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The Journal of Neuroscience, December 15, 1998, 18(24):10525-10540
The Connection from Cortical Area V1 to V5: A Light and Electron
Microscopic Study
John C.
Anderson1,
Tom
Binzegger1,
Kevan A. C.
Martin1, and
K. S.
Rockland2
1 Institute for Neuroinformatics, 8057 Zürich, Switzerland, and 2 University of Iowa
Hospitals and Clinics, Iowa City, Iowa 52242
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ABSTRACT |
Area V5 (middle temporal) in the superior temporal sulcus of
macaque receives a direct projection from the primary visual cortex
(V1). By injecting anterograde tracers (biotinylated dextran and
Phaseolus vulgaris lectin) into V1, we have examined the
synaptic boutons that they form in V5 in the electron microscope.
Nearly 80% of the target cells in V5 were spiny (excitatory). The
boutons formed asymmetric (Gray's type 1) synapses with spines (54%), dendrites (33%), and somata (13%). All somatic targets and some (26%) of the target dendritic shafts showed features characteristic of
smooth (inhibitory) cells. Each bouton formed, on average, 1.7 synapses. The larger boutons formed multiple synapses with the same
neuron and completely enveloped the entire spine head. On most
dendritic shafts and all somata the postsynaptic density en
face was disk-shaped but in about half the cases the
reconstructed postsynaptic densities of synapses on spines appeared as
complete or partial annuli. Even in the zones of densest innervation
only 3% of the asymmetric synapses were formed by the labeled boutons. Although the V1 projection forms only a small minority of synapses in
V5, its affect could be considerably amplified by local circuits in V5,
in a way analogous to the amplification of the small thalamic input to
area V1.
Key words:
visual cortex; area MT; corticocortical; synapse
morphology; postsynaptic target; 3-D reconstruction
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INTRODUCTION |
The best-studied extrastriate area
is that first discovered by Zeki (1969) , who used anatomical methods to
define an area in the posterior bank of the superior temporal sulcus in
macaque monkey that received an input from area 17. The homologous area in the new-world monkey is the middle temporal area (MT) (Allman and
Kaas, 1971). From the earliest recordings, it was evident that the
neurons of this area, now called MT or V5, were particularly sensitive
to the direction and velocity of motion of the stimulus (Dubner and
Zeki, 1971).
There are both direct and indirect projections from V1 to area V5
(Zeki, 1969; Ungerleider and Mishkin, 1979; Maunsell and Van Essen,
1983; Fries et al., 1985). The neurons that project directly from V1
have been identified as spiny stellates and pyramidal cells in layer 4B
and large pyramidal cells in upper layer 6 (Lund et al., 1975; Shipp
and Zeki, 1989). Their afferent axons form large boutons in a patchy
distribution in layers 3, 4, and 6 of V5 (Rockland, 1989, 1995). The
receptive fields of the V1 neurons that project to V5 have also been
studied. The projecting neurons were identified by antidromically
activating them from V5 (Movshon and Newsome, 1996). They had
fast-conducting axons and all were binocular, complex cells, with
high-contrast sensitivity and contrast-independent direction
preferences. They responded at least as well to short stimuli as to
long stimuli. Such complex cells are referred to as special (Palmer and
Rosenquist, 1974) and have the largest receptive fields, the highest
velocity preference, and the highest spontaneity of striate
cortical cells (Gilbert, 1977). This degree of uniformity in
physiological properties seems to indicate that MT uses this output
from V1 for further stages of specific processing. Movshon and Newsome
(1996) suggested that these V1 neurons form the first stage of motion
processing, in which the motion of the individual components of a
pattern is extracted (Adelson and Movshon, 1982; Movshon and Newsome,
1984). The second stage occurs outside V1, in areas such as V2 and V5,
where the motion of the entire pattern is computed.
If the V1 output to V5 is blocked, by making lesions or cooling V1 or
blocking the magnocellular pathway, some activity persists in V5, but
it is greatly diminished from normal. Although these experiments do not
distinguish between the direct and indirect projection from V1 to V5,
it is likely that the output from V1 is significant for V5.
Anatomically, however, the picture is unclear. As yet there are not
even qualitative estimates of the numbers of synapses involved in the
V1 to V5 projections. In the present study we assessed the synaptic
connections formed in V5 by the V1 projection neurons. Although the
manner of connection of the V1 afferents to neurons in V5 was
morphologically distinct, quantitatively they formed only a few percent
of the synapses within their major termination zones in V5.
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MATERIALS AND METHODS |
The materials examined in this study were obtained from two
adult Macaca mulatta monkeys in the laboratory of K.S.R.
(University of Iowa Hospitals and Clinics). The monkeys were prepared
for surgery with a premedication of ketamine (11 mg/kg). Surgery was performed under deep anesthesia induced by intravenous delivery of
Nembutal (25 mg/kg) and supplemented as required. All procedures were
performed under sterile conditions in accordance with institutional and
federal guidelines as specified in approved Animal Care and Use
Research Forms.
One animal received a single microinjection of 10% biotinylated
dextran amine (BDA) (Molecular Probes, Eugene, OR) in 0.0125 M PBS into cortical area V1. The second animal
received a similar microinjection of BDA and a second injection of
2.5% Phaseolus vulgaris leucoagglutinen (PHA-L) (Vector
Laboratories, Burlingame, CA) in 10 nM phosphate buffer
(PB) (see Fig. 1). Each injection was ~0.5 µl. After an 18 d
survival period the animals were anesthetized and perfused
transcardially with a solution of paraformaldehyde (4%) and
gluteraldehyde (0.25%) in saline, followed by sucrose solutions of 5, 10, and 15% in 0.1 M PB. After the brain was removed from
the skull, a block of tissue (~15 mm in dorsoventral extent) was
trimmed through the superior temporal sulcus (STS) intended to include
cortical area V5 or MT. The selected block of cortex was bisected and
vibratome-sectioned in the horizontal plane at 50 µm. Sections were
collected in 20% sucrose in PB and kept overnight in the
refrigerator. Sections were then immersed in liquid nitrogen for rapid
freezing as a way of promoting penetration. After thawing, the sections
were processed according to a Vector ABC kit (Elite) protocol to
visualize labeled axons. We used 3, 3diaminobenzidene tetrahydrochloride (DAB) to reveal peroxidase activity. For tissue injected with BDA, this followed 24 hr in ABC solution. For
PHA-L-injected tissue, this followed 2 d in anti-PHA-L (1:2000)
and repeated 70 min steps through secondary antibody and ABC
solutions. Selected sections were treated with a 0.5-1% osmium
tetroxide solution in 0.1 M PB. Dehydration through an
ascending series of alcohols (including 1% uranyl acetate in the 70%
alcohol) and propylene oxide preceded flat mounting in Durcupan (Fluka
ACM, Buchs, Switzerland) onto glass slides.
The labeled axons were reconstructed by light microscopy. Regions of
special interest were photographed and re-embedded for correlated
electron microscopy (EM). Serial ultrathin sections were cut at 70 nm
thickness and collected on Pioloform-coated single-slot copper grids.
Labeled profiles were photographed at 21,000× magnification. The
classification of synapses as symmetric or asymmetric was made on the
basis of conventional criteria (Gray, 1959; Colonnier, 1968). The
presence of reaction product in the presynaptic bouton obviously
compromises the visibility of the presynaptic membrane. However, the
unlabeled boutons in the adjacent neuropil indicated marked differences
in the thickness of the postsynaptic density of symmetric and
asymmetric synapses (see Fig. 4). This allowed us to make definitive
classifications of the synapses formed by labeled boutons. Serial
electron micrographs of labeled synaptic boutons were digitized and
reconstructed using an in-house computer system (Trakem). From the
digitized and reconstructed data, we measured structures such as
postsynaptic specializations and bouton area. Trakem generates a series
of wire frame profiles, which gives a three-dimension (3-D) impression.
The "object" can be rotated to offer different views of synapses.
We enhanced this effect by fitting a skin to the digitized structure
and then rendering the surface. The skin fitting used Nuages (B. Geiger, 1996), and the rendering used Geomview and Blue Moon
Rendering Tools (L. Gritz).
To reconstruct and measure the area of postsynaptic densities of
labeled boutons in 3-D, we developed our own computer software. A
semiautomated process first grouped the serially sectioned components of synapses. The postsynaptic surface was represented as a 3-D grid
that was created by interpolating points between the components. The
area was measured by summing the areas of the triangles that appeared
between the interpolated points. To represent the reconstructed synapses in two dimensions, we selected an appropriate plane on which
to project each synapse.
We used the physical disector method of Sterio (1984) to obtain a
stereological estimate of the percentage of labeled synapses in area
MT. Serial ultrathin (70 nm) sections were cut and collected as above.
Reference and look-up sections were separated by one intervening
section. Each section was photographed in the EM at 11,500×. Only
those synapses that appeared in the reference section but not in the
look-up section were counted here.
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RESULTS |
Light microscope observations
Pressure injections of biotinylated dextran amine (one animal) or
BDA and PHA-L (one animal) were made into the parafoveal representation
in area 17 (V1) (Fig. 1). After
processing, the injections were found to be 1-2 mm in diameter and
confined to the gray matter. At one site the white matter was damaged
by the injection needle itself. In area V5, labeled axons arborized in layers 3, 4, and 6. The lack of labeled cell bodies in extrastriate cortex was taken as evidence that the transport was anterograde only.
The largest diameter (3 µm) axons showed the label only at the cut
ends and not in the middle of the section. Presumably this is because
the avidin complex could not penetrate the thick myelin sheath
surrounding the labeled axons, despite the freeze-thaw procedure.
These heavily myelinated fibers, originating from V1, arose from the
white matter and branched to produce collaterals of decreasing diameter
as they ascended through the cortical layers. The smaller-diameter
collaterals retained a myelin sheath, which was not so thick as to
prevent penetration of the reagents. The axons appeared unmyelinated in
the light microscope (LM) at the point where boutons formed. This was
confirmed in the electron microscope. Long, uninterrupted
lengths of very fine collaterals also criss-crossed the termination
zones. The boutons formed by these fine collaterals were very small and
infrequent (<1/100 µm). The fine collaterals were not from a
separate population of afferents, but branched from axons that bore
large boutons. All boutons selected for this study were located in
layers 4 and 6 of area V5.
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Figure 1.
Summary schematic of macaque brain showing
position of three injections of neuronal tracers (2 of BDA and 1 of
PHA-L) into primary visual cortex, area V1, of two animals.
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Short strings of large boutons (up to 3 µm diameter) of both en
passant and terminaux morphology were gathered in local
clusters. Some of these boutons formed close appositions with somata.
From the LM views (Fig.
2A; see also Fig.
5A) we chose collaterals that formed clusters of boutons
that were more or less parallel to the face of the section. This
selection somewhat reduces the number of serial ultrathin sections from
unmanageable to very large. Long lengths of collaterals, including the
interbouton segments, were also traced through the serial ultrathin
sections. We examined 86 boutons (32 from layer 6 and 54 from layer 4)
in the electron microscope. Each bouton formed at least one asymmetric (Gray's type 1) synapse.
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Figure 2.
Correlated light and electron micrographs of
BDA/PHA-L-labeled electron dense axon and boutons. A,
Photomontage of an axon collateral located in lower layer 4 of area V5;
b1, b2, b3, and b4 are selected boutons. The associated synapses of
these boutons are shown in the following electron micrographs.
B, Bouton b1 forms an asymmetric synapse (solid
arrowhead) with a spine head (sp1) that can be
traced back to the parent dendrite (d) in a
single section. The dendrite produces a second spine
(sp2), which receives an asymmetric synapse
(small arrow) from an unidentified bouton.
C, A myelinated axon collateral
(m) gives rise to a bouton (b2)
forming an asymmetric synapse (solid arrowhead) with a
spine (sp). D, A large bouton
(b3) packed with vesicles and mitochondria forms an
asymmetric synapse (solid arrowheads) with a spine
(sp). The spine profile has been completely embraced by
the filled bouton. The postsynaptic density does not appear as a
continuous structure but instead is perforated or complex.
E, Another large bouton (b4) forms
asymmetric synapses (solid arrowheads) with a spine
(sp) and the shaft of a dendrite
(d). The spine apparatus is clearly visible. The
dendrite also forms an asymmetric synapse (small arrow)
with an unidentified bouton. Scale bars: A, 10 µm;
B, 1 µm; C-E, 0.5 µm.
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Electron microscopy
Synapses formed with dendritic spines
Figure 2 illustrates a correlated LM and EM sample from
labeled collaterals in layer 4. The reaction end-product was very intense in many of the boutons. Where this reaction end-product was
most intense it produced "chatter" over the labeled regions in the
ultrathin section. The heavy labeling made the details of the
presynaptic structures difficult to see, but the synaptic cleft and the
postsynaptic specialization were unaffected by the labeling. The
synaptic vesicles and mitochondria within the bouton did not contain
any significant deposit of reaction end-product. The labeled boutons
were usually packed with mitochondria and round vesicles (Fig.
2B-E). Some of the boutons and parts of the axon
contained vacuoles (e.g., Fig. 2C-E). These were clearly artifacts of the labeling process, because unlabeled structures in the
adjacent neuropil were intact. Similar vacuoles have been reported with
the use of wheat-germ agglutinin as a tracer (LeVay, 1986). Vacuoles
might contribute to the distinctive size of boutons when viewed in an
LM, although the largest boutons in our sample contained no vacuoles.
The collaterals that gave rise to strings of boutons were frequently
myelinated, but not so strongly that the avidin complex could not
penetrate (Fig. 2A,C). The boutons along the
collaterals formed asymmetric (Gray's type 1) synapses (Figs. 2,
3). The standard ultrastructural
criteria, particularly spine apparatus (Peters et al., 1991) in
conjunction with serial section reconstruction of the bouton and its
target, helped to identify the target. Sometimes the spine neck could
be traced back to the parent dendrite, either over several sections or
occasionally in a single section (Figs. 2B,E,
3A). This was made possible because in some cases the spines had a relatively thick spine neck that was only a little smaller in
cross section than the spine head. The majority of spines, unfortunately, could not be traced to their parent dendrites despite care being taken in collecting serial sections.
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Figure 3.
Electron micrographs of BDA/PHA-L-labeled boutons
found in layer 6 of area V5. A, Bouton filled with
mitochondria forming two asymmetric synapses (solid
arrowheads) with the same target neuron. One of the targets is
clearly a spine (sp; note the spine apparatus) that can
be traced back to the parent dendrite (d). The
second synapse forms on a region of the dendritic shaft that projects
slightly into the neuropil. Serial section reconstruction showed this
projection to be a sessile or "neckless" spine. B, A
spine (sp) containing spine apparatus forms an
asymmetric synapse with the labeled bouton, which shows a complex
postsynaptic density (solid arrowheads) within the
spine. Scale bars, 0.5 µm.
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Some spine heads were actually embedded deep in the bouton itself (Fig.
2D) and in many cases the bouton wrapped around the spine head (Figs. 2B,C,E, 3A,B). Further
detailed reconstructions are given below. The postsynaptic densities
were not single entities but were frequently divided into a number of
separate zones, as indicated in Figures 2D and
3B, arrowheads. The labeled bouton was the only
source of asymmetric synapse to the spine. In three cases the spine
formed a second, symmetric (Gray's type 2) synapse with an unlabeled
bouton. When reconstructed, eight of the boutons that formed a synapse
with a spine also formed synapses with the parent dendrite or a sessile
spine from the parent dendrite. One bouton formed synapses with two
spines and a dendritic shaft; all three targets belonged to the same
neuron (b1 in Fig. 2A,B).
Synapses formed with the shafts of dendrites
Dendritic shafts were identified by their numerous mitochondria
and microtubules, and in some cases by their projecting spines. Figure
4 illustrates the synapses formed with
dendritic shafts. Figure 4A illustrates a bouton
sampled from layer 4 that forms a synapse (large, filled
arrowhead) with a large caliber dendrite (d) and a
spine (sp, large, filled arrowhead). The dendrite forms numerous synapses from unlabeled boutons (small and unfilled
arrowheads). The dendrite contained many large mitochondria,
particularly in the dendritic varicosities or "beads." The beads
(one is evident in the Fig. 4A) were more evident in
serial reconstruction. No spines emerged from the shaft. These features
are all characteristic of the dendrites of smooth neurons (Somogyi et
al., 1983; Peters and Saint Marie, 1984; Kisvárday et al., 1985;
Ahmed et al., 1997). Some of the adjacent unidentified synapses on the
same target dendrites were symmetric (Fig. 4A,
open arrowheads), although the majority were asymmetric
(Fig. 4A, small, filled arrowheads). Smaller-diameter dendrites with no symmetric synapses but otherwise similar features were also the targets of labeled boutons. One dendrite
of small diameter formed a synapse with a labeled segment of axon. This
contact was not anticipated, because at the LM level there was no
suggestion of a bouton. Despite lengthy serial sections of axons, this
was the only synapse discovered, indicating that such axonal synapses
must be exceedingly rare.
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Figure 4.
Electron micrographs of labeled synaptic boutons
in contact with dendrites of smooth cells. A,
Characteristically large bouton, filled with vesicles, located in lower
layer 4 forms two asymmetric synapses (large, solid
arrowheads), one with a dendrite (d) and
the other with a spine (sp). The dendrite contains
numerous mitochondria and forms many synapses of the symmetric
(open arrowheads) and asymmetric (small, solid
arrowheads) types with unidentified boutons. When the target
dendrite was reconstructed over several serial sections, it became
clear that the variations in diameter, which can be visible in the
micrograph, were caused by a varicose or beaded morphology. These
features are consistent with those of neurons with smooth dendrites, which contain
GABA. In subsequent sections of the series, the same labeled bouton
formed another two synapses with the dendrite. The spine target is
almost completely enveloped by the bouton. The extensive postsynaptic
density within the spine indicates that the synapse has been sectioned
at an oblique plane. B, Large bouton located in layer 6 with a dendritic target similar to the one described above (Fig.
3A). The labeled bouton forms an asymmetric synapse
(large, solid arrowhead) with the dendrite, which also
forms symmetric synapses (open arrowheads), and other
asymmetric synapses (small, solid arrowheads) from
unidentified boutons. C, A higher-power micrograph of
the adjacent section in the series of the bouton shown in Figure
3B. Detail of the unlabeled symmetric synapse
(open arrowhead) and the labeled and unlabeled
asymmetric synapses (large, solid and small,
solid arrowheads, respectively) can be compared. Scale bars:
A, B, 1 µm: C, 0.5 µm.
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The bouton illustrated in Figure 4B (sampled from
layer 6) formed a synapse with a smooth beaded dendritic shaft similar
to that described above (Fig. 4A). In the
higher-power view of the synapse (Fig. 4C), the additional
unlabeled boutons that form synapses with the dendrite were packed
together. One formed symmetric synapses (open arrowheads),
one formed asymmetric synapses (small, filled arrowhead).
Approximately 26% of the dendritic shafts in V5 that formed synapses
with the V1 afferents were of this smooth neuron type.
Synapses formed with somata
Neuronal somata in both layers 4 and 6 also formed synapses with
V1 afferent boutons. Figure 5 illustrates
a soma in layer 4 that forms synapses with four separate boutons (Fig.
5B, b1-b4) that arose from one myelinated axon
collateral. Contacts between somata and the darkly labeled boutons of
the V1 axons were easily visible at the LM level (Fig.
5A,B). The postsynaptic somata contained large, deeply
invaginated nuclei, and the cytoplasm was packed with mitochondria
(examples indicated by arrows in low-power electron micrograph in Fig. 5C) and rough endoplasmic reticulum.
Crystalline inclusion bodies were sometimes found within the
mitochondria of contacted somata (Fig. 5H). These
have previously been seen within the mitochondria of Meynert cells in
area V1 of monkey cortex (Chan-Palay et al., 1974). The Meynert cells
project to V5 (Lund et al., 1975; Maunsell and Van Essen, 1983; Fries
et al., 1985). All of the synapses formed with the soma by unlabeled boutons were asymmetric. However, the postsynaptic density of somatic
synapses was far less prominent than those formed with spines and
shafts. The ultrastructural features of the somata are characteristic
of GABAergic smooth neurons (Peters and Saint Marie, 1984).
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Figure 5.
Correlated light and electron micrographs of
BDA/PHA-L-filled boutons in synaptic contact with a soma
(S) located in layer 4 of area V5.
A, Low-power light micrograph of an identified axon
collateral (large, solid arrow) rising through lower
layer 4. The soma (S) and some of the boutons
(small arrows) appear at higher magnification in
B. B, Numbered boutons (b1, b2,
b3, b4) all form contacts with the soma
(S). A large-diameter myelinated axon
(m) is indicated for reference. C,
Low-power electron micrograph of the soma (S)
seen in B. The myelinated axon (m)
referred to in B can be seen in close contact with the
soma. Some of the mitochondria appear to be lightly stained
(small arrows) , thus enabling the soma to be seen at the light microscopic
level. The large filled axon (ax) gives rise to the
boutons (b2, b3) after losing its myelin sheath (not
illustrated). D, High-power electron micrograph of
boutons b2 and b3. The bouton b2 forms an asymmetric synapse
(solid arrowhead) with the soma. Electron dense
mitochondria (mit) can be seen within the cytoplasm of
the soma. E, F, In sections after that shown in
D, boutons b3 and b1 can be seen to form asymmetric
synapses (solid arrowheads) with the soma.
G, The asymmetric synapse (solid
arrowhead) of the fourth bouton (b4) in
contact with the soma can be compared with the asymmetric synapses
(small arrows) formed by an unlabeled bouton also in
contact with the soma. H, High-power micrograph of a
crystalline inclusion (i) within the body of a
mitochondria found in the soma contacted by the above boutons. Scale
bars: A, B, 10 µm; C, 5 µm;
D-H, 0.5 µm.
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Three-dimensional reconstructions of synapses
One intriguing aspect of the large labeled boutons became evident
when we reconstructed them and their synaptic targets more fully.
Because the geometry might be of significance for the diffusion of
transmitter to receptors from the presynaptic release sites (Uteshev
and Pennefather, 1997; Rusakov and Kullmann, 1998a,b) we have made
complete reconstructions of some boutons to illustrate the feature
(Figs. 6, 7). The asymmetric synapses
formed with spines were made by boutons that enveloped the spine head,
which formed a virtual pocket within the volume of the bouton (Fig. 6A,B). Sometimes the bouton completely engulfed the
spine head (Fig. 6C,D). Almost half (46%) of the spine
heads in our sample were enveloped by the bouton. When synapses were
formed, as these examples show, the synapse itself could be located at
the tip of the spine or close to the point of penetration of the spine into the bouton, or both. The large bouton illustrated in Figure 7 formed synapses with four spines and a
dendritic shaft. This exceptional bouton formed eight distinct
synapses. The entire heads of two bulbous spines were almost completely
enveloped by the bouton. The other two spines were also less completely
enveloped. The entire circumference of a dendritic shaft was enveloped
by the bouton. It formed three synapses with the labeled bouton. The
target dendrite was varicose, packed with mitochondria, and formed one
other synapse with an unidentified bouton. It had no spines in the
reconstructed section and most likely was a dendrite of a smooth
neuron.
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Figure 6.
Three-dimensional reconstructions from serial
ultrathin sections of filled boutons in area V5 showing how targets are
enveloped by the bouton. A, B, Two views of a bouton
(blue) found in layer 6. In A a clear
depression or pocket can be seen at the top (uppermost
pole) of the bouton. The edge of the postsynaptic density
(yellow) can be seen at the lip of the
depression. In B the bouton was rotated around a
vertical axis (~180°) to provide a better view of the postsynaptic
density. C, D, A large layer 6 bouton
(blue) that forms synapses with three spines
(brown). In C the bouton and spines have
an opaque skin, and in D the skin is transparent. The
two spines on the right are deeply embedded within the
bouton. The postsynaptic surface (yellow) is
shown in apposition with the spines. Axes, 0.5 µm.
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Figure 7.
Reconstruction of a large bouton
(blue) in layer 4 of area V5 that forms synapses with
four spines and a dendrite. A, When the bouton is
rendered with an opaque skin it can be seen to wrap around the dendrite
(d in B). This dendrite forms three
synapses with the identified bouton. The spines (s in
B) are all deeply embedded in the bouton.
B, Both bouton and targets are rendered transparent,
showing the postsynaptic density. The asymmetric synapses are
yellow. One of the spines also forms a symmetric synapse
(red) with an unidentified bouton. Axes, 0.5 µm.
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The detail of the postsynaptic membrane was sufficiently well preserved
that we could reconstruct the complete postsynaptic surface of
synapses. It became clear that different types of target formed
synapses of different size and shape. The shape of the postsynaptic
disk could vary because of perforations in the postsynaptic density
(Peters et al., 1991). This results in a "complex" synapse. Figure
8 depicts the two-dimensional projection
of the postsynaptic densities from the sample of complete 3-D
reconstructions of the synapses. Postsynaptic densities in the form of
annuli and horseshoe shapes were more frequent on spines. Dendritic
shafts and somata in particular had the least perforated or complex
postsynaptic densities. It was a matter of opinion whether the
numerous, small synapses formed with somata were in fact one synapse,
but because each synaptic site could be separated by up to seven
sections they were considered to be individual sites. However, the
arrangement of vesicles above these multi-synaptic sites was of a
continuous dense cloud of vesicles. Sessile spines could form synapses
with regular or complex postsynaptic densities.
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Figure 8.
Two-dimensional projection of the
reconstructed postsynaptic densities found on spines, soma, and
dendrites postsynaptic to labeled boutons in area V5. The densities are
from individual synapses and are ordered by increasing surface area
calculated from the 3-D reconstructions. Scale bar, 1 µm.
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The histogram of synaptic areas (Fig. 9)
indicates that synapses made by the boutons contacting somata were the
smallest: mean area 0.031 µm2 (SEM = 0.008).
Spine synapses were the largest on average (0.127 µm2; SEM = 0.011), and those with dendritic
shafts were intermediate (0.071 µm2; SEM = 0.07). The synapse areas of the three groups are significantly different from each other (p = 0.01; Wilcoxson
paired rank test). Most synapses extended over only two or three
sections; however, one bouton could provide up to five small
synapses.
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Figure 9.
Histogram of the distribution of postsynaptic
areas (µm2) formed by labeled boutons in area
V5.
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Mitochondria in boutons
Evidence is accumulating that because of their role in calcium
metabolism, the mitochondria in axonal boutons may have a significant influence on the dynamics of synaptic transmission (Nicholls and Åkerman, 1982; Herrington et al., 1996; Tang and Zucker, 1997; Xu et al., 1997; Peng, 1998). We have thus fully reconstructed several
boutons to examine the details of their mitochondrial contents more
completely. These 3-D reconstructions indicated that the region
adjacent to the synapse had the greatest accumulation of vesicles.
Mitochondria of variable diameter filled out the remainder of the
volume of the bouton. Up to 12 mitochondrial profiles, sometimes
branched, could appear in any single ultrathin section.
Three-dimensional reconstructions of mitochondria have rarely been made
in such boutons, so the actual number of individual mitochondria is
unknown. After reconstructing some of these structures (Fig.
10) we discovered that there was
considerable variation in the organization of mitochondria. In some
boutons the densely packed mitochondria were relatively short (0.5 µm) and straight, and in others they formed continuous loops within
the volume of the bouton (Fig. 10). Hence the same mitochondrion was
sectioned many times in each ultrathin section. As the bouton volume
increased, so the number of mitochondrial profiles increased, but not
necessarily the total number of mitochondria. We could follow
individual mitochondria as they streamed from the bouton into the axon
[e.g., the mitochondria represented in dark orange and
pale green (Fig. 10C,D)]. The reconstructions also showed that the mitochondria accumulated at one side of the bouton, away from the synapses themselves. This space immediately around the synapses was filled with vesicles. Measurements in three
fully reconstructed boutons revealed that the mitochondria occupied an
average volume of 0.57 µm3, which was fully 22%
of the entire average volume of the boutons. The total surface area of
the mitochondria within a bouton was approximately equal to the surface
area of the bouton.
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Figure 10.
Two different views of two boutons reconstructed
and rendered with transparent skins to show solid mitochondria and
postsynaptic specializations (yellow). The
mitochondria are color-coded for identification of individual
structures. For both boutons the right-hand image (B, D)
is rotated (~180°) about the horizontal axis of the left-hand
image. A, B, Bouton found in layer 6 that formed
synapses with two spines and contained four mitochondria
(pink, blue, orange, and green).
The longest mitochondrion (pink) was branched and
formed three loops. Both synaptic surfaces
(yellow) are presented at oblique, nonoptimal
elevations but appear as incomplete annuli. C, D, Layer
4 bouton formed synapses with two spines and contained nine
mitochondria. One mitochondrion (pink) is
branched and forms loops. The synaptic specializations
(yellow) become superimposed on each other in
these views. One synapse is horseshoe-shaped and the second is composed
of two small patches. Axes, 0.5 µm.
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Targets of synapses
Data from layers 4 and 6 were pooled because no significant
differences between the two samples were observed. The principal synaptic targets of the V1 afferents were spines, which formed 54% of
targets. Dendritic shafts formed a large proportion of targets (33%),
about a quarter of which originated from smooth neurons. Somata,
probably also of smooth neurons, formed the remaining 13% of targets
(Fig. 11). Thus, ~78% of the V1
afferent synapses were formed with spiny excitatory neurons in V5. Most
boutons formed only one asymmetric synapse (Fig.
12). However, some formed two or more
synapses, so that on average each V1 afferent bouton formed 1.7 synapses.
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Figure 11.
Histogram of synaptic targets of boutons in area
V5 originating from neurons labeled in area V1.
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Figure 12.
Histogram of the number of synapses (1, 2, 3, 4, 5 or more per bouton) formed by individual labeled
boutons in layers 4 and 6 of area V5.
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Proportion of asymmetric synapses formed by V1 afferents in layer 4 of V5
When sections were viewed in the LM it was clear that the labeled
processes were not distributed evenly (Rockland, 1989) but formed zones
of higher and lower density. From one monkey we selected two patches of
particularly dense labeling within layer 4 for a stereological
assessment by the unbiased "disector" method (Sterio, 1984) of the
proportion of labeled synapses. This method entails the use of serial
sections. Synapses appearing in the "reference" section but not in
the "look-up" section are counted. We found that 2.7% (5 of 185)
and 3.5% (3 of 85) of boutons with disappearing synapses (Sterio,
1984) were labeled. The area (micrometers squared) of all labeled and
nonlabeled boutons with disappearing synapses (n = 270)
was measured. The size distribution of the unlabeled and labeled
profiles was broad (Fig. 13). Labeled
profiles did not occupy the lowest end of the distribution but were
evident among the largest measured. In the same tissue we could assess labeled versus nonlabeled myelinated axon profiles. Labeled myelinated axons occurred with about the same frequency (2.8%) as labeled synapses. The largest diameter axons (~2 µm) in layer 4 were both labeled and nonlabeled. All of the large-diameter axons were covered with a myelin sheath. The wall of the myelin sheath associated with
these large fibers ranged from 0.12 to 0.4 µm thick.
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Figure 13.
Histogram showing the distribution of area
(µm2) of labeled (black bars) and
nonlabeled (white bars) synaptic boutons in area V5.
Only those boutons with disappearing synapses were measured.
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DISCUSSION |
In the present experimental work we have studied the direct
projection from the parafoveal regions of V1 to V5 with the view of
establishing a structural basis for the transmission of the component
motion signal from V1 to V5. The source of the V1 afferents is known to
be the spiny stellates and pyramidal cells of layer 4B and large
pyramidal cells (Meynert cells) of layer 6 (Lund et al., 1975; Fries et
al., 1985; Shipp and Zeki, 1989; Elston and Rosa, 1997). These neurons
formed asymmetric (Gray's type 1) synapses with their targets. Nearly
80% of the synaptic targets of V1 afferents in V5 were the spines and
dendritic shafts of excitatory cells. The remaining synapses were with
the dendrites and somata of smooth (GABAergic inhibitory) neurons.
The similarity between the thalamic and feedforward intracortical
pathways has been noted by Johnson and Burkhalter (1996) in their
comprehensive study of feedforward and feedback connections in the rat
visual cortex. Both the thalamic afferent projection in the primate and
the V1 to V5 projection have similar terminal laminae and have spiny
neurons as their major target. Compared with the projection from V1 to
V5, the projections from V1 to lateral extrastriate cortex in the rat
show some minor variations in the cells of origin and in the laminar
distribution of the terminals. However, the types and proportions of
synaptic targets are quite comparable to those found here, with
pyramidal cells forming 90% of the targets and GABAergic interneurons
the remainder (Johnson and Burkhalter, 1996). Comparable distributions
were also found for the projection from the area 17-18 border to the posterior lateral suprasylvian visual area in the cat (Lowenstein and
Somogyi, 1991). This suggests a conservation of function of the
interareal feedforward projections in mammalian visual cortex. The
commissural and "feedback" connections differ in that a much smaller proportion (~2-4%) of synapses is formed with smooth
neurons (Jones and Powell, 1970; Czeiger and White, 1993; Johnson and Burkhalter, 1996).
The injection sites in V1 included all layers, so it is very likely
that the sample of boutons included those originating from both the
large pyramidal cells (solitary cells of Meynert) on the border between
layers 5 and 6 in monkey V1 and the layer 4B neurons. The layer 5/6
Meynert cells are outnumbered by the layer 4B neurons that project to
V5 by ~10:1 (Maunsell and Van Essen, 1983; Shipp and Zeki, 1989).
Although the terminal arborizations of these two neuronal types have
yet to be distinguished, the marked differences in the numbers of these
two types that project to V5 suggest that most of the terminals we
sampled would have originated from the 4B neurons. Qualitatively,
however, we did not get any hint that we might be sampling from two
quite distinct populations, at least in terms of their morphology and
synaptic targets, and we have therefore treated the V5 population as
one homogenous sample. The appearance of anatomical homogeneity may reflect the physiological homogeneity seen by Movshon and Newsome (1996). Six of the neurons they recorded were from layer 4B, and six
were from the layer 5/6 border, i.e., probably Meynert cells. All 12 had similar receptive field properties, including strong contrast-independent direction selectivity, and all had fast-conducting axons. This relation of projection pattern to physiology led Movshon and Newsome (1996) to follow Zeki (1974) and characterize V1 as a vast
"clearing house" that selectively distributes different specific
information to different visual areas.
Identification of target neurons and likely synaptic efficacy
The pyramidal cells in V5 are more highly branched and more spiny
than those of V1 pyramidal cells (Elston and Rosa, 1997). This is
perhaps a consequence of the higher density of neurons in V1 that may
prevent elaboration of the dendritic tree. The number of excitatory
synapses formed on these V5 pyramidal cells is probably in the range of
5000 to 10,000, as in other cortical areas. For the basket cells the
number is probably similar (Ahmed et al., 1997). Our estimates clearly
suggest that the afferents of V1 can only provide a few percent of
these excitatory synapses. The unbiased method of counting (Sterio,
1984) makes it likely that we have correctly assessed the percentage of
labeled synapses in layer 4 of V5. However, there are possible
technical issues that would lead to an underestimate of the actual
number. One obvious issue is that not all of the projecting neurons at
the site of injection might have taken up and transported the label, or
that the transport is incomplete. A more significant issue concerns the
divergence and convergence of the V1 to V5 projection. If a
single region of V5 receives its input from a very large region of V1,
our injections in V1 would have failed to label all of the neurons
projecting to the sampled terminal region in V5. However, if not all of
the boutons were labeled, this estimate cannot be greatly in error,
given that the V5 projection neurons in V1 are sparse andhave to map
retinotopically in V5. It is likely that each target neuron in V5 must
form at most only a few hundred synapses with V1 afferents. This raises
the interesting functional problem for the computation of pattern
motion. If V1 does form the first stage of the motion computation and
V5 the second, then the signal is being conveyed from V1 to V5 by a
small fraction of the synapses. How then does the V1 signal avoid being
swamped or corrupted by the activity of all of the other excitatory
synapses simultaneously active on the dendritic tree of the recipient
V5 neuron?
One possible answer to this question is that the V1 afferent synapses
are particularly powerful relative to the other excitatory synapses
formed with the V5 neurons. The size of the synaptic potentials in V5
neurons has yet to be measured. However, the size of the postsynaptic
densities of the V1 afferent synapses may provide an approximate
indication of the number of receptors that could be located in the
postsynaptic membrane of the V5 recipient neurons. For example, 1000 AMPA receptors of 10 nm diameter could be packed side by side into 0.1 µm2. Our measurements of the area of the
postsynaptic densities of the synapses formed by the labeled boutons
indicate that the largest (those on spines) are slightly smaller (0.13 µm2) than those of the thalamocortical synapses in
the cat, which are ~0.18 µm2 (Dehay et al.,
1991; Friedlander et al., 1991). These postsynaptic densities in V5 are
at least threefold larger in area than those observed at comparable
excitatory synapses formed with spines of CA1 pyramidal cells in the
mouse and rat hippocampus (Harris and Stevens, 1989; Schikorski and
Stevens, 1997). They are slightly smaller than the individual synapses
formed by single mossy fiber boutons with the branched dendritic spines
of the CA3 pyramids (Chicurel and Harris, 1992).
Individual geniculocortical fibers in the cat visual cortex provide
average EPSP amplitudes of nearly 2 mV, which would require >400 postsynaptic ion channel openings (Stratford et al., 1996). The
extraordinarily low coefficient of variation of 8% suggests that they
could arise from multiple release sites with an extremely high release
probability or single large synapses (Stratford et al., 1996). By
analogy with these cat data, the size of the V5 synapses studied here
suggest that the mean amplitudes of the unitary AMPA receptor EPSPs
will be ~1-2 mV. This remains to be tested. We also found evidence
of multisynaptic connections from single afferents to single neurons in
V5. Unfortunately, our method cannot provide an estimate of the
frequency of such multisynaptic connections, but such multiple synapses
would lead to larger EPSPs and so provide V5 with a functionally
powerful input from V1 despite the small number of synapses that the V1
projection provides in V5.
Influence of synapse morphology on transmitter diffusion
The 3-D reconstruction of the postsynaptic elements revealed that
the postsynaptic densities formed with dendritic shafts were single
disks. By contrast the synapses formed with spines had perforated
postsynaptic densities that formed complex horseshoe or circular
arrangements when viewed en face. In addition, the larger
presynaptic boutons enveloped the entire head of the postsynaptic spines and occasionally dendritic shafts. We do not yet understand why
the spine synapses in the V1 to V5 projection form such elaborate morphology, but presumably one reason is to secure more efficient and
reliable transmission with the fewest possible synapses. Previously we
have observed such embedding of the postsynaptic target in the
presynaptic bouton in the magnocellular boutons of the thalamic afferents in V1 of the macaque monkey (Freund et al., 1989) and in
boutons of the nondeprived Y-axons in area 18 of long-term monocularly
deprived cats (Friedlander et al., 1991). Elsewhere, the giant mossy
fiber boutons in CA3 of the hippocampus also appear to embed their
targets (Chicurel and Harris, 1992). An additional feature of such
large boutons is that they form multiple synapses. Such arrangements
could have some significance for the spillover of neurotransmitter
between receptor domains within a single postsynaptic or perisynaptic
complex and for spread of transmitter between synapses.
The diffusion of glutamate neurotransmitter from the synapse has been
modeled in some detail (Uteshev and Pennefather, 1997; Rusakov and
Kullmann, 1998a,b). Kullman and Asztely (1998) have reviewed the
relevant experimental literature. The modeling shows that both the
viscosity (determined by the diffusing molecules interactions with cell
walls and macromolecules in the extracellular space) and geometry of
the tissue have a significant effect on diffusion. Rusakov and Kullman
(1998a) show that NMDA receptors, but not AMPA receptors, located at a
distance of ~500 nm from the center of the synaptic cleft will be
activated by the release of one vesicle. The synapses formed by the
multisynaptic boutons in V5 tended to have intersynaptic distances in
excess of 500 nm. This, together with the envelopment of the synapses
by the bouton, would restrict the cross-talk between these synapses. However, this same morphology might increase the concentration of
neurotransmitter in the region surrounding the synapse and so increase
the probability of the activation of receptors on both the postsynaptic
and the presynaptic membrane. The "gap" between the synaptic
densities in the perforated synapses averaged ~300 nm (range, 168 to
476 nm) (Fig. 8), which is within the effective diffusion range of the
contents of a single vesicle (Rusakov and Kullman, 1998a). The same
constraints of diffusion studied by Rusakov and Kullman (1998a,b) of
course applies to the movement of extracellular ions, such as
Ca2+, into the boutons. If the diffusion of
Ca2+ is slow, then it may become locally depleted
and so modify synaptic activity (Montague, 1996).
Little attention has been paid to the correlation between synaptic
morphology and physiology in the visual cortex. Nevertheless, there are
some quite distinct differences in the synaptic physiology of the
inputs to layer 4 of cat visual cortex, as noted above. In particular
the thalamocortical synapses in the cat produce EPSPs of large
amplitude and exceedingly low variance (Stratford et al., 1996). This
is most easily explained if only a single release site were
active. However, the contents of a single vesicle [~5000
molecules of neurotransmitter (Riveros et al., 1986)] may not contain
enough transmitter to produce 100% double occupancy (saturation) of
the receptors that such low variance implies (Larkman et al., 1991).
Thus, multiple release sites, each having a high probability of
release, may invariably be involved in such synapses if the diffusion
distance between any single release site and target receptors is to be reduced.
Synapses with multiple release sites that have large-amplitude
low-variance synaptic currents have been observed in the multi-site glutamatergic synapses that mossy fibers form with granule cells in the
rat's cerebellum (Silver et al., 1996). At these sites the synaptic
current appears to be limited by the number of postsynaptic channels
rather than by the amount of neurotransmitter released. This is
probably because the transmitter released from neighboring sites
overlaps and so changes both the concentration and length of occupancy
of the transmitter in the cleft (Faber and Korn, 1988). Importantly
too, the perisynaptic concentration of transmitters would also be
raised. One possibility is that there are receptors located in the
perforation of the postsynaptic density for example, the metabotropic
glutamate receptors (Nusser et al., 1994; Baude et al., 1995).
Extrasynaptic receptors located at the center of the horseshoe would be
at a relative advantage because they would receive a higher
concentration of spillover neurotransmitter from multiple release sites
than receptors located outside the ring. Unfortunately the
neurochemistry and distribution of V5 receptors is unknown.
The smallest postsynaptic densities seen in the present study were
formed mainly with dendritic shafts. In the cat, the smallest postsynaptic densities are also on the dendritic shafts, but their source is local layer 6 pyramidal cells. (Ahmed et al., 1994, 1997).
These layer 6 pyramidal cell synapses show strong paired-pulse facilitation and not the paired-pulse depression seen with the layer 4 spiny stellate synapses or the thalamic afferent synapses (Stratford et
al., 1996; Tarczy-Hornoch et al., 1998). The mechanisms that
determine this dynamic behavior of synapses are largely unknown. However, it has recently been discovered that the mitochondria may have
an important role in the dynamics of synaptic function.
Mitochondria in the synaptic boutons
A striking morphological feature was the considerable volume
occupied by mitochondria within the terminal boutons of the V1 afferent
neurons. In a previous study in the cat it was noted that the number of
mitochondrial profiles seen in a single cross section of a thalamic
afferent bouton scaled linearly with the size of the bouton
(Friedlander et al., 1991). By making the 3-D reconstructions, it
became clear that the many mitochondrial profiles seen in a single
cross section actually arose from the tortuous folding and branching of
individual mitochondria within the bouton. The functional role of the
mitochondria in the presynaptic terminals is beginning to be understood
from work in invertebrates and lower vertebrates. In the crayfish, the
axon terminals of phasic motoneurons contain fewer mitochondria and
show marked synaptic depression, whereas the terminals of tonic
motoneurons have more mitochondria, more oxidative activity, and a
greater resistance to synaptic depression. The synapses of the tonic
motoneurons also show strong frequency-dependent facilitation (Nguyen
et al., 1997). These data suggest the possibility that the V1 neurons
projecting to MT have a tonic activity and that one role of the
mitochondria in the boutons is to prevent or reduce depression in this synapse.
Mitochondria play an important role in calcium metabolism in cells
(Nicholls and Åkerman, 1982; Herrington et al., 1996; Xu et
al., 1997), but despite the importance of calcium in synaptic transmission, the significance of mitochondria for synaptic
transmission has only recently been recognized. Calcium enters the
synaptic bouton through voltage-gated channels, and it is the key ion
in producing the release of neurotransmitter. Increasing the calcium concentration within the bouton increases the probability of
transmitter release. In the bullfrog sympathetic ganglia (Peng, 1998),
calcium entering the bouton is taken up by the mitochondria, but in a frequency-dependent manner. If the terminal is activated at low rates,
then the calcium transporters in the mitochondria can provide adequate
buffering. If the rates of activation are high, the mitochondria cannot
buffer adequately, and calcium accumulates within the bouton. This
uptake during repetitive stimulation also explains the development of
post-tetanic potentiation at synapses. Tang and Zucker (1997) have
found that mitochondria in the neuromuscular junction of the crayfish
also slowly release the calcium they accumulated during tetanic
stimulation. It is this maintained higher concentration of calcium
within the bouton that is responsible for post-tetanic potentiation.
Although the role of mitochondria at central synapses in mammals has
still to be studied, it is most likely that their role in the boutons
studied here includes the mechanisms referred to in the above studies
of invertebrate and lower vertebrate synapses. Thus, the quantification
of the contribution of mitochondria within the synaptic boutons of
identified neurons may provide another important link between
morphology and function of these synapses.
Common basic circuits in V1 and V5
On anatomical grounds, therefore, it seems that both the light and
electron microscopic pattern of innervation of the V1 afferents in V5
does not differ markedly from that of the dLGN input to V1. Johnson and
Burkhalter (1996) have suggested schemes of how the thalamic and
interareal feedforward and feedback circuits might connect into the
local recurrent microcircuits of the visual cortex (Douglas et al.,
1989a,b). Models of component direction selectivity have been simulated
on such recurrent microcircuits (Douglas and Martin, 1991; Suarez et
al., 1995). In principal, the extraction of global motion should be
possible using the same machinery as is used for the component motion
analysis. Simoncelli and Heeger (1998) have developed an abstract
linear model to explain how global motion in V5 can be derived from a
component motion input from V1 by means of essentially the same
algorithm. Although their model does not map directly onto the known
circuitry and biophysics of the cortex, they pointed out that the
commonality of their computation for V1 and V5 is conceptually related
to the structural commonality of circuits in different cortical areas. It is this same principle of multifunctionality of a basic circuit that
is captured in the concept of the recurrent microcircuit (Douglas et
al., 1989a,b; Douglas and Martin, 1991).
Future prospects
The present study has raised some important questions about the
possible role of the V1 input to V5. Obviously, V5 receives other
inputs from areas such as V2, V3, VP, MST, and the thalamus. Blocking
all of the activity transmitted from V1 either by cooling appropriate
retinotopic regions (Girard et al., 1992) or by ablation (Rodman et
al., 1990) leaves V5 quite impaired but still partly functional. The
remaining active neurons are directional. Combined lesions of the
colliculus and V1 eliminate activity in V5 (Rodman et al., 1990). These
alternative routes are not evidence against the hypothesis of Movshon
and Newsome (1996) that the computation of pattern motion takes place
in V5. However, considerable work still needs to be done to settle the
matter. Clearly, in view of the results obtained in this study, it will
be important to establish whether V5 is built of recurrent circuits
comparable in structure and function to those of V1.
| |
FOOTNOTES |
Received June 23, 1998; revised Aug. 27, 1998; accepted Sept. 14, 1998.
This work was supported by a Schweizer Schwerpunkt Programme Swiss
National Science Foundation grant to Professor R. J. Douglas and K.A.C.M. and by National Institute of Mental
Health/National Science Foundation Grants MH 53598 and IBN 9421970 to
K.S.R. We are grateful to Professors W. T. Newsome and M. N. Shadlen for their critical reading of an earlier draft of this manuscript.
Correspondence should be addressed to John C. Anderson and Kevan
A. C. Martin, University/ETH Zürich,
Winterthurerstrasse 150, 8057 Zürich, Switzerland.
| |
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Abstract
Introduction
