassler’s layer IV, and this nomenclature emphasizes
the fact that our analyses involved comparable populations of cells in the
different areas. Furthermore, our laboratory is also involved in studies in
various New World monkey species, for which the Hassler (1966)
nomenclature of cortical layers is usually employed; thus, we opted for
the use of a consistent nomenclature between studies.
Identification of Layer III
In case RM 16 we specifically targeted neurons in transverse sections in
V1 that were retrogradely labelled from area MT, and therefore known to
be located in sublamina IIIc (Shipp and Zeki, 1989a; Vogt Weisenhorn et
al., 1995). The remaining cases were sectioned tangential to the cortical
layers. In V1 we identified the upper limit of sublamina IIIc in
f lat-mounted sections by cytochrome oxidase patterns, using the
characteristic cytochrome oxidase ‘honeycomb’ of sublamina IIIb as a
criterion (Hendrickson et al., 1978). Furthermore, in all areas the basal
limit of layer III was determined by the distinct cytoarchitecture of layers
III and IV. To determine cytoarchitecture, the slices were incubated for
10 min in 10–5 M 4,6-diamidino-2-phenylindole (DAPI; Sigma D9542) in
0.1 M phosphate buffer, which enabled the visualization of somata by
f luorescence microscopy. By focusing through the thickness of the slice,
we found that layer III was characterized by relatively large, sparsely
spaced somata of pyramidal cells, whereas layer IV contained the
relatively small, closely packed cell bodies of granule cells (Fig. 3).
Furthermore, injection of the densely packed DAPI-labelled neurons
below the transition point yielded only the small multipolar neurons
characteristic of the granular layer (unpublished results). In V2 only layer
III neurons whose somata were located within the thick cytochrome
oxidase-rich stripes (Tootell et al., 1983; Livingstone and Hubel, 1987;
Shipp and Zeki, 1985, 1989b) were included for analysis. Similar to other
studies using appropriately f lattened sections of V2 (e.g. Shipp and Zeki,
1989b; Abel et al., 1997), we found that the morphological distinction
between thick and thin cytochrome oxidase-rich stripes was relatively
obvious.
Intracellular Injection
Pre-labelling the tissue with DAPI facilitated rapid impalement of a large
number of cells in a single slice. Neurons in layer III were injected in a
semiregular array, which minimized any possible effects of experimenter
bias. The injection apparatus consisted of a fixed stage Zeiss microscope,
equipped with a ×40 long focal length immersion lens, a Leitz
micromanipulator and a perspex tissue chamber mounted on the stage.
The tissue was immersed in 0.1 M phosphate-buffered saline (pH 7.2).
The slices were mounted such that the layer closest to the pia mater was
uppermost in the injection chamber. Somata within ∼100 µm of the
surface of the tissue, but superficial to the layer III/ IV interface, were
injected to reveal a short portion of the apical dendrite and the complete
extent of the basal skirt of dendrites. A similar technique has been
employed in previous studies addressing tangential dendritic field areas of
pyramidal cells in sensory cortex (Hübener and Bolz, 1992; Matsubara et
al., 1996; Elston et al., 1996, 1997). Micropipettes (250–300 MΩ) were
pulled on a Sutter microelectrode puller (Brown Flaming P77, San
Francisco, CA). Individual neurons were injected with Lucifer Yellow
(Sigma L 0259; 8% in 0.05 M Tris buffer) by hyperpolarizing current (a
maximum of 100 nA for 10–20 s, subject to visual determination).
Following cell injection, the tissue was processed with an antibody to
Lucifer Yellow (kindly supplied by Dr David Pow) at a concentration of
Figure 3. (A) Photomicrograph showing a cross-section of extrastriate cortex that was
labelled with DAPI (4,6-diamidino-2-phenylindole), as viewed with UV excitation
(341–343 nm). (B, C) Sections through layers III (B) and IV (C) of flat-mounted tissue.
Note that in both cross-sectional and flattened preparations the granular layer was
easily distinguished from the supragranular layer due to the large number of closely
spaced neurons with relatively small somata. Scale bar (in C) = 200 µm for (A) and 75
µm for (B) and (C). The arrow in (A) points to the upper limit of layer IV.
Cerebral Cortex Jul/Aug 1997, V 7 N 5 435
1:400 000 in stock solution [2% bovine serum albumin (Sigma A3425), 1%
Triton X-100 (BDH 30632) and 5% sucrose in 0.1 M phosphate buffer].
The primary antibody was detected by a species-specific biotinylated
secondary antibody (Amersham RPN 1004; 1:200 in stock solution for 2
h) then serially processed with a biotin–horseradish peroxidase complex
(Amersham RPN 1051; 1:200 in 0.1 M phosphate buffer). Labelling was
revealed using 3,3′-diaminobenzidine (DAB; Sigma D 8001; 1:200 in 0.1 M
phosphate buffer) as a chromagen (Fig. 4).
Fluoroemerald injection in MT, and 428 DAPI-labelled neurons
in areas V1, V2, MT, LIPv and 7a. Of these, 41 retrogradely
labelled V1 cells were successfully reconstructed in transverse
sections to a level of detail that allowed their classification as
pyramidal or multipolar, and 183 DAPI-labelled pyramidal cells
in f lat-mounted tissue were successfully filled as per the three
criteria specified in Materials and Methods.
Analysis
Injected cells were classified as pyramidal or multipolar (Lund, 1984;
Nieuwenhuys, 1994; Elston et al., 1996), and were drawn with the aid of
a camera lucida. In order to be included for further analysis injected
neurons in f lattened sections had to satisfy three criteria: (i) they had to
be located at the base of layer III; (ii) they had to be unambiguously
identified as pyramidal cells by the presence of the characteristic apical
dendrite, which could be visualized by focusing through the thickness of
the slice (see fig. 3 of Elston et al., 1996); and (iii) they had to be
successfully filled, thus revealing their complete basal dendritic
arborization as well as fine details such as dendritic spines (Fig. 4). The
applicability of the last criterion to individual cells was judged by tracing
each terminal dendrite to an abrupt, well defined tip which usually had a
spinous process (see Matsubara et al., 1996). Cells in which the distal
branches had an uneven or beaded appearance were excluded. The
serendipitous nature of the slice boundaries relative to the cortical layers,
and the desire to inject cells that were both away from the upper limit of
the slice and above the limit of layer IV, meant that we were not able to
obtain samples from each area in each monkey (Table 1). Instead, we
concentrated the analyses in the best slice obtained from each area across
cases, as judged by the appropriate laminar position of cells and quality of
the cell filling. Note, however, that in every case in which we had more
than one sample, they were not distinguishable in terms of morphological
parameters such as dendritic field area.
In f lattened sections, basal dendritic field areas were estimated by
scanning the original drawings of the cells and drawing a polygon (hull)
joining the outermost distal tips of each terminal dendrite (examples of
drawn polygons are illustrated in Fig. 11). The area of the resulting
polygon was then calculated using standard features of NIH-IMAGE (NIH
Research Services, Bethesda, MD), which was run on a Macintosh Quadra
950 computer (Apple Computer Inc., Cupertino, CA). Other analyses of
basal dendritic fields were performed on a sample of 20 neurons from
each area, including cells whose dendritic fields ranged from the smallest
to the largest (explained in detail in the Results section). These included a
Sholl analysis (cf. Hübener and Bolz, 1992) aimed at comparing the
complexity of the branching pattern in different areas (estimated by the
number of intersections between dendritic branches and concentric
circles of increasing radius centred on the cell body). We chose to use
equal sample sizes rather than investigating each cell in each area, in
order to improve the reliability of multifactorial ANOVA design employed
in this analysis. The same sample was studied to explore the possibility of
significant directional asymmetries in the distribution of dendritic
branches, by determining the distribution of intersections as a function of
the radial position of the dendrite relative to the cell body. Spine densities
were calculated by drawing a selection of 20 basal dendrites of pyramidal
neurons from each area at high power (using a Zeiss ×100 immersion
objective and an Axioplan microscope). Because of the nature of the
immunocytochemical stain employed, spines were visible by focusing
across the dendritic shafts when viewed by high power light microscopy.
Thus, correction factors for hidden spines, which may be needed for
more opaque Golgi-stained material (Feldman and Peters, 1979), were
deemed unnecessary. No distinction was made between different spine
types, such as sessile or pedunculated (Jones and Powell, 1969). Cell
bodies were also drawn at high power in order to test for differences
between peak cell body cross-sectional areas, and for a correlation
between cell body size and basal dendritic field area. Again, areas were
calculated using standard features of the NIH-IMAGE software.
Neurons in V1 That Project to MT
The main objective of this study was to compare morphological
features of a single cell type (pyramidal) in different visual areas
that comprise the occipitoparietal pathway. In V1, specific
populations of neurons project from layer IIIc to area MT (Tigges
et al., 1981; Shipp and Zeki, 1989a). However, there is confusion
as to whether pyramidal neurons in layer IIIc contribute to these
projections. Thus, because we intended to compare neurons at
different levels of the occipitoparietal pathway, it was first
necessary to ascertain whether the study of pyramidal cells in
layer IIIc of V1 was appropriate. The injection of the tracer
Fluoroemerald in MT revealed retrograde labelling in V1 that
extended from the medial portion of the occipital operculum to
the dorsal bank of the calcarine sulcus [corresponding to the
representation of eccentricities between 5 and 30° of the lower
visual field (Daniel and Whitteridge, 1961)]. A ll of the
retrogradely labelled neurons were located at the base of layer III
(sublamina IIIc of Hassler, 1966).
Of 41 MT-projecting cells that were successfully reconstructed, 28 (68.3%) were pyramidal (including modified
pyramidal) and 13 (31.7%) were spiny multipolar neurons (Figs
5–7). Thus, pyramidal cells in layer IIIc form a substantial
portion of the MT-projecting population. Pyramidal neurons
were distinguished from spiny multipolar neurons by the
presence of an unambiguous apical dendrite and a characteristic
‘skirt’ of basal dendrites (Figs 5B, D and 6). Spiny multipolar
neurons lacked an apical dendrite, and their dendrites projected
radially from the cell body (Figs 5C and 7B–D). Two types of
spiny multipolar cells were retrogradely labelled from MT: one
with a relatively small soma and relatively thin dendrites (Fig. 7C)
and another with a very large cell body and relatively thick, wide
ranging dendrites (Figs 5C and 7B–D). A number of neurons that
were retrogradely labelled and subsequently injected had
morphological characteristics intermediate between those of
pyramidal and multipolar neurons. For example, these cells had
an ‘apical’ dendrite with daughter segments which in many cases
ref lected back toward the infragranular layers, and, instead of
the characteristic ‘skirt’ of basal dendrites, they had multiple
dendritic processes issuing from the soma in a radiate fashion
(Fig. 7A). Such modified pyramidal neurons have been reported
variously in sensory cortex (Lorente de No, 1938; Lund, 1973,
1984; Jones, 1975; Simons and Woolsey, 1984; Nieuwenhuys,
1994).
In many of the injected neurons the initial part of the axonal
trajectory was visible (Figs 6 and 7). In all cases where the axon
could be visualized, it issued from the surface of the soma closest
to the white matter. In most pyramidal neurons in which the
axon was visible, the main segment, which projected directly
toward the white matter, gave rise to horizontal collaterals
within layer III (e.g. Fig. 7A).
Results
Intracellular injection of Lucifer Yellow was performed on
47 neurons in V1 that were retrogradely labelled from a
436 Supragranular Pyramidal Cells in Visual Cortex • Elston et al.
Basal Dendritic Fields of Layer III Pyramidal Cells
Our conclusions related to the tangential area of basal dendritic
fields of pyramidal neurons, measured in f lat-mounted slices of
five visual areas, are summarized in Figures 8 and 9. The main
Cerebral Cortex Jul/Aug 1997, V 7 N 5 437
point revealed by this analysis can be qualitatively assessed
simply by observing a sample of injected pyramidal neurons in
different visual areas (Fig. 8): the basal dendritic fields of V1 and
V2 neurons were generally smaller than those of cells in MT, LIPv
and area 7a, but there was no obvious difference in area between
dendritic fields of cells in the last three visual areas. In order to
illustrate this point, cells in each area were ranked in ascending
order of tangential basal dendritic field area. Cells corresponding
to equivalent percentiles of the distributions of dendritic field
area were then drawn, with dendritic fields collapsed to a single
plane (Fig. 8). Figure 9 and Tables 3 and 4 provide graphic and
statistical summaries of the entire data set. A one-way analysis of
variance (Table 4) revealed a highly significant difference
between areas (P < 0.0001). In spite of a large overlap between
the basal dendritic field areas of pyramidal neurons located in V1
and in the thick stripes of V2 (Fig. 9), there was a statistically
Figure 5. Photomicrographs illustrating Fluoroemerald-labelled MT-projecting neurons in sublamina IIIc of V1 (A) and neurons that were subsequently injected with Lucifer Yellow
(B–D). Cells B and D have an apical dendrite characteristic of pyramidal neurons. Cell C, which lacks an apical dendrite, was classified as a spiny multipolar neuron by virtue of the
radiate fashion in which dendrites issue from its soma (note the exceptionally large soma). Scale bar = 200 µm for (A) and 100 µm for (B–D).
Figure 4. Photomicrographs of three extrastriate pyramidal neurons (A–C) located at the base of layer III, injected with Lucifer Yellow and processed for a DAB
(3,3′-diaminobenzidine) reaction product. The plane of focus includes the cell body and the initial segments of the basal dendrites. The apical dendrite and distal tips of dendrites
could only be visualized by focusing through the thickness of the section (not shown). Dendritic spines were clearly outlined, even at low power. There is little leakage of dye around
the cell body, thus allowing the unambiguous determination of somal area and number of primary dendrites. (D) is a magnified view of (C), illustrating the dendritic spines in greater
detail. Scale bar = 50 µm in (A–C), 20 µm in (D).
438 Supragranular Pyramidal Cells in Visual Cortex • Elston et al.
Figure 6. (A–D) Drawings of four MT-projecting neurons located in sublamina IIIc of V1. These cells had the characteristic morphology of pyramidal neurons. Part of the axon was
revealed and is illustrated in fine ink. Inserts show the position of cells relative to the pial surface (solid line) and the top of layer IV (dashed line). Scale bar = 100 µm.
Cerebral Cortex Jul/Aug 1997, V 7 N 5 439
Figure 7. Drawings of MT-projecting V1 neurons. Cell (A) was classified as a modified pyramidal neuron due to the ambiguous ‘apical’ dendrite, the daughter segments of which
project basally, and to the fact that the remaining dendrites radiated from the cell body in all directions, rather than forming the classical basal ‘skirt’. Neurons (B–D) were all classified
as spiny multipolar (stellate). Two types of multipolar neurons project to area MT, one with a very large cell body and thick, long-ranging dendrites (B and D), the other with a relatively
small cell body and restricted dendritic field (C). Axons are illustrated in fine ink. Inserts show the position of the cell relative to the pial surface (solid line) and the top of layer IV
(dashed line). Scale bar = 100 µm.
significant difference (P < 0.05) between the populations in
these two areas (Table 4). The distributions of dendritic field
area of cells in MT, LIPv and area 7a were clearly different from
440 Supragranular Pyramidal Cells in Visual Cortex • Elston et al.
those observed in V1 and V2 (Figs 8 and 9), a result which was
confirmed to be statistically significant (Tables 3 and 4). Finally,
there were no significant differences in basal dendritic field area
Figure 8. Basal layer III pyramidal neurons from V1, V2 (cytochrome oxidase- rich thick stripes), MT, LIPv and area 7a, viewed in the plane of section tangential to the cortical layers.
The neuron with the smallest basal dendritic field area from each visual area is illustrated on the left of each row; the one with the largest is on the right of each row. The other neurons
in each row represent 20% rank increments along the distribution of dendritic field area (i.e. the 20th, 40th, 60th and 80th percentiles). Scale bar = 100 µm.
of pyramidal neurons located at the base of layer III between
areas MT, LIPv and 7a (P > 0.05 in all cross-comparisons; see
Table 4). In summary, there is evidence for a stepwise increase in
dendritic field area of layer III pyramidal neurons at early stages
of the proposed cortical hierarchy (V1, V2 and MT), but not
between MT, LIPv and 7a.
Complexity of the Branching Patterns
A study of Figure 8 suggests that layer III pyramidal neurons in
rostral visual areas may have more complex basal dendritic
branching patterns than those in V1. However, a comparison of
the number of primary dendrites issuing from the cell body
showed no significant difference between areas (not illustrated).
In order to investigate this issue quantitatively, we performed a
Sholl analysis on the dendritic fields of cells from each area. The
result of this analysis (Fig. 10) confirmed that pyramidal cells in
layer III of all areas studied had similar branching patterns near
the cell body (in a ring 25 µm away from the centre). However,
the overall pattern of branching complexity was significantly
different between areas (Table 5). Cells in V1 and V2 had very
similar branching patterns throughout, both quantitatively and
qualitatively. In both areas the number of intersections peaked in
a relatively narrow region ∼50 µm from the centre of the cell
body, and then gradually decreased with very few branches
reaching beyond 150 µm. This pattern contrasts with the one
observed in areas MT, LIPv and 7a, where the peak number of
intersections occupied a broader region, ∼50 µm wide, centred
around 75 µm from the cell body. As shown in Figure 10, the
complexity of the dendritic tree in these areas, evaluated by the
number of intersections at equivalent distances from the cell
body, was greater than in V1 and V2. This conclusion is true even
if a comparison is restricted to the region where maximal
complexity was found in V1 and V2 (50 µm ring). Finally, while
the higher number of intersections suggested that the dendritic
trees in LIPv were more complex than those in either MT or area
7a (Fig. 10), this result was not statistically significant (Table 5).
Cerebral Cortex Jul/Aug 1997, V 7 N 5 441
Table 3
Basal dendritic field areas of pyramidal neurons in deep layer III
Visual area
n
Mean
SD
SEM
Minimum
Maximum
(× 103 µm2) (× 103 µm2) (× 103 µm2) (× 103 µm2) (× 103 µm2)
V1
V2
MT
LIPv
7a
25
45
34
40
39
35.8
44.8
83.8
88.1
84.7
5.5
10.4
10.6
16.0
23.3
1.10
1.55
1.82
2.53
3.74
27.3
17.8
56.1
60.0
37.2
48.5
65.8
104.2
149.6
145.5
Table 4
One-way ANOVA: dendritic field area of neurons in different visual areas
Source
Sum of squares
10
Between
8.55 × 10
areas
Within areas 3.99 × 1010
Total
1.25 × 1011
DF
4
178
182
Mean squares
10
2.14 × 10
F-ratio
Probability
95.34
<0.0001
8
2.24 × 10
One-tailed between-groups comparison: Dunnett’s t procedure
V2
MT
LIPv
7a
V1
V2
MT
LIPv
2.42*
12.18**
13.70**
12.75**
11.46**
13.29**
12.17**
1.22
0.25
1.00
*P < 0.05; **P < 0.01.
Figure 9. Frequency histograms of basal dendritic field areas of layer III pyramidal
neurons in V1, V2, MT, LIPv and area 7a in the macaque monkey. Basal dendritic fields
of layer III pyramidal neurons in V1 were smaller than those of V2, which were smaller
than those of cells in MT. There was no significant difference in basal dendritic field area
of layer III pyramidal neurons between areas MT, LIPv and 7a. Arrows indicate the
means for each area.
442 Supragranular Pyramidal Cells in Visual Cortex • Elston et al.
Dendritic Field Asymmetries
We also investigated whether basal dendrites of pyramidal cells
were symmetrically arranged around the cell body or demonstrated bias towards particular axes. The vast majority of the
dendritic trees were circular or gently elliptical, with the cell
body occupying the approximate centre. Few cells had somata
that were obviously eccentric relative to the tangential extent of
the dendritic trees (e.g. in area 7a; see the bottom row of cells in
Fig. 8). However, a closer inspection of the data also suggested
that, in some cases, dendritic branches tended to cluster in some
directions relative to the cell body, even though this did not
result in an overall elongation of the dendritic field. To evaluate
this possibility, we subdivided the counts of intersections used
for the Sholl analysis in arbitrary 30° polar angle sectors centred
on the cell body, and generated polar plots (Fig. 11) that indicate
the number of intersections in different directions (including
cumulative data for distances ranging from 25 to 150 µm from
the cell body). The result of this analysis (Fig. 12) demonstrates
that dendritic biases existed, and that the nature of the bias
varied in the different areas.
In order to compare the dendritic biases in different visual
areas we selected 20 neurons from each visual area, representing
regular increments along the distributions of basal dendritic field
area. We classified the polar plots of dendritic branches versus
direction from the cell body in four categories. By analogy with
the well-known polar plots of direction selectivity of neuronal
responses (e.g. Maunsell and Van Essen, 1983b; Albright, 1984),
dendritic fields of cells were classified as ‘morphologically
orientation-biased’ if dendritic branches tended to cluster in two
diametrically opposed directions away from the cell body. To fit
this category, the counts of number of intersections in the
preferred directions had to be similar (within 50%), and their
sum had to be at least twice as large as that of the orthogonal directions (Fig. 11A). ‘Morphologically direction-biased’
Table 5
Two-way ANOVA, repeated measures (n = 20), on Sholl analysis (5 × 6 design) area (V1, V2,
MT, LIPv, 7a) by distance from cell body (25, 50, 75, 100, 125, 150 µm)
Source
Sum of squares
Mean squares
F-ratio
Probability (P)
Area
Distance
Area ×
distance
Within cells
13052.82
16605.07
3738.14
DF
4
5
20
3263.20
3321.01
186.91
158.61
161.42
9.08
<0.01
<0.01
<0.05
11726.81
570
20.57
Post-hoc comparison between pairs of areas (Newman–Keuls)
V2
MT
LIPv
7a
V1
V2
MT
LIPv
n.s.
P < 0.05
P < 0.05
P < 0.05
P < 0.05
P < 0.05
P < 0.05
n.s.
n.s.
n.s.
degrees of clustering (Fig. 11B, C). Some neurons had dendrites
that radiated more or less homogeneously in all directions from
the cell body, forming a symmetrical polar plot. These cells were
termed ‘nonbiased’ (Fig. 11E). Finally, a few cells could not be
categorized according to the above criteria. For example, some
neurons had two clusters of dendrites that were not
diametrically opposed and could therefore not be classified as
‘morphologically orientation-biased’, and some cells had three
clusters of dendrites radiating from the cell body (Fig. 11D).
These cells were classified as ‘morphologically complex’.
Analysis of the data revealed significant differences between
cortical areas in the relative proportions of cells that fitted each
category (Table 6). In V1 layer IIIc, but in no other area, a
relatively large proportion of cells (45%) had morphologically
orientation-biased dendritic fields (Fig. 12A). Another 40% of the
V1 sample showed morphological direction-bias, mostly in a
narrow range of directions (Fig. 12B). Only 15% of the V1 sample
showed no directional clustering of dendrites. In sharp contrast,
within the thick stripes of V2 a majority of cells (75%) had
morphologically direction-biased dendritic fields (Fig. 12C), with
the remaining cells showing either a mild morphological
orientation-bias or no directional preference (Fig. 12D). No
consistent relationship was detected between the axes of
dendritic clustering in different cells and the visuotopic
representations in V1 or V2. Morphologically orientation-biased
cells were rare in the higher-order areas of the occipitoparietal
pathway: only 10–20% of the cells sampled in MT, LIPv and area
7a showed any evidence of morphologically orientation-biased
dendritic fields. In these three areas, the majority of neurons had
either slightly morphologically direction-biased or nonbiased
dendritic fields (Fig. 12E–H).
Figure 10. Sholl analysis of dendritic trees of layer III pyramidal neurons in five visual
areas of the visual occipitoparietal pathway.
dendritic fields were those in which the dendritic branches
clustered in a particular direction, resulting in an intersection
count that was at least twice as large as that in the diametrically
opposed direction. This category included cells with different
Spine Densities Along Basal Dendrites
Our analysis of dendritic spine density demonstrated a similar
trend in all areas studied. Generally (Fig. 13), spine density was
low within 40 µm of the cell body, reached a peak in the region
40–130 µm away from the cell body (according to the cortical
area) and gradually decreased towards the distal tips. This
variability meant that calculations of average spine density were
not particularly meaningful, and we therefore decided to do
statistical comparisons of the peak spine densities between
areas. In order to do this we ascertained, from the graphs
showing mean spine density as a function of distance from the
cell body (Fig. 13), which combination of five consecutive 10 µm
Cerebral Cortex Jul/Aug 1997, V 7 N 5 443
20
10
15
15
20
10
15
Figure 11. Classification of dendritic tree biases. Five examples of cells from areas MT,
LIPv and 7a are illustrated on the left. The corresponding polar plots of dendritic tree
intersections (from Sholl analysis) as a function of direction from the cell body are
illustrated on the right. (A) Morphologically orientation-biased dendritic field; (B)
narrowly morphologically direction-biased dendritic field; (C) broadly morphologically
direction-biased dendritic fields; (D) morphologically complex dendritic field, with
dendrites clustering in three non-diametrically opposed directions; (E) nonbiased
dendritic field. The radial dimension in the polar plots was computed by adding the
number of intersections with imaginary circles 25, 50, 75, 100, 125 and 150 µm from
the cell body, in 30° polar angle intervals. Each division along the axes equals 5
intersections. The directions relative to the cell body are arbitrary. The basis of the
classification is explained in the main text. The polygons around the cells (fine dashed
line) illustrate the criterion for delimitation of basal dendritic field areas (e.g. Young and
Vaney, 1991). Scale bar = 100 µm.
444 Supragranular Pyramidal Cells in Visual Cortex • Elston et al.
segments of dendritic shaft yielded the highest spine count
(arrowheads in Fig. 13), and calculated ‘peak spine density’
as the average of all counts within this 50 µm segment. We
found, by means of a one-way analysis of variance and post-hoc
analysis (Table 7), that the peak density of basal dendritic spines
of neurons in V1 (mean ± SD: 5.79 ± 2.59 spines per 10 µm
dendritic segment) was significantly lower than that in V2 (7.42
± 2.09 spines/10 µm), that the peak spine density in V2 was not
different from that in MT (7.95 ± 2.04 spines/10 µm) and LIPv
(7.68 ± 2.06 spines/10 µm), and that the peak spine density in
area 7a (9.83 ± 2.46 spines/10 µm) was greater than that in all
other areas.
The total number of spines in the basal dendritic field was
calculated for the ‘average’ neuron in each visual area. In order
to calculate this, the average number of dendritic branches at
different distances from the cell body (in 25 µm increments),
obtained from the Sholl analysis, was multiplied by the average
number of spines along the corresponding 25 µm sector of
dendritic shaft. Using this approximation, it was apparent that
the increase in tangential area and complexity of dendritic fields,
combined with the larger spine densities in rostral areas of the
occipitoparietal pathway (as compared with V1), resulted in a
gradual increase in the number of spines per neuron. While
neurons in V1 had on average 799 spines along their basal
dendritic trees, in V2 they had 1201 spines, in MT 2077 spines,
in LIPv 2316 spines and in area 7a 2572 spines. Thus, although
the peak spine density of area 7a neurons was 1.7 times that of
neurons in V1, there was a 3.2-fold difference between the total
number of spines in the basal dendritic fields of the ‘average’
neuron in these areas.
Somal Areas of Layer III Pyramidal Cells
The peak cross-sectional areas of cell bodies of pyramidal
neurons were compared between areas, using the tangentially
sectioned tissue. One hundred and seventy-seven of the 183
neurons were used for this analysis. Six neurons were excluded
due to leakage of dye around the soma. A nonparametric test
(Kruskal–Wallis) across the entire sample revealed significant
differences between areas, as summarized in Table 8. Mann–
Whitney U-tests demonstrated significant differences for all
comparisons between pairs of areas, with the exception of the
comparison between MT and area 7a (Fig. 14; Tables 8 and 9).
There was no apparent correspondence between cell body size
and position in the proposed anatomical hierarchy: somal areas
of pyramidal neurons at the base of layer III in V1 were larger
than those in the corresponding layer of V2, although there was
a large overlap between populations, and areas of neurons in
LIPv were larger than those of neurons in area 7a. When somal
cross-sectional areas were compared with dendritic field areas of
individual neurons, a significant relationship between these two
variables was revealed (Fig. 15).
Discussion
The present study was designed to compare the morphology of
a specific neuronal type (pyramidal cell) in the homologous
layer (basal layer III) of areas that are known to be functionally
interrelated in the occipitoparietal pathway. Pyramidal cells in
layer III are the principal originators of ‘feedforward’ corticocortical projections, as well as participating in interhemispheric
connections and, to a lesser extent, ‘feedback’ projections to
other areas (for a review see Felleman and Van Essen, 1991).
Basal dendritic fields of layer III pyramidal neurons in extrastriate areas MT, LIPv and 7a were compared with those in
Figure 12. Examples of polar plots showing dendritic bias in different visual areas. The illustrated directions are arbitrary. (A) Five morphologically orientation-biased V1 dendritic
fields; (B) five morphologically direction-biased V1 dendritic fields; (C) five morphologically direction-biased V2 dendritic fields; (D) five non-biased or mildly orientation-biased V2
dendritic fields; (E) four broadly morphologically direction-biased MT dendritic fields; (F) four non-biased MT dendritic fields; (G) four broadly direction-biased LIPv dendritic fields; (H)
three non-biased and one morphologically complex LIPv dendritic fields. See also legend to Figure 11 for details.
Cerebral Cortex Jul/Aug 1997, V 7 N 5 445
Table 6
Percentages of neurons with different categories of dendritic biases in visual areas*
Visual area
V1
V2
MT
LIPv
7a
Type of dendritic tree
Morphologically Morphologically
orientation-biased direction-biased
Morphologically
complex
Nonbiased
45
5
15
10
10
–
–
–
15
15
15
20
50
35
20
40
75
35
40
55
*n = 20 for each area.
regions at the origin of the occipitotemporal pathway, including
V1 and the thick cytochrome oxidase-rich stripes of V2. Whereas
there is general agreement that MT occupies a position
intermediate in the proposed anatomical hierarchy, between V2
and posterior parietal areas LIPv and 7a, there is still controversy
regarding the relative level of the latter two areas: some authors
regard LIPv and area 7a as occupying the same hierarchical level
(Boussaoud et al., 1990), whereas others suggest that area 7a is at
a higher level than LIPv (Andersen et al., 1990; Felleman and Van
Essen, 1991). Moreover, while V1, V2, MT and, to a lesser extent,
LIPv are well-defined areas, it is unclear whether the subdivision
of area 7a within the superior temporal sulcus is part of the same
functional area as the subdivision occupying the caudal inferior
parietal lobule, which has been most intensively studied in
terms of connections (Andersen et al., 1990; Boussaoud et al.,
1990). These caveats should be kept in mind when, in the
following sections, we try to establish whether or not there are
correlations between dendritic morphology and the proposed
anatomical hierarchy.
Neuronal Types Forming the V1 Projection to MT
Various cell types, typically in layer III, have been demonstrated
to form ‘feedforward’ projections between visual areas. These
are typically classified within two main groups: pyramidal
neurons and spiny multipolar (stellate) neurons (Tigges et al.,
1981; Diamond et al., 1985; Shipp and Zeki, 1989a; Vogt
Weisenhorn et al., 1995). We were particularly concerned with
the cells forming the projection from V1 to MT, which, as
indicated by our data, form a mixed population with a
predominance of pyramidal neurons. Previous reports differ in
their conclusions on this subject. This discrepancy may, in part,
be attributed to species differences. For example, in the
marmoset monkey all MT-projecting neurons in V1 are reported
to be pyramidal (Vogt Weisenhorn et al., 1995), while in the
squirrel monkey and the macaque multipolar neurons have been
reported to form a substantial part of this projection (Tigges et
al., 1981; Shipp and Zeki, 1989a). The relative proportions of
pyramidal and multipolar neurons in different species do not
bear any obvious relationship with either habits or phylogeny: in
both the macaque (a diurnal Old World monkey) and the squirrel
monkey (a diurnal New World monkey) many stellate cells are
reported to project to MT, but the same is not true in the
marmoset, another diurnal New World monkey. This variability
suggests a developmental process regulating dendritic
morphology, rather than a clear-cut separation between two
categories. In fact, we obser ved many modified pyramidal
neurons among the population of V1 cells that project to MT,
some of which resembled multipolar cells in that their basal
dendrites issued in all directions from the soma. Other authors
have proposed that spiny multipolar cells represent an extreme
446 Supragranular Pyramidal Cells in Visual Cortex • Elston et al.
Figure 13. Spine density as a function of distance from the cell body along basal
dendrites of pyramidal neurons in five visual areas. The illustrated means and standard
deviations are based on examination of 20 randomly selected basal dendrites from
neurons in each area. Spine density was calculated per 10 µm increment of dendrite,
and the arrowheads define the five consecutive segments that yielded the highest
cumulative counts.
Table 7
One-way ANOVA: peak spine density in different visual areas
Source
Sum of squares
DF
Mean squares
F-ratio
Probability
Between areas
Within areas
Total
755.69
2407.61
3163.30
4
475
479
188.92
5.07
37.27
P < 0.0001
One-tailed between-groups comparison: Dunnett’s t procedure
V2
MT
LIPv
7a
V1
V2
MT
LIPv
5.11**
6.85**
5.80**
12.09**
1.69
0.79
7.24**
0.85
5.71**
6.36**
** P < 0.01.
Table 8
Statistical comparison between cell body cross-sectional area
Kruskal–Wallis test (DF = 4, n = 177, H = 114.32, P < 0.0001)
Visual area
Mean rank
V1
V2
MT
LIPv
7a
60.25
30
100.77
139.59
113.31
Mann–Whitney U-tests
Comparison
U′
z
V1 versus V2
V1 versus MT
V1 versus LIPv
V1 versus 7a
V2 versus MT
V2 versus LIPv
V2 versus 7a
MT versus LIPv
MT versus 7a
LIPv versus 7a
913
648
915
843
1268
1726
1640
1019
732
1126
–4.94*
–4.68*
–6.03*
–5.31*
–6.50*
–7.58*
–7.14*
–4.63*
–1.51
–3.94*
* P < 0.001.
case of modified pyramidal neurons, involving the loss or
reduction of the apical dendrite (Lund, 1984; Nieuwenhuys,
1994; Vogt-Weisenhorn et al., 1995).
Methodological factors may also complicate the interpretation
of the results of these different studies. For example, both Vogt
Weisenhorn and colleagues (1995) and the present study, in
which cellular morphologies were established by intracellular
injection of retrogradely labelled neurons, concluded that the
bulk of the projection from V1 to MT is formed by pyramidal
neurons, whereas studies in which neuronal classification was
based on retrograde labelling alone concluded that spiny
multipolar neurons predominate (Tigges et al., 1981; Shipp and
Zeki, 1989a). In addition, it is possible that the relative
proportion of pyramidal and spiny multipolar neurons varies in
different portions of V1 (Shipp and Zeki, 1989a). Note, for
example, that Vogt-Weisenhorn and collaborators (1995), who
found that only pyramidal cells give rise to projections to MT,
dealt primarily with injections in the peripheral representation,
whereas Tigges and colleagues (1981), who found mainly
Figure 14. Frequency histograms of somal cross-sectional area of layer III pyramidal
neurons in five visual areas. Somal areas of neurons in V1 were larger than those in V2
but smaller than those of neurons in MT, LIPv and area 7a. Somal areas of neurons in MT
and area 7a were similar, but significantly smaller than those of neurons in LIPv. Arrows
indicate means.
Cerebral Cortex Jul/Aug 1997, V 7 N 5 447
Table 9
Somal cross-sectional areas of layer III pyramidal neurons in different visual areas
Visual area
n
Mean (µm2) SD
SEM
Minimum
(µm2)
Maximum
(µm2)
V1
V2
MT
LIPv
7a
24
44
31
39
39
170
126
218
283
233
6.9
6.0
6.0
12.0
6.8
129
71
160
172
167
279
259
297
586
334
34
39
33
76
42
multipolar neurons, studied the connections of the central field
representation.
In spite of the variable morphology, the laminar location (just
above the granular layer) of the MT-projecting neurons in V1 is
remarkably constant across primates, including prosimians
(Diamond et al., 1985). This constancy suggests that the unifying
feature across species is the ability to sample extensively, both
directly and indirectly, from magnocellular inputs which
terminate primarily in layer IVa (Fitzpatrick et al., 1985;
Florence and Casagrande, 1987; Freund et al., 1989; Lachica et
al., 1993). As recently demonstrated, pyramidal neurons in
sublamina IIIc are inf luenced by parvocellular inputs onto their
apical dendrites (Sawatari and Callaway, 1996); thus, the
variability in the proportion of pyramidal cells may have
functional consequences related to the amount of parvocellular
input that reaches MT.
In all retrogradely labelled pyramidal neurons in V1 in which
we were able to visualize part of the axonal tree, there were
horizontal radiations that projected laterally from the main
axonal segment. At present it is unclear whether the horizontal
axonal radiations of MT-projecting pyramidal neurons also
contribute to clustered intrinsic connections in V1 (Rockland
and Lund, 1983; Livingstone and Hubel, 1984; Amir et al., 1993;
Lund et al., 1993). Intracellular injection studies in cat visual
cortex (area 18) have revealed that intrinsically projecting
pyramidal neurons have significantly smaller basal dendritic field
widths than those that form extrinsic projections (Matsubara et
al., 1996). Further experiments need to be performed to
establish if this is also the case in the primate.
Variation of Basal Dendritic Fields Between Areas, and
Possible Functional Correlates
Previous studies have demonstrated that anatomical parameters
of intrinsic connections, such as the cross-sectional area and
centre-to-centre spacing of intrinsic patches and the lateral
extent of connectivity, increase from caudal to rostral visual
areas (Yoshioka et al., 1992; Amir et al., 1993; Lund et al., 1993).
Furthermore, Lund and colleagues (1993) have suggested that
changes in cross-sectional area of intrinsic axonal patches are
paralleled by changes in basal dendritic territories of
supragranular pyramidal neurons. The present data confirm that,
in some cases, basal dendritic fields of layer III pyramidal
neurons increase with rostral progression along the proposed
anatomical hierarchy of visual areas. However, they also
demonstrate that a change of level in the hierarchy is not
necessarily paralleled by changes in basal dendritic field area.
Accepting that the size of basal dendritic fields of layer III
pyramidal neurons is correlated with characteristics of intrinsic
connectivity (Lund et al., 1993; Malach, 1994), we would expect
to see a similar pattern of intrinsic axonal clusters in MT, LIPv
and area 7a. It will certainly be worthwhile comparing the
448 Supragranular Pyramidal Cells in Visual Cortex • Elston et al.
Figure 15. Dendritic field area as a function of somal cross-sectional area for
pyramidal neurons in basal layer III in V1, V2, MT, LIPv and area 7a. Regression analysis
revealed a significant correlation between these variables (r2 = 0.59).
intrinsic connectivity of these areas in order to establish whether
the proportional relationship between dendritic fields and
intrinsic patches is maintained.
Due to their morphology and location within the cortical
column, layer III pyramidal neurons in extrastriate areas have the
potential to sample from different types of inputs. As well as the
intrinsic axonal clusters, pyramidal neurons at the base of layer
III are in a position to sample ‘feedforward input’ (Rockland and
Pandya, 1979; Felleman and Van Essen, 1991), feedback
projections (some of which terminate in layer III, e.g. Boussaoud
et al., 1990) and callosal projections (Cusick et al., 1984;
Kennedy et al., 1986; Beck and Kaas, 1994). At present, little is
known about the relative size and possible overlap of the
multiple sources of inputs to cells in the rostral visual areas. In
terms of connectional diversity (see Malach, 1994) it is feasible
that neurons in layer III of rostral visual areas sample intrinsic
and extrinsic input in different proportions. If we assume that
the patchy nature of intrinsic and extrinsic connections is
common to all visual areas (e.g. Rockland and Pandya, 1979;
Montero, 1980; Van Essen et al., 1981, 1986; Pandya and Seltzer,
1982; Livingstone and Hubel, 1984; Caminiti and Sbriccoli,
1985; Seltzer and Pandya, 1989; Krubitzer and Kaas, 1990;
Yoshioka et al., 1992; Amir et al., 1993; Lund et al., 1993; Shipp
and Zeki, 1995; Fujita and Fujita, 1996; Leichnetz and
Gonzalo-Ruiz, 1996; Seltzer et al., 1996), extrinsic inputs may
form terminal patches that are smaller than, equal to or larger
than the intrinsic axonal clusters in a given visual area. Thus,
simply by varying the geometry of axonal patches and dendritic
fields, neurons in different areas may be able to attribute
different weights to different inputs and perform different
functions.
Aside from a correlation with intrinsic axonal patch size, it is
possible that the observed increase in basal dendritic field areas
of layer III pyramidal neurons between V1, V2 and MT, and the
relative constancy beyond MT, are consequences of the fact that
the present analysis was based on comparison of randomly
sampled neurons in different areas, instead of specifically
focusing on cells with similar projection patterns (intrinsic,
extrinsic or interhemispheric). Current evidence suggests that
high-order visual areas are generally connected to more cortical
areas than are lower-order visual areas. For example, while layer
III cells in V1 only form three main ‘feedforward’ cortical
projections (Zeki, 1978; Weller and Kaas, 1983; Van Essen et
al., 1986; Krubitzer and Kaas, 1993), with minor or variable
pathways to a few other targets (Felleman and Van Essen, 1991;
Rockland and Van Hoesen, 1994), cells in layer III of areas such
as MT and area 7a may project to 10–20 other areas (Ungerleider
and Desimone, 1986; Andersen et al., 1990; Felleman and
Van Essen, 1991). Therefore it is possible that the proportion of
neurons in layer III that form extrinsic projections is greater
in higher-order areas than in V1 or V2. Given that interareal
projection neurons tend to have larger dendritic trees than
intrinsic projection neurons, as reported in cats (Matsubara et
al., 1996), higher-order areas may have a greater percentage of
large layer III pyramidal neurons. However, this explanation does
not seem entirely satisafctory, as in areas MT and LIP the smallest
neurons are bigger than the largest neurons in V1. Secondly,
callosally projecting pyramidal neurons in layer III, which are on
average larger than intrahemispheric projecting pyramidal
neurons (Matsubara et al., 1996), are widespread in rostral visual
areas such as MT, MST, LIP and area 7a. In contrast, in V1 and, to
a lesser extent, V2 these cells are concentrated in the vicinity
of the representation of the vertical meridian and fovea (Van
Essen and Zeki, 1978; Newsome and Allman, 1980; Van Essen et
al., 1982; Cusick et al., 1984; Kennedy et al., 1986; Olavarria
and Abel, 1996). Thus, the greater number of inter- and intrahemispheric connections in rostral visual areas may, in part, be
responsible for the overall trend towards large basal dendritic
territories.
(e.g. Leventhal and Schall, 1983), recent studies in cortex have
emphasized the role of biased intrinsic axonal projections (e.g.
Lund et al., 1995; Sincich and Blasdel, 1995; Fitzpatrick, 1996).
We find it striking that a large proportion of cells with narrow
morphologically orientation- and direction-biased dendritic
fields were found in V1, where this property is first generated in
the monkey cortex, but not in the other areas. These
obser vations suggest the possibility that, in addition to the
excitatory and inhibitory effects of intrinsic lateral connections
(Vidyasagar et al., 1996), the orientation selectivity of layer III
cells may be sharpened by sampling inputs from underlying layer
IV along given axes of the visuotopic map. For dendritic field
biases to be of any value in contributing to orientation
selectivity, it is necessary that the given cortical area has a
well-defined visuotopic map. This is certainly the case in V1,
where cells in layer IV which provide inputs to layer IIIc form a
fine-grained map of the visual field (Blasdel and Fitzpatrick,
1984), but not in the other areas studied (Rosa, 1997). In
extrastriate areas the observed dendritic field biases may be
more related to functional compartmentalization (cf. Katz et al.,
1989; Hübener and Bolz, 1992; Kossel et al., 1995). For example,
the cortical projections to these areas often terminate in a
patchy pattern (Livingstone and Hubel, 1984, 1987; Ungerleider
and Desimone, 1986; Cavada and Goldman-Rakic, 1989;
Andersen et al., 1990; Boussaoud et al., 1990; Leichnetz and
Gonzalo-Ruiz, 1996). Even though a cell body in these areas may
be located relative to patches of axonal projection in a position
to sample from different sources of input, its directional
dendritic field could bias its responses in such a way as to ref lect
mainly one of the inputs.
Dendritic Field Complexity and Directional Biases
Our results suggest that neurons in layer III of V1 and V2 differ
from those in higher-order areas not only in that they have
smaller basal dendritic field areas but also in the relative
complexity of these fields. Even in the region of cortex 50–75 µm
from the cell body, well within the lateral extent of the dendritic
fields of V1 and V2 cells, there are relatively more dendritic
branches per unit area in MT, LIPv and area 7a than in V1 and V2.
The fact that maximal complexity was observed among neurons
in LIPv, rather than in area 7a, suggests that there is no simple
correlation between dendritic field branching complexity and
position in the proposed hierarchy (although, as mentioned
above, the level of the subdivision of area 7a that we studied is
still uncertain). The fact that neurons in LIPv had, on average, a
larger cell body than that of neurons in MT and area 7a (which
were similar) may be related to the greater complexity of the
dendritic field in the former area. The overall trend towards
pyramidal neurons with more complex basal dendritic
projections in areas MT, LIPv and area 7a may partially ref lect the
relatively high proportion of neurons forming contralateral
projections in these areas. For example, in area 18 of the cat,
neurons that form callosal projections have more complex
dendritic fields than those that form intrinsic or ipsilateral
interareal projections (Matsubara et al., 1996).
Our results suggest that there are significant orientation biases
in dendritic fields of most neurons in layer IIIc of V1, that most
neurons within the thick bands in V2 display some degree of
dendritic field directional bias, and that these biases are less
marked in neurons in higher-order areas (MT, LIPv and area 7a).
While some models of generation of orientation selectivity in the
nervous system suggest that dendritic field asymmetries may in
part be responsible for the generation of this response property
Spine Densities
Our results indicate the existence of a significant variability in
spine density along the basal dendrites of layer III pyramidal cells
in all visual areas tested. These data are in agreement with
previously published studies in the visual cortex of the rat
(Larkman, 1991). Spine density of the basal dendrites of layer III
pyramidal cells also differed markedly across areas (Fig. 13). We
have shown significant differences in peak spine density
between different visual areas of single animals, and between
areas in animals of similar age. For example, neurons sampled
from V1 and area 7a in a single 20-month-old animal (RM 13)
were significantly different. Furthermore, the peak spine
densities of V1 neurons in this animal were significantly
different from those in areas MT and LIPv in another
20-month-old monkey (RM 14). Thus, any age-related causality
for the different spine densities (Lund et al., 1977; Boothe et al.,
1979; Courns et al., 1996) is unlikely. By combining the data
from the Sholl and spine-density analyses we were able to
calculate the average total number of basal dendritic spines for
layer III pyramidal neurons in different visual areas. In spite of
the approximate nature of the obtained figures, they serve as a
relative measure for comparisons between areas, demonstrating
a dramatic increase along the occipitoparietal pathway.
It has been established that symmetrical synapses, which are
thought to be excitatory, form the vast majority of contacts to
dendritic spines (for review see Shepherd, 1996). We believe
that the peak spine density, the spine distribution and the total
number of spines, which are characteristic of different areas,
may have functional significance. In the simplest case, one might
consider two pyramidal cells with similar dendritic fields but
different spine densities. In this case, it is reasonable to expect
that the firing rate of the cell with greater spine density may be
Cerebral Cortex Jul/Aug 1997, V 7 N 5 449
inf luenced by a greater diversity of excitatory input, particularly
in view of the fact that only a small number of EPSPs may be
needed to reach firing threshold in pyramidal neurons (Deuchars
et al., 1994). This could allow the cell to sample from
superimposed sources of differing origin; for example,
juxtaposed feedforward input from multiple ‘low’ order areas, as
well as intrinsic, feedback, thalamic and callosal projections.
Furthermore, dendritic segments with greater spine densities
may achieve greater summation of excitatory postsynaptic
potentials, more easily depolarizing the target neuron even if
only a subset of the dendritic tree is stimulated. However, the
exact physiological effect of the varying spine density along
dendrites is difficult to estimate, given a number of uncertainties
regarding active versus passive centripetal propagation of
electric activity in neocortical dendrites (for review see Johnston
et al., 1996).
Conclusions
We have tested the hypothesis that there is a stepwise change in
basal dendritic field morphology of supragranular pyramidal
neurons from ‘lower’ to ‘higher’ stations of the proposed
anatomical hierarchy which processes motion and spatial
relationships. Our results yield some support to this view, in that
the expected serial increase in basal dendritic field area is
observed between areas V1, V2 and MT. However, the
relationship breaks down at ‘higher’ stations such as LIPv and
area 7a, as the basal dendritic field area of neurons in these areas
is similar to that observed in MT. We have also observed other
cytological correlates for the proposed hierarchy in the form of
increases in the complexity of basal dendritic branching pattern
and the density and total number of dendritic spines. These
features may enable cells in the rostral areas to sample a greater
diversity of inputs. A precise interpretation of the functional
significance of these results will depend on a better
understanding of the geometry of the pattern of axonal
terminations from multiple origins onto single pyramidal cells.
Acknowledgments
We thank Dr David Vaney for allowing free access to his laboratory and,
in particular, his intracellular injection apparatus. We are also greatly
indebted to Dr David Pow for allowing us access to his superb antibody to
Lucifer Yellow. Finally, we would like to thank Rowan Tweedale for
assistance with the Sholl analysis, and Rowan Tweedale, Dr Mike Calford
and Prof. Jack Pettigrew for comments on the manuscript. Supported by
Project Grants from the Australian National Health and Medical Research
Council.
Address correspondence to Guy Elston, Vision, Touch and Hearing
Research Centre, Department of Physiology and Pharmacology, The
University of Queensland, Queensland 4072, Australia.
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