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The Journal of Neuroscience, August 1, 1998, 18(15):5723-5745
Mechanisms Underlying the Early Establishment of Thalamocortical
Connections in the Rat
Zoltán
Molnár,
Richard
Adams, and
Colin
Blakemore
University Laboratory of Physiology, Oxford OX1 3PT, United Kingdom
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ABSTRACT |
We labeled axonal projections using carbocyanine dyes in the
developing rat brain to study cellular interactions that might underlie
the establishment of thalamocortical connectivity. By embryonic day 14 (E14), groups of neurons in the ventral diencephalon and the
primitive internal capsule have established projections to the dorsal
thalamus, and thalamic fibers pass in topographic order among them.
Simultaneously, axons from the early-born cells in both subplate and
marginal zone (i.e., the original cortical preplate) establish an
ordered array that fills the intermediate zone. Thalamic axons and
preplate fibers meet in the lateral part of the internal capsule (at
E15 for occipital cortex and dorsolateral thalamus). Subsequently,
selective labeling of corresponding thalamic and early corticofugal
projections reveals thalamic fibers growing in association with early
corticofugal axons, right up to the cortical subplate. A small
carbocyanine crystal implanted at any point in the cortex shortly after
the arrival of thalamic axons (E16 for the occipital cortex) labels a
single, tight bundle containing both descending and ascending fibers,
rather than two separate tracts, providing further evidence for
intimate topographic association of the two axon systems. Crystals
placed in a row, parasagittally or coronally along the hemisphere,
reveal separate, topographically distributed, discrete fiber bundles
throughout the pathway, leading to spatially ordered groups of
back-labeled thalamic cells. These results indicate that the topography
of thalamic axons is maintained throughout the pathway and that they
reach the cortex by associating with the projections of a number of
preexisting cells, including the preplate scaffold.
Key words:
cortex; thalamus; rat; pioneer axons; development; subplate; perireticular nucleus; preplate; "handshake" hypothesis; internal capsule; thalamic reticular nucleus; axon guidance
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INTRODUCTION |
The adult mammalian neocortex
consists of numerous areas, differing in connectivity and functional
properties (Peters and Jones, 1985 ), many of which have a distinctive
cytoarchitectural appearance (Brodmann, 1909). Thalamic axons, the
carriers of most afferent information to the cortex, arrive before the
majority of cortical neurons are born (Lund and Mustari, 1977; Rakic,
1977; Wise and Jones, 1978; Shatz and Luskin, 1986). These early
afferent projections, if they are topographically distributed and
distinctive for each particular area, could play a part in initiating
regional differentiation (O'Leary, 1989). In that case, unraveling the mechanisms by which thalamic axons are directed toward their target regions is important in understanding how cortical organization is
determined.
The representation of the thalamus on the cortex and the topographic
distribution of thalamic axons have been studied extensively in
postnatal and adult rodents (Caviness and Frost, 1980; Frost and
Caviness, 1980; Crandall and Caviness, 1984; Bernardo and Woolsey,
1987; Caviness, 1988; Agmon and Connors, 1991; Agmon et al., 1993,
1995; Mitrofanis and Guillery, 1993). All areas of the rodent neocortex
receive a thalamic input. In general, neighborhood relationships are
preserved in the pattern of projection, but, in some cases, contiguous
neocortical fields receive input from nonadjacent thalamic nuclei.
However, studies of the mature projection do not directly illuminate
its initial topography or the means by which connections are
established. The spatial relationships of thalamic cell groups might
change substantially, and thalamic axons themselves might undergo local
rearrangement, after their projection to the cortex.
The capacity to trace axons in fixed embryonic tissue offered by the
carbocyanine dye technique has stimulated many recent studies of early
axonal outgrowth between cortex and thalamus for several species and
various parts of the projection system (McConnell et al., 1989;
Blakemore and Molnár, 1990; Molnár and Blakemore, 1990;
Catalano et al., 1991, 1996; De Carlos and O'Leary, 1992; Erzurumlu
and Jhaveri, 1992; Ghosh and Shatz, 1992; Miller et al., 1993; Clasca
et al., 1995; Métin and Godement, 1996). However, no coherent
picture has emerged of the relationships between growing thalamic axons
and other fiber systems or of the mechanisms by which thalamic axons
navigate to their appropriate target regions.
McConnell et al. (1989) drew attention to the fact that axons from
early postmitotic neurons of the cortical subplate (Marin-Padilla, 1971; Luskin and Shatz, 1985a,b) pioneer the pathway from the cortex
toward subcortical targets, before neurons of layers 5 and 6 have
migrated into position. They suggested that these early corticofugal
axons might play a part in guiding subsequent descending and ascending
projections.
Here we characterize the entire period of development of early
corticofugal and thalamocortical projections in the rat and the degree
of order that both sets of fibers maintain. We describe the cellular
elements that thalamic axons encounter as they grow out from the
diencephalon, and specifically we present evidence that they associate
closely with axons emanating from early cortical neurons.
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MATERIALS AND METHODS |
Hooded Lister rats, whose gestation period is 21-22 d, were
time-mated at the University Laboratory of Physiology (Oxford, UK).
Assessment of the embryonic age of the fetuses was based on the plug
date, defined as embryonic day 0 (E0). Fetuses were removed by cesarean
section under pentobarbital anesthesia (100 mg/kg, i.p., to the
pregnant female) at various stages, from the 13th to the 21st or 22nd
postconceptual day. They were immediately chilled on ice and
transcardially perfused with 4% paraformaldehyde in 0.1 M
phosphate buffer, pH 7.4, delivered through a glass micropipette under
a dissecting microscope. Postnatal animals of various ages were
anesthetized by chilling [up to postnatal day 4 (P4)], or by overdose
of intraperitoneal sodium pentobarbital (if older than P4),
before perfusion.
The brains were removed, and, after overnight post-fixation (in 4%
buffered paraformaldehyde), single crystals (0.1-0.3 mm diameter) of
fluorescent carbocyanine dyes [1,1'-dioctadecyl
3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI),
4-[4-(dihexadecylamino)styryl]-N-methylpyridinium iodide
(DiA), 4-[4-(didecylamino)styryl]-N-methylpyridinium
iodide (DiAsp), and 3,3'-dipentyloxacarbocyanine iodide (DiO);
Molecular Probes, Eugene, OR] were inserted into different parts of
the cortex or diencephalon under an operating microscope (Godement et
al., 1987).
For each cortical placement, a single carbocyanine crystal was inserted
into a tiny slit made in the cortex with the tip of a pair of fine
forceps or, in embryonic brains, with a thin stainless steel wire. The
depth to which the crystal was inserted into the cortex varied with
age, from as little as 0.1 mm at E13 to 0.5 mm in the oldest animals.
In half of these brains, we made deposits of a number of different
dyes, at points across the cortex, usually spaced 1-2 mm apart in
either a parasagittal or a coronal row in one hemisphere. For most of
these experiments, we used alternating placements of the two dyes that
are best transported, DiI and DiA, which are clearly distinguishable
under different wavelengths of fluorescent illumination.
For crystal placements in the thalamus [usually in the vicinity of the
putative lateral geniculate nucleus (LGN)], we first transected the
brainstem coronally, rostral to the superior colliculus, to expose the
posterior, dorsal thalamus. For insertions into deeper or more rostral
thalamic nuclei (e.g., the ventrobasal complex), we bisected the brain
sagittally and cut away the medial part of the diencephalon to expose
the relevant part of the thalamus. Then a single carbocyanine crystal
was inserted into a small slit made in the tissue with the tip of a
fine pair of forceps under an operating microscope.
In other specimens, the diencephalon was removed from the hemisphere by
means of a parasagittal cut through the internal capsule, and a small
crystal of carbocyanine dye was inserted into the exposed telencephalic
face of the internal capsule, to stain descending connections reaching
that region from other parts of the hemisphere, as well as any
ascending axons that had already passed through the internal capsule.
Table 1 lists the number of brains (both hemispheres) used for the various studies at each developmental stage
(E13 to P8).
After crystal insertion, the brains were stored in fixative or in
PBS containing 0.1% Na azide, to prevent contamination, at room
temperature (22°C) or at 37°C. They were incubated for periods
ranging from 1 to 10 weeks, depending on age and temperature (Table
2), to allow diffusion of the dye. We
came to prefer using room temperature because of the slightly lower
background labeling. At the end of the incubation period the brains
were embedded in 5% agar, and coronal or horizontal sections, 75-250
µm thick, were cut on a vibratome (Oxford Instruments). All sections
were counterstained with bisbenzimide (10 min in 2.5 µg/ml solution in PBS; Riedel-De Haèn AG, Seelze-Hannover, Germany) or acridine orange (10 µg/ml in PBS; Molecular Probes). The sections were coverslipped under Hydromount (National Diagnostics) or
glycerol-phenylenediamine solution (0.1%
p-phenylenediamine, 10% PBS, and 90% glycerol, made up and
stored according to the method of Johnson et al., 1982).
For the three-dimensional serial reconstructions, we coverslipped the
sections under PBS, sealed the coverslips with Paraseal (Raymond A. Lamb, London, UK), and imaged the sections within 24 hr to
prevent differential shrinking and distortion of the individual members
of the series. From the serial sections, double- or triple-exposure
color micrographs were taken (Kodak Ektachrome 400 ASA color slide
film), and camera lucida drawings were made under
epifluorescent illumination, using different filters to reveal the
various dyes.
In early experiments, some sections from each brain (and from
other control brains not used for dye tracing) were subsequently stained with cresyl violet to help with identification of the major
divisions of the thalamus and to reveal the full thickness and
lamination pattern of the cortex. Counterstaining with chromatin stains
(bisbenzimide and acridine orange) helped us interpret the sections by
revealing the outlines and boundaries of obvious cell groups (e.g.,
cortical layer I and layers in the hippocampus). Observation of nuclear
chromatin staining with high-power objectives and/or obtaining fine
optical sections with confocal microscopy enabled us to confirm whether
labeled profiles were cell bodies.
Some sections, selected in conventional fluorescence microscopy, were
subsequently examined and photographed with a laser-scanning confocal
microscope (CLSM-Fluovert; Leica, Heidelberg, Germany). Digitized fine
optical sections (up to 64 sections) were stacked to generate
high-resolution extended focus images (see Fig. 10F). Stereo pairs (with ±7° disparity) could be constructed from these three-dimensional data sets (see Fig. 10G).
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RESULTS |
This study is based on a total of 285 axon-tracing experiments,
covering the period from E13, before the arrival of the first cells in
the cerebral cortex, to P8, when the organization and innervation of
the cortex appear virtually fully mature (see Table 1). For the sake of
clarity we present the results in five parts:
(1) We describe the timing of outgrowth of thalamocortical fibers,
their pattern of organization and initial topography, their arrival at
the cortex, and invasion of the cortical plate, based on anterograde
labeling from one or more dye crystals implanted in the dorsal
thalamus.
(2) The cells of origin of the earliest corticofugal projections were
labeled and examined after placement of crystals in the primitive
internal capsule before thalamic fibers approach the cortex.
(3) The timing of outgrowth and topography of these early corticofugal
projections is then described, on the basis of anterograde tracing from
dye crystals placed in the cortex, before the arrival of thalamic
axons.
(4) We then describe the relationship between early corticofugal and
thalamocortical fibers at their meeting point in the primitive
internal capsule, within the intermediate zone, and as the thalamic
axons approach the cortical subplate (from E14.5 to E16 for different
parts of the cortex). This is based partly on double labeling (with
different dyes placed in corresponding regions of thalamus and cortex)
and partly on the labeling of both sets of axons and cortical cell
bodies from crystals placed in the internal capsule.
(5) Finally, single- and multiple-crystal placements in the cortex,
shortly after the arrival of thalamic axons, provided further
information about the relationship between afferent and efferent axons
and about the global topography of the pathway established between
thalamus and cortex.
Development of thalamocortical projections
Thalamocortical fibers grow out in topographic order among cell
groups that project to the thalamus
Implantation of a small crystal of carbocyanine dye into the
dorsolateral part of the thalamus (including the presumptive LGN) on
E14 reveals anterogradely labeled thalamic fibers that have already
grown down through the diencephalon and are passing through the
primitive internal capsule. The example in Figure 1 is representative of all 14 experiments
performed at this age. The thalamic axons run under and through the
anlage of the developing corpus striatum to reach the intermediate zone
of the ventrolateral telencephalon at approximately E15.5 (see Fig.
3).
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Figure 1.
A small crystal of DiI was implanted into the
dorsolateral part of the left thalamus of an embryonic rat brain fixed
during the first half of E14. After 4 weeks incubation at room
temperature, 100-µm-thick coronal sections were cut, counterstained
with bisbenzimide, and examined by both fluorescence and laser-scanning
confocal microscopy. A, Bisbenzimide staining of a
coronal section shows the crystal implantation site (unfilled
arrow) in the putative dorsal LGN. The interrupted
line indicates the border between dorsal thalamus
(DT) and ventral thalamus
(VT). B, DiI labeling in the same
field as A. The labeled fiber bundle extends from the
dorsal thalamus to the ventral diencephalon. There are numerous
back-labeled cells within the thalamic reticular nucleus of the ventral
thalamus (filled arrow below the implantation
site, which is indicated by an unfilled arrow) and a few
above, in the epithalamus (filled arrow above).
The mass of axons descending from the dorsal thalamus is presumably a
mixture of the ascending axons of thalamic reticular cells and
orthogradely labeled thalamofugal fibers from the presumptive LGN.
C, High-power view of back-labeled thalamic reticular
cells within the region indicated by the lower filled
arrow in B. D, Two sections away
from A, 200 µm anterior, where geniculofugal labeling
reaches its full lateral extent, within the primitive internal capsule
(IC), beneath the medial ganglionic eminence. This
double exposure shows both bisbenzimide-stained cells and DiI-labeled
axons. E, F, Medium- and high-power views in the region
of the fiber endings reveal a small number of back-labeled cells within
the primitive internal capsule (filled arrows), a
region called the perireticular nucleus by Mitrofanis (1992).
G, An image from the same region of the internal capsule
as E, showing both chromatin (blue) and
DiI staining (green). Two stacked sets of three
2-µm-thick confocal sections, taken at identical optical planes but
with different filters, have been combined. Many cell bodies
(blue chromatin staining) are not back-labeled, but some
certainly are. Two examples (outlined) are shown in
higher power in H and I, which are single
confocal sections through the middle of the thickness of the cell
bodies, in which the blue chromatin is clearly visible,
surrounded by DiI staining. Scale bars: A, B, D, 500 µm; C, E, 100 µm; F-H, 50 µm.
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DiI implantation in the dorsal thalamus, even early on E14, also labels
numerous cell bodies in the ventral diencephalon (presumably in the
thalamic reticular nucleus; Mitrofanis and Baker, 1993) and a few
within the primitive internal capsule, lying below both medial and
lateral ganglionic eminences (Fig. 1E,F), in
the region that Mitrofanis (1992) calls the perireticular nucleus. Even
where they are close to the tips of growing thalamocortical axons, the back-labeled perireticular cells can be convincingly distinguished from
the surrounding growth cones by their chromatin content, revealed by
matching fine confocal optical sections collected with different
filters to distinguish the carbocyanine dye and the bisbenzimide and/or
acridine orange counterstaining (Fig. 1G,H,L). The
fact that the cells of the thalamic reticular and perireticular nuclei
are densely stained with carbocyanine dye (even after the shortest
incubation periods) and are intermingled with entirely unlabeled cells
makes us confident that they were labeled retrogradely along axons
already projecting into the dorsal thalamus at E14, rather than by
transcellular diffusion of dye from labeled thalamic axons.
We obtained some information about the topography of the initial
outgrowth of thalamic fibers by tracing the axons labeled by two or
more crystals of different dyes, placed in the dorsal thalamus. The
entire thalamus is so small at this stage that these placements
necessarily had to be very close together. However, such experiments
reveal that fiber bundles originating from different thalamic regions
remain separate as they descend in the ventral thalamus, enter the
internal capsule, and grow through and under the developing striatum
(Fig. 2). The location of the
back-labeled thalamic reticular and perireticular cells was also
clearly dependent on the location of the crystal placement within the
dorsal thalamus, indicating at least some degree of topographic order
in the projection of those two cell groups to the thalamus at very
early stages.
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Figure 2.
One crystal of DiI and one of DiA were inserted
about 200 µm apart (DiI more lateral) into the left dorsal thalamus
of two E15 brains. After 3 weeks incubation at room temperature, one of
the brains was sectioned coronally (100 µm thick)
(A-D), the other was sectioned horizontally
(E-H). The sections were counterstained with
bisbenzimide. Fluorescence micrographs were taken by exposing the film
three times to reveal the three different fluorescent signals. The
serial coronal sections are presented in caudorostral sequence
(A-D), the horizontal sections dorsoventrally
(E-H). A, Crystal placement sites
in dorsal thalamus. The dense cores, presumably corresponding to the
major uptake sites, are indicated by black-outlined
arrowheads (filled, DiA;
unfilled, DiI). They are surrounded by large,
overlapping halos of dye. B, C, The two
labeled bundles of thalamic fibers appear largely segregated as they
descend in the ventral thalamus (white arrows,
unfilled for DiI, filled for DiA). Groups
of neurons back-labeled with DiI and DiA are visible in the thalamic
reticular nucleus of the ventral thalamus, below the implantation sites
(especially in B). D, At this level,
rostal to C, the growing tips of the axons are seen
within the primitive internal capsule. Here the two bundles largely
overlap each other, but examination of neighboring sections shows that
they are still segregated in the parasagittal plane, those from the
more lateral thalamic site lying more caudal and heading for a more
posterior region of the hemisphere. E, F, A similarly
labeled brain was sectioned horizontally, to demonstrate the
rostrocaudal separation of the two bundles of labeled thalamic fibers
as they pass into the internal capsule (H)
and up toward the intermediate zone (G, F, E). The
fibers labeled with DiI (unfilled white arrows) from the
more lateral thalamic implantation (unfilled black arrow
in E) head to a more posterior region then those labeled
with DiA (filled arrows) from the more medial
crystal placement (filled black arrow in
E). Scale bar, 500 µm.
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The examples in Figure 2 show serial coronal sections (Fig.
2A-D) and horizontal (Fig.
2E-H) sections from two brains in which a
pair of different carbocyanine dye crystals were placed into the dorsal
thalamus, only ~200 µm apart in the coronal plane, at E15. The
diffuse halos of dye around the individual crystals are large and
overlapping, but it is likely that uptake of dye was largely restricted
to the dense cores (Fig. 2A,E). In coronal sections,
two differently colored bundles of axons are seen descending side-by-side through the ventral diencephalon (Fig.
2A-C). In the most rostral coronal section (Fig.
2D) the advancing wave front of thalamic fibers is
passing through the primitive internal capsule, beneath the anlage of
the striatum. Here, the two axon bundles overlap each other in the
coronal plane, but the series of horizontally cut sections from a
similarly labeled brain (Fig. 2E-H) shows
that they are still quite separate in that plane. Fibers from the more
medial thalamic site run rostral to those from the more lateral
implantation. This suggests that the array of fibers from this region
of the dorsal thalamus rotates by ~90° but maintains its
topographic order as it leaves the diencephalon. Additional evidence
for both the topography of the axon array and its rotation comes from
retrograde labeling of the same axons from the cortex ~1 d later (see
below).
Growth through the primitive internal capsule and
intermediate zone
Between E15 and E16, thalamocortical fibers pass through the
primitive internal capsule (Fig. 3). The
homogeneous, topographic array of axons leaving the diencephalon (Fig.
4C) parcellates into distinct
fascicles, which open up in a fan-shaped pattern (Fig.
4D), as they pass within the anlage of the corpus
striatum. The fibers do not appear to cross each other extensively at
any point along their course, although some individual axons and small axon bundles can be seen switching from one fascicle to another within
the striatal anlage (Fig. 4B). Viewed in coronal
sections, the ordering of fibers is such that the inferior lateral
fascicles are destined for the ventral part of the cortex, whereas the
superior medial fascicles turn upward and head toward more dorsal
cortical areas (Fig. 3G,K).
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Figure 3.
The advance of the thalamocortical fibers toward
the cortex is revealed with a crystal placement into the putative LGN
in the left dorsal thalamus (DT) of brains at E15
(top row, A-D), E16 (middle row,
E-H) and E19 (bottom row, I-L). After
2-6 weeks incubation at 37°C, 100 µm coronal sections were cut and
counterstained with bisbenzimide. For each row, the first two
panels (A, E, I, DiI staining; B, F,
J, bisbenzimide counterstain) demonstrate the thalamic crystal
implantation sites (unfilled arrows). Numerous cells are
labeled in the thalamic reticular nucleus (filled
arrows) of the ventral thalamus (VT). The
right pairs of panels show the tips of
the thalamic fibers at a more rostral level (C, G, K,
DiI; D, H, L, bisbenzimide). The thalamic fibers are
growing into the intermediate zone of the ventral telencephalon at E15.
By E16, thalamic fibers have extended up into the convexity of the
occipital cortex and reached the subplate layer. Even by E19
(K) there is very little advance into the
cortical plate itself. Note that no cells of the cortex are
back-labeled from the dorsal thalamus at these stages. Higher-power
photomicrographs of the regions indicated by white outline
boxes in G and K are presented in
Figure 5. LV, Lateral ventricle; ST,
anlage of the corpus striatum; IC, primitive internal
capsule, demarcated by unfilled arrows in
D. Scale bar, 500 µm for all panels.
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Figure 4.
Thalamocortical axons show distinct patterns of
organization at different points along their path to the cortex.
Crystal placement into the left dorsal thalamus labels fibers that
reach the intermediate zone beneath the ventrolateral cortex at E15.5
(0.5 d before they reach the occipital pole itself).
A, Leaving the diencephalon, the fibers form fascicles,
which open up in a fan-shaped manner. Viewed here in a coronal section,
the order of the fibers is such that the inferior-lateral fascicles
are destined for the more ventrolateral part of the cortex, whereas the
superior-medial fascicles turn upward and head toward more dorsal
cortical areas. The trajectories of these fascicles remain
approximately parallel as they turn up into the intermediate zone; they
do not cross each other to any significant degree. B,
Higher-power view of the region where the fascicles break up into
(Figure legend continues)individual fibers as they enter the intermediate zone. To
examine the topography of fibers in more detail, the contralateral
hemisphere (with an identical crystal placement to that in
A) was cut perpendicular to the fiber path. C, D,
E, Sections at levels corresponding to the labeled
arrows in A. C, The fibers leave
the diencephalon through the primitive internal capsule. The bundle is
at its narrowest at this point. No fasciculation is apparent in this
region. D, As the fibers run under the anlage of the
corpus striatum, they form 30- to 50-µm-thick fascicles, slightly
separated from each other. The labeled fiber array loosens up and
expands slightly (compare with C). E, The
fiber bundles defasciculate as they reach the intermediate zone and
turn under the cortical plate. The right side of the section still
shows some fascicles, whereas the left demonstrates defasciculated
fibers running approximately parallel to each other in the intermediate
zone. Scale bars: A, 300 µm; B, 50 µm; C-E, 100 µm.
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At the border between the corpus striatum and the intermediate zone the
thalamic fibers defasciculate and form a fairly uniform array of
individual fibers (Fig. 4E). The trajectories of the axons
remain more or less parallel as they turn up into the intermediate zone. Fibers from the dorsolateral thalamus (primordial LGN) are advancing through the intermediate zone beneath the ventrolateral cortex at approximately E15.5, and they reach the subplate layer of the
occipital cortex itself at E16 (Figs. 3-5 are examples representative of all experiments; see Table 1). As with the fascicles from which they
emerge, the ordering of the individual fibers is such that the more
inferior and lateral ones are destined for the more ventral cortical
segment, whereas the more medial axons, situated deeper in the white
matter, head toward more dorsal cortical segments (Figs. 3,
5). The entire, ordered array of thalamic
fibers fills the full depth of the intermediate zone, from the cortical
subplate down to the subventricular zone (Fig.
3G,H).
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Figure 5.
Three stages of thalamic fiber growth in the
cortical target region of the left hemisphere, revealed by crystal
placement in the dorsal thalamus: E16 (top
row), E19 (middle row), and
P2 (bottom row). Left
panels, DiI labeling; right panels, bisbenzimide
labeling of the same section. The examples shown for E16
and E19 are higher-power fluorescence photomicrographs
from Figure 3, G and H and
K and L, respectively. The bottom
row demonstrates fiber labeling in the cortex of a P2 animal
after a similar crystal placement. Thalamic fibers reach the occipital
cortex at E16 but remain mainly restricted to the
subplate (SP) and intermediate zone (IZ).
At E19 some fibers have developed side branches, but the
vast majority still lie within the SP and have not yet entered the
cortical plate (CP). By P2, thalamic
axons have turned up into the cortical plate en masse. They take
irregular courses through layers 6 and 5, and the majority branch and
terminate in presumptive layer 4, ~300 µm below the pial surface,
directly underneath the dense cortical plate (DCP),
which consists of newly arrived, densely packed immature neurons. A few
axons are seen extending up to the marginal zone (MZ).
Note that, with discrete crystal placements restricted to the
dorsolateral thalamus, very few if any cell bodies are back-labeled in
the cortical plate or the subplate, even at E19 and
P2. Scale bar, 100 µm for all panels.
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Accumulation of thalamic fibers below the cortex
Although axons from the region of the LGN reach the
occipital cortex at approximately E16, they do not substantially invade the cortical plate for some time. Most of the labeled fibers accumulate in and remain restricted to the intermediate and subplate zones until
E19. During this period, thalamic fibers develop increasing numbers of
local branches along their length within the subplate layer. These
branches are nearly all directed upward toward the cortical plate, but
most are short and restricted to the subplate region, at least in the
occipital cortex. A few penetrate the lowest part of the cortical plate
(Fig. 5, E19), and very rarely some are seen reaching close
to the lower boundary of the dense cortical platethe tightly packed,
recently arrived, immature neurons that form the upper part of the
cortical plate.
The generation of cortical plate neurons in the ventricular and
subventricular zones is at its peak when thalamic axons arrive (Miller,
1988). Migrating cells therefore have to pass through the mass of
fibers accumulating in the subplate layer, as well as between the
subplate cells themselves.
Invasion of the cortex
Before approximately E19-E20, very few thalamic axons appear to
have grown into the cortical plate itself in the occipital region (Fig.
5). Invasion on a massive scale suddenly begins at approximately
E19-E20, just a couple of days before birth. The initial course taken
by these invading fibers is distinctive and highly ordered; most make
virtually a 90° turn from their trajectory in the white matter, and
the vast majority initially grow radially, straight up into the cortex.
A small proportion take more erratic initial routes, running somewhat
obliquely upwards (Fig. 6). The ingrowth
could partly originate from the side branches of thalamic axons, some
of which penetrate the cortical plate at earlier stages.
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Figure 6.
Photomicrographs and camera lucida drawings of
thalamic fibers entering the right occipital cortex of a P2 rat labeled
from a DiI crystal placed in the dorsal thalamus. A, The
thalamic fibers form a remarkably parallel array until they enter the
cortical plate, where they diverge and follow more irregular patterns,
arborizing as they ascend to the middle layers. The fibers projecting
to more lateral areas (right side) run superficial to
those destined for more dorsal areas. The more ventrolateral cortex is
innervated earlier, and the topographic ordering of fibers is reflected
in the chronological sequence of their arrival under the
cerebral cortex. B, The photomicrograph was taken
from the same section as A but focusing on a single
arbor extending over a large area, some 200 µm below the pial
surface. To illustrate the variety of individual thalamic fiber arbors
in the occipital cortex, just after they have entered the cortex,
camera lucida drawings were made of individual arbors seen in
200-µm-thick coronal sections of P2 cortex. In each drawing the
top continuous line marks the pial surface, whereas the
interrupted line below shows the upper boundary of the
white matter. It is quite likely that many individual arborizations
extended outside the thickness of the 200 µm section, because some
arbors extend as far as 500 µm within the plane of the section. Scale
bars: A, B, 200 µm; camera lucida drawings, 500 µm.
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In favorable preparations, at all stages from E15 onward, individual
thalamic axons can be seen forming an impressively ordered, parallel
array running up through the intermediate zone and into the subplate
layer below the cortex (Figs. 3G, 5, 6A).
Fibers heading for more ventral and lateral cortex run superficial to those destined for more dorsal segments. Because the earlier axons enter ventral cortical areas first, and these areas develop relatively early, the ordering of thalamic fibers mirrors the temporal order of
maturation of their cortical target areas.
Establishment of laminar termination patterns
By birth (P0, equivalent to E21-E22) and afterward, the
trajectories of thalamic fibers within the cortical plate appear far less regular and parallel than in the white matter (Figs. 5,
6A). Some individual thalamic axons have branches in
layer 6 of the cortical plate and may terminate there; but most of them
branch and appear to terminate above in putative layer 5 and especially in layer 4. By approximately P2, most axons have bifurcated at least
once near the top of layer 5 and have arborized quite widely in the
lamina that is differentiating into layer 4, directly below the dense
cortical plate, which is itself reducing in thickness as the upper
layers mature. Many axons now appear to have lost their growth cones. A
few fibers continue to grow all the way to the marginal zone, but the
vast majority terminate within a fairly narrow laminar range and avoid
the uppermost sector of the cortex.
Individual labeled axons were followed in 200-µm-thick sections and
drawn via a camera lucida (Fig. 6). At P2 their arborizations in
presumptive layer 4 typically extend over an area ~300 µm wide. This wide lateral spread of the terminal arbors of thalamic axons contrasts with the remarkable order with which they are arranged at the
junction between white and gray matter (Figs. 5, 6A).
Between P2 and approximately P8 (when the cortex has achieved its
mature lamination), layer 4 and the arborizations of thalamic afferents within it appear to be progressively displaced downward as the supragranular layers thicken.
Tracing from the internal capsule
Through the entire period of outgrowth of thalamocortical fibers,
their accumulation below the cortex and the initial invasion of the
cortical plate, small crystal placements in the thalamus itself
back-labeled very few cortical cells, even within the subplate, and
none was ever labeled from crystals restricted to the dorsal thalamus
(Figs. 3-6). Clasca et al. (1995) described similar findings in the
ferret; they suggested that early corticofugal projections from the
subplate and layer 6 are delayed for several weeks in the cerebral
white matter, and that axons from layer 5 are the first to pass through
the internal capsule into the diencephalon. We used small crystal
placements within the internal capsule itself to determine when
corticofugal projections reach the internal capsule in the rat and
which cortical neurons project through it at early stages.
At E14 a small crystal implanted into the primitive internal capsule
back-labels numerous cell bodies and their processes within the
presumptive perirhinal and lateral cerebral cortex. It also labels a
segment of the striatal radial glia above and below the implantation
site and some adjacent neurons within the internal capsule itself.
Figure 7 demonstrates two representative examples from six experiments. Many of the back-labeled cells within
the putative perirhinal cortex have a pyramidal shape and differ from
the typical polymorphous, polygonal morphology of the labeled preplate
cells in the lateral and dorsal cortex (Fig. 7, compare D,
E, respectively). Even by E14-E14.5, in the more mature lateral
segment of the cortex this population of first-generated cells is
already being split into marginal zone and subplate by newly arrived
migrating neurons of the emerging cortical plate (Fig. 7E).
Interestingly, back-labeled cells are seen in both subplate and
marginal zone of the lateral cortex at E14.5, indicating that some
neurons from throughout the depth of the original preplate have axons
in the primitive internal capsule at this age. Occasionally we observed
a few back-labeled cells within the cortical plate itself, at least
some of them with pyramidal morphology, but the vast majority are in
the preplate or its derivatives (Fig. 7).
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Figure 7.
Results of implanting a DiI crystal into the
primitive internal capsule on the left side at E14 (A, B,
D) and E14.5 (C, E), showing bisbenzimide
(A) and DiI (B-E)
labeling. A, B, Matched images show the crystal
placement (arrows) into the primitive internal capsule
(IC). Numerous presumed radial glia are stained in the
striatal anlage (ST), and back-labeled cells are
seen in perirhinal cortex and adjacent lateral neocortex. A large group
of cells was also labeled within the primitive internal capsule itself
(data not shown). C, DiI labeling in a matched
section from a similar experiment in an E14.5 fetus. Neurons are now
also back-labeled further dorsally in the cerebral wall.
D, A high-power view of the lateral part of
B (indicated by the outline box)
demonstrates back-labeled neurons, lying mainly at the base of the
cortical plate in presumptive perirhinal cortex. Note that some of the
cells in perirhinal cortex are pyramidal in form, with processes
extending up toward the pial surface. Their morphology is quite
different from the polygonal and polymorphic (presumed preplate) cells
labeled in the lateral cortex (seen in C, E).
E, Segment of cortex containing back-labeled cells
(viewed with higher magnification from the outline box indicated in
C). The majority of back-labeled cells in lateral
neocortex are situated in the subplate and marginal zones, but there
are also a few within the emerging cortical plate. CTX,
Cortex; MZ, marginal zone; CP, cortical
plate; SP, subplate. Scale bars: A-C,
500 µm; D, E, 100 µm.
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At E14.5, crystals placed in the internal capsule back-label cells only
in the most mature, lateral neocortical segments. At later ages the
territory of back-labeled cortical cells expands dorsally, indicating
that axons from the preplate of more dorsal areas reach the internal
capsule progressively later than those from more ventral regions.
Carbocyanine crystals implanted into the internal capsule after E16
back-label cells in the subplate but, interestingly, very few if any in
the marginal zone, implying that the cells of layer 1 that originally
projected to the internal capsule have either died or withdrawn their
axons. At this stage cortical lamination is more advanced. Back-labeled
pyramidal neurons are seen in both layers 5 and 6, showing that
corticofugal projection neurons in all these layers have now reached
the internal capsule. Because cells in the cortex can be labeled before
birth from the internal capsule but rarely if at all from the dorsal
thalamus, we suspect that axons of subplate cells and even those from
true corticothalamic neurons of layer 6 slow down or stop somewhere in
the ventrolateral part of the diencephalon, medial to the primitive
internal capsule, and do not enter the body of the dorsal thalamus
prenatally.
Anterograde labeling of the earliest corticofugal projections
E14-E15: establishment of the preplate "scaffold"
The exact appearance of the cerebral wall (and the state of axon
outgrowth, as revealed by crystal placements in the cortical surface)
varies with position across the hemisphere, at any particular age, in
line with the rostrolateral-to-caudomedial gradient of cortical
development (Bayer and Altman, 1991). The rostrolateral cortex is
~1.5 d ahead of the medial occipital cortex in its state of maturity
until several days after birth. Unless otherwise stated, the following
descriptions apply to crystal placements in the occipital cortex
(putative area 17).
Crystal implantation in the surface of the occipital cortex early on
E14 results in staining (to varying degrees) of the pial surface of the
cortex and of a population of radial processes extending from the
ventricular surface to the pial surface, with labeled bipolar,
nucleated cell bodies in the lower half of the cerebral wall (Fig.
8B). The radial
processes are presumably those of radial glia, and the somata probably
include both progenitor cells of the ventricular zone and postmitotic,
immature neurons, migrating toward the pial surface. In the most dorsal
areas of cortex the preplate has apparently not yet begun to form; no
cell bodies are present close to the pial surface. Nor are any
outgrowing axons seen at this early stage.
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Figure 8.
Outgrowth of the first corticofugal projections.
The photomicrographs were taken from the regions indicated in the
schematic drawing on the left: A and
B at E13; C at E13.5; and
D-F at E14.5. A, High-power
photomicrograph taken from a 10-µm-thick, cresyl-violet-stained
coronal section of the occipital cortex of an E13 embryo. At this early
stage the wall of the telencephalic vesicle consists of proliferative
ventricular zone (VZ) and subventricular zone
(SVZ), with a thin layer of postmitotic cells beginning
to accumulate to form the primordial plexiform zone or preplate
(PP). B, Coronal section of the
cerebral wall at E13 (slightly less mature than A),
stained with a small crystal of DiI inserted into the cortex nearby,
leading to intense local labeling of a few radial processes, presumably
of glia and/or germinal cells. At this very early stage, no cell bodies
or any axons are seen at the outer edge of the cerebral wall, but a
little later (C-E), a dense plexus of axons
grows out from the preplate. C, A crystal of DiI
inserted, at the point indicated by an asterisk, in the
convexity of the left hemisphere at E13.5 reveals the very first axons
(arrows) arising from the thin layer of postmitotic
cells forming the preplate (PP), between the heavily
stained pial surface and the subventricular zone. Radial processes are
seen, out of the plane of focus, below the placement site.
D, An E14.5 brain with a similar cortical DiI crystal
placement (asterisk) reveals early corticofugal axons
descending and turning medially into the primitive internal capsule
(PIC). Individual cells and their processes are
difficult to resolve by ordinary fluorescence microscopy because of the
intensity of labeling of the somata and the dense plexus of fibers.
E, Higher magnification of D. In dorsal
regions, where the preplate is very thin, fibers are seen coursing
among the early postmitotic cells. When the preplate is split by the
emerging cortical plate (first in more ventral regions), most
descending fibers are seen to belong to cells of the lower, subplate
zone, but some originate from neurons of the marginal zone. When the
crystal placement site is in the most ventral region of neocortex or in
the perirhinal cortex, occasional back-labeled cells are seen in the
primitive internal capsule, within the bundle of labeled axons. This
suggests that fibers originating from more dorsal cortex are purely
corticofugal, whereas those from perirhinal cortex consist of both
anterogradely labeled corticofugal fibers and retrogradely labeled
axons of "perireticular" cells lying within the primitive internal
capsule. F, In higher power, the labeled fiber bundle
from occipital cortex at E14.5 is seen entering the primitive internal
capsule. These fibers end in large and elaborate growth cones. Scale
bars: A-C, F, 50 µm; D, 350 µm;
E, 100 µm.
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During the 14th embryonic day, early-born cells begin to
accumulate below the surface of the occipital cerebral wall to form the
preplate. Crystal placement in the cortex at successively later times
on E14 produces progressively more intense superficial staining,
presumably concentrated in the membranes of these preplate cells. The
first axons are seen growing out from these cells at mid-E14 (Fig.
8C), and they appear, virtually without exception, to be
directed ventrally in the intermediate zone, heading toward the basal
telencephalon and the primitive internal capsule, which they begin to
enter during the second half of E14 (Fig. 8D).
Wherever the crystal is placed, a substantial population of axons is
labeled, and they form a fairly tight bundle of fibers running within
and then directly beneath the subplate zone.
All the axons at the advancing front of the stained bundle end in
expanded structures, which are indubitably growth cones rather than
cell bodies, because chromatin staining reveals no nuclei within them.
These growth cones exhibit complex filopodial morphology and are
exceptionally large (~10 µm across) as they descend in the nascent
intermediate zone (Fig. 8F). The bundle of axons
originating at any particular point in the cerebral wall maintains its
coherence quite tightly and follows a direct, smoothly curving path
toward the primitive internal capsule. As they approach the
perireticular cells below the lateral ganglionic eminence, the growing
tips of the axons are somewhat more dispersed than the diameter of the
bundle behind them, perhaps implying that they are entering territory
with different properties.
Crystal placements in the occipital cortex on E14 never resulted in the
back-labeling of thalamic cells, indicating that no thalamic fibers
have reached the site of crystal placement at that age, in agreement
with the results of anterograde labeling from the thalamus described
above.
Topography of the scaffold
Examination of brains after multiple dye placements, in
parasagittal or coronal rows across the cortical hemisphere, clearly demonstrated the temporal order of descending projections from the
preplate, matching the gradient of maturation of cortical lamination
(Bayer and Altman, 1991). Along each row, the more anterior and lateral
the site, the more advanced the fiber outgrowth. These multiple
injections also revealed the quite precise topographical organization
of these early corticofugal projections; each individual crystal
labeled a group of axons, forming a discrete, fairly tight bundle, and
the spatial arrangement of the separate labeled bundles was maintained
throughout their path.
Along the rostrocaudal axis, at these early stages, axons follow
straight or slightly curved trajectories toward the primitive internal
capsule and, hence, form a fan-shaped array converging on that region,
maintaining topography correlated with the spatial separation of their
sites of origin.
Topography is also preserved along the coronal axis (Fig.
9), despite the fact that all the fibers
from any mediolateral strip of cortex share the same narrow portion of
the intermediate zone. Figure 9, B and C, shows
the separate, approximately parallel trajectories of the bundles of
descending axons labeled by a coronal row of three crystals. At any
point in the intermediate zone, the descending axons derived from more
dorsal (later-maturing) parts of the hemisphere lie deep to those from
more lateral (earlier-maturing) regions; the full array appears to
extend through the entire thickness of the intermediate zone. Fiber
order is, then, correlated with the time of initial outgrowth. This
implies that, as new fibers grow down from progressively more dorsal
regions, they take their place immediately adjacent and deep to the
array of preexisting fibers, and that this order is preserved
throughout the pathway. Thus the array of preplate axons approaching
the ventral telencephalon forms a three-dimensional representation of
the entire surface of the hemisphere.
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Figure 9.
Carbocyanine crystal placements in the cortex at
E15 revealed quite strict topographic order in the corticofugal
projection within the intermediate zone and primitive internal capsule.
A, Coronal, bisbenzimide-counterstained section from the
left hemisphere of an E15 brain. A single DiI crystal (unfilled
star) labeled a discrete bundle of early corticofugal axons
descending through the middle of the intermediate zone toward the
primitive internal capsule (PIC), as well as diffusely
staining the cortical surface and labeling a column of radial
processes, mainly radial glia. B, Three carbocyanine
crystals, two DiI (unfilled stars), with a single DiO in
between (filled star in the
middle), were placed along a coronal line in the
cortical convexity of an E15 brain. Labeled fibers emanate from each
crystal, descending through the intermediate zone, the differentially
labeled fibers remaining separate and ordered along their path. Fibers
labeled from the more lateral cortical crystal placement sites are more
superficial in the intermediate zone and more advanced in their growth
compared with those from dorsal sites, indicating a spatial and
temporal ordering of outgrowth, in concordance with the
ventral-to-dorsal maturation gradient across the cortex.
C, Higher-power photomicrograph (taken from the region
of the more ventral intermediate zone indicated by the white
outline in B) showing mainly fibers from the
most lateral of the three crystals. A few of the deepest axons are
stained with DiO from the middle crystal. Note the strict ordering of
axons, those from more dorsal sites lying deep to those from more
lateral cortex. Scale bars: A, B, 500 µm;
C, 100 µm.
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Evidence that thalamic fibers grow over the scaffold of preplate
axons within the internal capsule and intermediate zone
Combined double labeling of cortical and thalamic axons
By late on E14, both advancing axon fronts, from occipital
preplate and dorsolateral thalamus, reach a region in the basal telencephalon, at the lateral edge of the primitive internal capsule. The outgrowths of the two corresponding sets of axons, corticopetal and
corticofugal, are remarkably synchronized and coordinated in their
timing and spatial patterns.
Following the suggestion of McConnell et al. (1989) that the preplate
pioneer fibers might play a part in subsequent axon guidance, we
proposed that, after thalamic axons reach the internal capsule, they
complete their navigation to the appropriate cortical region by growing
over fibers that have descended from their target areathe
"handshake" hypothesis (Blakemore and Molnár, 1990; Molnár and Blakemore, 1995). To examine this notion, we attempted to label corresponding regions of dorsal thalamus and occipital cortex
with different dyes to see whether the two fiber systems do literally
contact each other, intermingle, and appear topographicaly aligned
within the intermediate zone. Results from such experiments performed
at mid-E16 (Table 1) were rejected when we discovered that cell bodies
in the thalamus were retrogradely labeled with the dye placed in the
cortex. This was to be expected from the results of the thalamic
tracing experiments described above, which showed that thalamic axons
reach the occipital cortex at that age (Fig. 3). At even later stages,
the picture becomes further complicated by the outgrowth of fibers from
layers 6 and 5 of the true cortical plate (De Carlos and O'Leary,
1992; Miller et al., 1993). Our analysis with this double-labeling
technique was therefore restricted to the period from E14.5 to very
early E16.
The fundamental problem with this approach is that the exact
correspondence between developing thalamic and cortical loci cannot be
established empirically until thalamic fibers have actually reached
their target areas in the cortex, but after that point it is obviously
very difficult reliably to label fibers arising from cells in that
exact target region separately from the incoming thalamic axons. The
handshake hypothesis demands that thalamic axons should
intermingle only with axons from the corresponding region of the
cortex; therefore, any misalignment of dye placements in the two
structures would be expected to reveal spatially separate groups of
labeled fibers. Indeed, in 11 of 26 (42%) attempts to label matched
cortical and thalamic sites with small crystal placements, from E14.5
to E15.5, the bundles of differently labeled descending and ascending
axons lay close to each other within the intermediate zone but did not
overlap extensively, just as in the results of Miller et al. (1993).
However, because of the inherent difficulty in defining correspondence,
negative results in such experiments are to be expected; even a small
fraction of positive results would be definitive in demonstrating that
the two axon systems grow through the same compartment within the
intermediate zone.
In fact, in the majority of cases (5 of 8 at E14.5, 4 of 10 at E15, and
6 of 8 at E15.5) there was definite intermixing of a substantial
fraction of the bundles of labeled preplate and thalamic axons in the
ventral telencephalon and intermediate zone (Fig.
10 shows several representative
positive examples). This was also true in one case at early E16 in
which the labeled thalamic axons had not quite reached their target
area, and no cells back-labeled by the cortical dye were seen in the
thalamus. In several of these positive cases, the overlap of thalamic
and corticofugal axons was extensive.
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Figure 10.
Evidence that thalamic and early corticofugal
axons confront each other in the lateral internal capsule and share the
same growth compartment within the intermediate zone (the handshake
hypothesis; see Molnár and Blakemore, 1995). Shown are examples
of the intermixing of differentially labeled early corticofugal and
thalamocortical projections from four animals: at E15
(C), E15.5 (A, B, E, F),
and very early E16 (D). Crystals of two
distinguishable carbocyanine dyes were placed, one into the dorsal
thalamus and the other into the cerebral cortex of the same hemisphere,
at or shortly after the time when cortical and thalamic efferents are
converging at the lateral edge of the internal capsule. At these early
stages the labeled thalamic fibers did not extend all the way to the
cortex, and no back-labeled thalamic cells were seen in any of these
specimens (Figure legend continues), allowing the unambiguous identification of both sets of
fibers from their pure anterograde labeling. Coronal (A, B,
D-F) or horizontal (C) sections
through the region of overlap in the primitive internal capsule were
examined to study the relationship between the developing thalamic
projection and the earliest descending corticofugal axons.
A, In an E15.5 animal, a crystal of DiI was placed in
the dorsolateral thalamus (the putative LGN), and a DiO crystal was
placed into the presumed matching region of the occipital cortex
(putative area 17; crystal placement indicated with an
asterisk), so that both thalamocortical and preplate
fibers were independently labeled. In this coronal section, the leading
fronts of the DiO-labeled (green) preplate fibers
and the DiI-labeled (orange-red) thalamic axons appear
to be aligned and starting to intermingle at the point indicated by the
arrow. B, High-power view of the region
indicated by the arrow in A. The
fluorescence photomicrograph demonstrates that the DiI-labeled thalamic
fibers (orange-red) are indeed intimately associated
with the DiO-labeled corticofugal axons (green)
in the same plane of focus. C, An E15 brain was
sectioned horizontally and imaged perpendicular to the trajectories of
the thalamic fibers (labeled with DiI, appearing red),
which have passed under the developing striatum and turned up into the
ventral intermediate zone. At the same time the earliest corticofugal
fibers (labeled with DiA, appearing green) have run down
through the intermediate zone and turned medially, under and through
the developing corpus striatum. Individual thalamocortical and early
corticofugal fibers are seen in intimate contact
(arrows) as they run in opposite directions. In some
cases the two types of fibers are so close that the green and red
fluorescence is optically fused to form yellow.
D, A slightly older example, early E16 (but before
arrival of thalamic fibers at the cortical crystal site in this case),
sectioned coronally, in which the two differently colored fibers
systems are mixed within the same fascicles (arrow),
crossing the developing striatum. E, This example at
E15.5 shows thalamic fibers slightly more advanced in their growth into
the intermediate zone than in A, but still no thalamic
fibers had reached the cortical crystal placement site. The entire
depth of the array of DiO-labeled (green)
corticofugal fibers is closely associated with thalamic axons, labeled
with DiI (red), in the same plane of focus.
F, To examine more precisely the proximity and
three-dimensional relationships of thalamocortical and early
corticofugal fibers within these bundles, a region of the section shown
in E (marked with an arrow) was examined
by confocal microscopy. Thalamic fibers appear red;
corticofugal fibers appear green. This extended focus
image, reconstructed from 32 individual 1-µm-thick optical sections,
demonstrates beyond doubt that individual fibers running in opposite
direction are intimately associated within the same fascicles. Red and
green fibers were seen side by side in many of the individual 1 µm
sections. G, This stereo pair (±7° disparity) was
prepared from the three-dimensional data set of F. It
can be fused by voluntary divergence of the eyes or by viewing with
appropriate prisms or a stereoscope. Red and
green axons are seen closely approximated throughout the
depth of the section. Scale bars: A, 250 µm; B,
E, 100 µm; C, D, 50 µm; F, 10 µm.
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In these 16 successful double-labeling experiments, conventional
fluorescence microscopy showed the two fiber arrays to be mixed
together within individual 100-µm-thick sections, with differently colored fibers lying side-by-side in the same plane of focus. This was
also the case in the thin optical sectioning afforded by confocal
microscopy. For example, individual red and green fibers were seen
close together in many of the 32 individual 1-µm-thick sections that
were stacked to create the extended focus image of Figure
10F. The stereo pair derived from this data set (Fig. 10G) clearly shows corticofugal and thalamic fibers
co-resident in single, bidirectional fascicles.
Cross-sectioning of the mixed bundles, in the intermediate zone lateral
to the striatal anlage, confirmed the very close association of the two
populations, with individual descending and ascending axons often lying
side-by-side, literally in contact. In Figure 10C, for
instance, there is a considerable area of overlap, with some red
(thalamic) and green (corticofugal) axons so close together that they
appear optically fused as yellow.
Labeling of cortical neurons and thalamic axons as they
approach the subplate
Between E15 and E16, a dye crystal in the primitive internal
capsule labels not only early corticofugal axons and their cell bodies
(retrogradely) but also thalamic axons growing toward the cortex
(anterogradely). In 42 such cases we examined the relationship between
ascending and descending axons that pass through the same region of the
internal capsule, especially in brains that were cut in a plane
(near-coronal) chosen to be parallel to the axon arrays close to the
labeled region of cortex.
Figure 11 shows results from an E15.5
brain in which a crystal of DiI had been placed in the medial part of
the primitive internal capsule. At the rostrocaudal level of the
crystal site, an ordered array of intensely stained, thick thalamic
axons, identified by the growth cones at their tips, is advancing into
the basal part of the intermediate zone. A number of thinner axons,
intermingled with this mass of thalamic fibers, extend all the way back
to the cortex, some of them unequivocally identified as corticofugal by
being followed back, in serial confocal sections, to labeled cell
bodies in the subplate (Fig. 11C). Further rostral, where the entire pattern of growth is more advanced, a dense array of thin
axons without growth cones leads back to a population of back-labeled
cells in the cortical subplate and some in the lowest part of the
cortical plate. Many of these fine, labeled axons can be traced back
individually to their cells of origin. Among this ordered descending
array of corticofugal axons, thicker axons, presumed to be thalamic,
because many of them end in definite growth cones within the section,
are seen growing up toward the cortex, running parallel to and in
intimate association with corticofugal fibers (Fig.
11D). There is no tendency for the two types of axons to separate from each other, even as the thalamic axons enter the
subplate layer.
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Figure 11.
To examine the relationship between early
corticofugal axons and thalamocortical afferents as the latter approach
the target region of the cortex, a single DiI crystal was placed in the
internal capsule of an E15.5 brain. 200-µm-thick sections were cut in
a vertical plane, angled 45° forward from the coronal plane,
approximately parallel to the axon pathway between the internal capsule
and the cortex in the middle of the hemisphere. Confocal microscopic
reconstructions from these specimens reveal that thalamic fibers
(labeled anterogradely) and corresponding early corticofugal efferents
(labeled retrogradely from the same dye crystal) occupy the same region
of the intermediate zone, and that the thalamic fibers grow among the
corticofugal axons right up to the target region. A,
Low-power view of a section through the crystal placement site
(asterisk) stained with acridine orange.
B, Fluorescent micrograph of the same view as in
A reveals the mass of DiI-labeled fibers extending from
the primitive internal capsule (PIC) into the
intermediate zone and up toward the lateral wall of the telencephalon.
C, Higher-power confocal microscopic reconstruction of
the outlined area in B from the
intermediate zone under the lateral sector of the cortex. This extended
focus projection reveals anterogradely labeled thalamocortical fibers
as well as retrogradely labeled corticofugal axons and their cell
bodies. The thalamocortical fibers, many of which were unequivocally
identified by following them to growth cones at their tips (examples
are marked with unfilled arrows), form a broad array as
they enter the intermediate zone; note that they appear relatively
thick and intensely stained. Mixed with these thalamic axons in the
basal telencephalon is another array of much finer axons, hardly
visible at this magnification, extending all the way up to the cortical
subplate (filled arrows), many of which were
definitively identifiable as corticofugal because they could be traced
back to labeled cell bodies in the cortical subplate or occasionally in
the very lowest part of the cortical plate (filled
arrowheads). D, Higher-resolution confocal
reconstruction from directly below the lateral cortex in a slightly
more anterior section, where thalamic axons have advanced further
toward the cortical subplate. A large number of neurons
(filled arrowheads) are back-labeled in the
subplate and in the lowest part of the cortical plate itself, some even
with pyramidal morphology. The most advanced of the thalamic fibers are
approaching this region, ending in large growth cones (open
arrows), just 100 µm below the cortex. These thalamic fibers
are running parallel to and in close association with finer,
retrogradely labeled early corticofugal projections
(filled arrows), many of which can be traced back
to their labeled somata. This intimate mixture of thalamocortical and
corticofugal axons runs as a broad swathe of parallel fibers through
the intermediate zone and into the subplate, with no obvious
segregation into two separate compartments. E, Acridine
orange counterstaining of D demonstrates cell layering
in the cerebral wall. Scale bars: A, B, 500 µm;
C, 100 µm; D, E, 50 µm.
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Figure 12 is an enlarged view of
the region of the confocal image of Figure 11D, in
which the outlines of several identified corticofugal axons have been
filled in black, together with their cells of origin, whereas the
outlines of some of the definite thalamic fibers, identified by their
growth cones, are shaded white. The two types of identified axons run
parallel, in close association, filling the same compartment of the
intermediate zone, as the thalamic fibers approach within 100 µm of
the cortex.
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Figure 12.
The trajectories of individual thalamic and
cortical axons in the region shown in Figure 10D
were separately traced within the three-dimensional data set. Two masks
were constructed by marking the suprathreshold pixels of seven
corticofugal axons, traced to somata in the subplate or lower cortical
plate (black), and of five thalamic axons, with
identified terminal growth cones (white). These profiles
were finally projected and merged with the original data set. In one
case, a cortical axon and a thalamic axon, identified by their
emergence from a cell body and terminal growth cone, respectively, were
so closely juxtaposed that they could not be separately resolved
(interrupted line). Scale bar, 50 µm.
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Shared topography of early corticofugal and
thalamocortical projections
Although exactly corresponding thalamic and preplate
axons cannot reliably be separately labeled after thalamic fibers have reached the subplate layer, further support for the handshake hypothesis can be drawn from experiments involving crystal placement in
the cortex alone at slightly later ages. Fibers from the putative LGN
reach the subplate of the occipital pole of the cortex at E16 (Fig.
3G is representative for all experiments; n = 8). In accordance with this, a single dye crystal in the occipital
cortex at E16 leads to the labeling not only of fibers, extending
through the telencephalon and diencephalon, linking the two structures (Fig. 11C), but also of cell bodies in the LGN itself
(especially clear after relatively long incubation periods). Therefore
the fibers labeled from any such single site in the cortex must be a
mixed population of anterogradely labeled, corticofugal
axons and retrogradely labeled, thalamocortical fibers. And, by
definition, they must be spatially corresponding. If these two sets of
axons were to take very different routes through the telencephalon, any
single crystal should stain two or more distinctly separate bundles.
We therefore examined the axon arrays labeled by small dye placements
at various points on the cortex, at ages from E16 onward, following the
axons in serial coronal or horizontal sections with conventional
fluorescence and confocal microscopy. As well as viewing these serial
sections, we made three-dimensional reconstructions from thin confocal
optical sections at various magnifications to reveal more of the fine
and gross topography.
Unfortunately, even very small crystal placements lead to a wide halo
of label around the uptake site itself, especially after the relatively
long incubation periods that we used to obtain good retrograde labeling
of thalamic cell bodies. This halo consists partly of intrinsic cell
and fiber staining in the cortex itself, partly of direct labeling of
radial processes and cells in the subadjacent intermediate and
subventricular and ventricular zones, and partly of background dye that
has presumably diffused radially around the crystal. The fact that the
total bundle of fibers emerging from such a halo and running down
toward the internal capsule is always much smaller than the diameter of
the halo implies that the effective uptake site for fiber labeling is
very much smaller than the halo, as with most axonal tracers.
Although the labeled fibers could not be traced reliably within the dye
halo itself, very close to the uptake site, they could be clearly
resolved over the remainder of the pathway, throughout most of the
intermediate zone, the internal capsule, and right into the thalamus.
The mixed descending and ascending axons labeled by any small crystal
placement in the cortex at E16 form a discrete bundle rather than two
separate fiber tracts. This single, tight bundle is maintained
throughout its trajectory within the intermediate zone and primitive
internal capsule, over the region containing both sets of axons. The
stained bundle narrows as it approaches and runs through the internal
capsule, where, for a small crystal site, it can be as tiny as 50 µm
in diameter (Fig. 13B). A
fine ribbon of stained fibers extends into the diencephalon and follows a smoothly curving trajectory to the appropriate region of the thalamus, ending in a cluster of back-labeled cells.
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Figure 13.
Further evidence for common topography and close
association of early corticofugal and thalamocortical projections comes
from combined anterograde and retrograde tracing from the cortex at and
after E16. A, B, Small crystals of three different dyes
(DiA, DiI, and DiAsp) were placed at points in a parasagittal row along
the right hemisphere at E20, and the three distinct bundles of axons
(containing both corticofugal and thalamocortical axons) were followed
in 100-µm-thick horizontal sections. (Rostral is to the
left, lateral is up). There was no
evidence for substantial mixing or crossing of these bundles at any
point in the pathway between cortex (ctx) and thalamus
(thal). A is slightly superior to
B and shows the three bundles within the ventral
telencephalon (top), converging toward the internal
capsule and diverging again within the diencephalon
(bottom), heading toward different points in the
thalamus. B, at the level of the hippocampus
(hip), shows the three distinct bundles funneling
through the narrow junction between telencephalon and diencephalon.
C, A single crystal of DiI placed in the left occipital
cortex of an E16 brain produced a large halo of diffuse stain around
the implantation site (asterisk) but nevertheless
labeled a single, quite narrow fiber bundle. This double exposure shows
both DiI labeling (red) and bisbenzimide
counterstaining. The bundle of fibers runs in the intermediate zone,
turns medially, narrowing as it passes through the internal capsule,
and then turns quite sharply upward as it enters the diencephalon,
sweeping up to the dorsolateral part of the thalamus. In higher power,
a small group of back-labeled cells is seen in the LGN
(arrow). It is likely that the segment of the bundle
within the thalamus itself consists only of thalamocortical axons (see
tracing from the internal capsule in Results). D,
Five separate crystals of carbocyanine dye were placed in a
parasagittal row along the left hemisphere of an E20 animal (indicated
by arrows in the inset diagram of a
dorsal view). Bundles of axons linking cortex and thalamus (containing
both corticofugal and thalamocortical axons) were revealed in coarse,
250-µm-thick coronal sections. Each ran to a different region of the
thalamus, ending in a small group of back-labeled cells. At the level
of the section drawn here, the five distinct bundles were all clearly
visible, passing through the primitive internal capsule without obvious
mixing or crossing; the tip of each bundle is marked with an
arrow. Back-labeled cells associated with the two
bundles from the most caudal crystal placements lay in this plane; the
bundles labeled by the more anterior crystals terminated more ventrally
and medially in the thalamus. Scale bars: A, B, 500 µm; C, 250 µm; D, 1 mm.
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From serial confocal microscopic optical sections, we made
high-resolution reconstructions to reveal the fine topography of stained fibers within the entire bundle between thalamus and cortex. This technique did not quite allow us to follow individual axons all
the way from the implantation site to the thalamus, but it did show
that the bundle consists of an approximately parallel set of axons
throughout its course, with very little crossing over within the array
(just as in the images of separately labeled thalamic and preplate
axons seen at earlier stages; Fig. 10F).
Because this method does not allow discrimination of the two classes of
fibers, it is impossible to be sure whether the segment of the labeled
bundle within the diencephalon contains corticofugal axons. However,
the bundle usually appears thinner and yet less densely packed within
the diencephalon (Fig. 13C), suggesting that it contains
fewer fibers. And the fact that we never succeeded in back-labeling
subplate cell bodies with small dye placements restricted to the dorsal
diencephalon (see above) also implies that their axons do not reach the
relay nuclei of the thalamus itself. Therefore, the fine bundle of
stained axons running within the thalamus, toward the group of
back-labeled thalamic neurons, probably consists entirely of the
retrogradely stained axons of those cells.
It is important to note that the topographic arrangement of the fiber
bundles in the diencephalon and the cell clusters in the thalamus
itself, after cortical placement at E16, is entirely compatible with
the picture derived from the implantation of crystals in the thalamus
~1 d earlier, shortly after axon outgrowth (compare Figs. 2,
13D). This strengthens the conclusion that outgrowing thalamic axons form a highly ordered array, even within the
diencephalon itself.
Taken together with the double-labeling experiments and those in which
both descending and ascending axons were labeled from the internal
capsule, these results suggest that (1) the topography of the scaffold
of early corticofugal axons within the intermediate zone and basal
telencephalon is extremely similar in form to that of the
thalamocortical fibers; (2) each set of fibers appears to be
distributed, in order, within the entire depth of the intermediate zone; and (3) ascending and descending axons share the same compartment and are intermingled, through an extensive region of the ventral telencephalon, to within a very short distance of the target area for
the thalamic fibers.
It might be argued that some mixing of the two sets of axons is
inevitable if they share a single extracellular growth compartment, especially as they approach the constriction of the internal capsule. However, compelling evidence for global, topographically ordered intermeshing of the two axon arrays comes from the experiments involving multiple cortical dye placements (Figs. 13A,B,D,
14), in which the individual bundle of fibers labeled by each crystal can be followed all the way to the thalamus. At E16, each distinct bundle is unitary and clearly separate from the others, even as they
converge and pass through the primitive internal capsule. This is most
clearly shown in Figure 14,
C and D, which is a three-dimensional reconstruction of the entire trajectory of two closely spaced fiber
bundles running between frontal cortex and anterior thalamus.
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Figure 14.
A, B, Bundles of closely mixed
corticofugal and thalamocortical axons were revealed by applying three
crystals of carbocyanine dye (DiI, DiA, DiI) in a parasagittal row
along the right hemisphere and two crystals along a coronal line in the
left hemisphere (DiA ventral to DiI) of the same E16 brain. A,
B, Multiple-exposure fluorescence photomicrographs of the left
(A) and right (B)
hemispheres taken from a single 100 µm horizontal section just
superior to the junction of telencephalon and diencephalon.
A, Right hemisphere (top, rostral;
left, medial). The three distinct bundles
(a-c, from rostral to caudal) are clearly visible
within the ventral telencephalon (top right). The
corresponding groups of back-labeled thalamic cells, visible on the
left, form anteroposterior slabs
(a'-c'). Note the 90° rotation in topography between
telencephalon and thalamus, which occurs in the ventral diencephalon;
the anterior-to-posterior sequence of cortical loci
(a-c) corresponds to a mediolateral sequence of
thalamic slabs (a'-c'). B, Left
hemisphere (top, rostral; right, medial).
In the ventral telencephalon (top left) the two fiber
bundles appear partly superimposed, although they are separate in the
orthogonal plane; the bundle from the more ventral crystal
(a, DiA; yellow-green) lies deep to that
from the more dorsal crystal (b, DiI;
orange). The two corresponding back-labeled cell groups
(a', b'), although clearly segregated, line up along a
continuous anteroposterior slab in the thalamus (bottom
right). The ventrodorsal sequence along the coronal line on the
cortical convexity corresponds to an anterior-to-posterior sequence of
thalamic cells along one continuous slab. Scale bar: A,
B, 300 µm. C, D, Three-dimensional
reconstructions of the entire pathway between thalamus and cortex at
E16 to show the shared topology of early corticofugal and thalamic
projections. Frontal cortex was selected because the pathway is
relatively straight. Two crystals of DiI were placed on a coronal line
near the frontal pole of the left hemisphere, labeling both thalamic
and preplate axons (probably with very little involvement of fibers
from cells of the true cortical plate at this early age). After 6 weeks
incubation, to allow complete anterograde and retrograde diffusion,
75-µm-thick horizontal sections were cut and counterstained with
acridine orange. Each section was imaged in a fluorescent microscope
with both rhodamine and fluorescein filters, and a series of 43 images
was digitized, aligned, and superimposed for the reconstruction of a
three-dimensional data set. C, The reconstructed stack
of superimposed sections has been rotated around its sagittal axis to
tilt the left hemisphere up slightly; the tilted brain is viewed from
above (rostral to the right). To expose all the DiI
labeling, which appears green, surrounding parts of the
brain have been "dissected" away in the reconstruction, with the
central part "scooped out," right down to the internal capsule. The
two labeled axon bundles are clearly visible, running backward,
medially and down in the intermediate zone, through the internal
capsule and into the anterior thalamus. D, In this
enlarged view of the DiI labeling alone, the two fiber bundles are seen
to be clearly discrete, running parallel to each other, even through
the constriction of the internal capsule (between the filled
arrows). The back-labeled cells in the thalamus form a
continuous slab (unfilled arrow), oriented
anteroposteriorly. Scale bar (on D): C, 2 mm; D, 1 mm.
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Within the resolution of this method, the apparently single bundle
labeled from any one crystal could conceivably represent closely
adjacent but nonoverlapping strands of thalamocortical and corticofugal
axons. But obviously, close proximity of corresponding parts of the two
axon arrays without overlap of any part is incompatible with the
overall topography and closely packed fiber ordering seen within
each array. Ascending thalamic axons appear, then, to follow the
same topographically ordered paths through the telencephalon as do
their counterparts descending from the subplate. Either the two arrays
are systematically very slightly misaligned from each other,
or, as demanded by the handshake hypothesis, they are spatially
corresponding.
Retrograde labeling of thalamic neurons: global topography
of the projection
Although it is impossible to resolve the individual components of
the mixed fiber population labeled from a cortical implantation site,
important inferences about their interrelationships and topography can
be drawn from the patterns of retrograde labeling of thalamic cell
bodies. In horizontal sections, the thalamic cells back-labeled from
any single crystal in the cortex tend to form an anteroposteriorly
elongated slab. Different crystals placed in a parasagittal line along
the hemisphere at E16 revealed that more anterior cortical regions are
innervated by more medial slabs within the developing thalamus, whereas
more posterior cortical regions receive from more lateral thalamic
slabs (Fig. 14A). In concordance with this, any two
adjacent cortical implantation sites arranged along a coronal line
produced a pair of elongated groups of back-labeled thalamic cells
aligned to form a more-or-less continuous anteroposterior slab, the
rostral portion of which projects more ventrally in the cortex (Fig.
14B).
Other corticofugal fiber systems
De Carlos and O'Leary (1992) showed that pyramidal neurons of the
lowest part of the true cortical plate (presumably layer 6) start to
extend axons into the white matter as early as E15 in the rat.
Interestingly, at E16, axon bundles labeled from the cortex were always
unitary throughout their visible course, although the telencephalic
portion should have contained at least some axons of layer 6 cells as
well as those of preplate cells and thalamocortical fibers. This surely
means that all these axon systems run close to each other. And the
separate, ordered arrangement of bundles resulting from multiple dye
placements implies not only that all the component axon types share the
same growth compartment but also that they are distributed according to
the same overall topography.
For the portion of the pathway running through the intermediate zone,
this general pattern persists even after E18-E19, when there is also
significant extension of axons from layer 5 of the cortical plate,
ultimately directed toward structures below the diencephalon (De Carlos
and O'Leary, 1992; Mitrofanis and Guillery, 1993). Again this implies
topographically ordered mixing of all the fiber systems within the
telencephalon. However, the capacity of the single-crystal technique to
demonstrate divergence of axon populations when they do exist is
established by the fact that, at these later ages, each single dye
placement in the cortex does indeed reveal a fiber bundle (presumably
consisting of layer 5 cell axons) that diverges from the main labeled
tract into the posterior extension of the primitive internal capsule to
form the putative cerebral peduncle (Fig.
15). At this stage, dye placed in the
posterior part of the primitive internal capsule exclusively labels
cell bodies in layer 5 of the cortical plate itself, presumably the
cells of origin of corticotectal, corticopontine, and corticospinal projections. Thus, it seems that these nonpioneering, later-descending fibers from layer 5 share the same gross topography as the earlier ascending and descending projections within the telencephalon (although
possibly systematically slightly displaced from them), but that the
layer 5 axons follow radically different trajectories in the ventral
telencephalon, presumably responding to guidance cues in that
region.
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Figure 15.
Divergence of corticofugal projections at later
ages. Two different carbocyanine crystals were placed 3 mm apart along
a parasagittal axis in the convexity of the left hemisphere of a P2 rat
(DiI in the putative visual cortex, DiAsp in the presumed somatosensory
cortex), as shown in the inset diagram.
A, B, Double-exposure fluorescence
micrographs of two adjacent 100-µm-thick coronal sections
(A rostral to B) from the region of the
ventral diencephalon, just medial to the internal capsule
(IC), indicated by the box in the camera
lucida drawing on the left. Through the telencephalic
portion of the pathway, each dye labels a discrete, coherent bundle of
fibers, and the two bundles do not mix as they sweep through the
internal capsule, implying that all the projections labeled at this
late stage (corticofugal, from layers 6 and 5, as well as preplate and
thalamocortical) share the same overall topography. But on the
diencephalic side of the internal capsule, each labeled bundle divides
into two: a component that continues into the thalamus (thalamocortical
and corticothalamic fibers) and a ventral branch (presumably containing
the axons of the corticotectal, corticopontine, and corticospinal
projections), which descends into the cerebral peduncle. This
divergence is visible as early as E18-E19. A, Axons
labeled with DiI (orange) turn up toward the LGN,
whereas those labeled with DiAsp (green) run
medially toward the ventrobasal thalamus (VB). Each
bundle has a ventral branch that diverges into the cerebral peduncle
(CP). Note that the differently colored descending
branches remain segregated from each other, suggesting that fiber order
is maintained in this portion of the brainstem projection.
B, Just posterior, each thalamic component is
approaching the group of back-labeled cells in its own nucleus. As this
level the thalamic reticular nucleus (TRN) lies
directly below the ventrobasal thalamus. CTX, Cortex.
Scale bars: camera lucida drawing, 1 mm; A,
B, 300 µm.
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DISCUSSION |
Topographic order of thalamic axons
The arrangement of the bundles of thalamic axons labeled by
crystal placements in the thalamus, shortly after their outgrowth (Fig.
2), or in the cortex, immediately after their arrival (Figs. 13D, 14C,D), suggests that they maintain ordered
topography, running as an approximately parallel array along their
entire route, with a single 90° rotation of the array as it leaves
the ventral diencephalon (Fig. 2). Fibers from parasagittal slabs of
thalamic cells innervate loci lying along approximately coronal lines
across the cortex; more ventral cortex is innervated by cells lying
more rostral in the thalamic slab, and the more anterior the coronal
strip, the more medial (and slightly ventral) is the corresponding
thalamic slab (Fig. 14). The global topographic relationship between
thalamus and cortex appears very simple at this stage, before the clear segregation of thalamic nuclei. The entire volume of the thalamus maps
continuously onto the cortical sheet.
The initial pattern of thalamocortical innervation could subsequently
be altered through differential growth in thalamus or cortex, irregular
ingrowth of thalamic fibers, selective growth and retraction of their
side branches (Naegele et al., 1988; Ghosh and Shatz, 1992), or
activity-dependent redistribution of thalamic afferents in the cortex
(see Shatz, 1990; Molnár, 1998).
Initial guidance of thalamic outgrowth
The ordered outgrowth from the thalamus might depend
simply on intrinsic interactions between neighboring thalamic fibers. However, a number of other neural systems in this region could be
players in the process of guidance. Like Mitrofanis and Baker (1993), we saw cells of the thalamic reticular nucleus
back-labeled from the dorsal thalamus at E14 and also small numbers of
back-labeled neurons within the primitive internal capsule, presumably
belonging to the "perireticular" cell group of Mitrofanis (1992)
but projecting into the thalamus earlier than previously described
(Mitrofanis and Baker, 1993; Mitrofanis and Guillery, 1993). Thus, at
least two populations of fibers have reached the thalamus when thalamic axons are emerging, and they could conceivably play a part in guiding
that initial outgrowth.
The "handshake" hypothesis: possible guidance by
preplate axons
Axons from preplate cells pioneer a pathway through the
intermediate zone (McConnell et al., 1989; Blakemore and Molnár, 1990; De Carlos and O'Leary, 1992; Erzurumlu and Jhaveri, 1992; Miller
et al., 1993; Molnár et al., 1998). Rows of dye crystals placed
in the cortex as early as E15 show that axons form a highly ordered
array, reflecting the spatiotemporal wave of maturation that sweeps
across the developing cortex. Moreover, this array fills the
intermediate zone, through which thalamic axons subsequently grow in
the opposite direction as a similar topographic array. Back-labeling
from the internal capsule at this stage (Fig. 7) shows that cells in
both divisions of the original preplatemarginal zone as well as
subplatecontribute to the early corticofugal axon array.
Corresponding thalamic and cortical axons enter the lateral part of the
internal capsule approximately simultaneously, from opposite sides
(McConnell et al., 1989; Molnár and Blakemore, 1990; Bicknese and
Pearlman, 1992; De Carlos and O'Leary 1992; Erzurumlu and Jhaveri,
1992; Molnár et al., 1998). Preplate axons could constitute a
scaffold (McConnell et al., 1989) over which thalamocortical fibers
grow toward the appropriate cortical areathe handshake hypothesis
(Molnár and Blakemore, 1995).
The most direct test of the handshake hypothesis is to use double
labeling to see whether axons from corresponding cortical and thalamic
regions do meet and intermingle. There is a narrow window of
opportunity, when the two axon systems can indubitably be separately
labeled from the cortex and the thalamus (E14.5 to early E16 for the
geniculocortical pathway). But, paradoxically, it is impossible to be
certain of the precise spatial correspondence between thalamic and
cortical regions until thalamic axons actually reach their target
areas.
Miller et al. (1993) did not see obviously intermixed thalamic and
cortical axons in any of their nine double-labeling experiments in the
hamster. But the handshake hypothesis predicts that even slight offset
in double labeling should result in nonoverlapping fiber bundles, so
negative results are not definitive. We too saw little or no overlap in
42% of 26 such experiments between E14.5 and E15.5 in the rat.
However, in 58% of cases, substantial fractions of the axon bundles
were clearly mixed, with thalamic and preplate fibers juxtaposed,
following the same trajectories, even in thin confocal optical sections
and in stereo reconstructions (Fig. 10). Ascending and descending axon
systems, at least for the dorsolateral segment of the cortex, share the
same compartment within the internal capsule and the intermediate zone.
Bicknese and Pearlman (1992) and Erzurumlu and Jhaveri (1992), using
double labeling in the mouse and the hamster, also reported that
thalamic and early corticofugal fibers interdigitate in the internal
capsule.
The full handshake hypothesis proposes that thalamic fibers navigate
all the way to the cortex along the axons of subplate cells. However,
Bicknese et al. (1994) described thalamic axons in the rat extending
within the subplate layer, whereas fibers of subplate and cortical
plate cells ran below the subplate. On the basis of these observations,
they suggested that ascending and descending axons run in separate,
adjacent compartments close to the target area. On the other hand,
labeling with small crystals in the internal capsule between E15 and
E16 clearly shows a broad array of identifiable thalamic axons running
in close association with corticofugal fibers, right up to the subplate
(Figs. 11, 12). These findings, together with the positive results of
double labeling, strongly suggest that many thalamic fibers follow
preplate axons right to the target area. Although some thalamic axons
may continue to grow further within the subplate layer, the two
projection systems appear to share the same growth compartment and to
run along similar trajectories within the intermediate zone, at least toward the dorsolateral segment of the cortex.
Further powerful evidence that descending and ascending arrays are at
least approximately locked in correspondence comes from the arrangement
of individual, discrete bundles of mixed afferent and efferent axons
seen after multiple crystal placements in the cortex at E16 (Fig.
14D). The results of similar experiments at later
ages, when the axons of cells of layers 6 and 5 are also labeled,
suggest that they follow the same basic global topography as the
earlier projections, until the point at which layer 5 axons peel off at
the medial edge of the internal capsule to form the cerebral peduncle
(Fig. 15). Thus the pioneering preplate scaffold may play some part in
guiding not only afferent thalamic axons but also other descending
corticofugal projections, as suggested by McConnell et al. (1989).
In the adult rat, corticofugal axons from the dorsal occipital (visual)
cortex run some 150 µm deep to the corresponding thalamocortical axons in the white matter (Woodward and Coull, 1984; Woodward et al.,
1990). Perhaps postnatal loss of subplate axons leads to the appearance
of this distinct misalignment of afferent and efferent fibers in the
adult.
Of course, even close association of corresponding preplate and
thalamic axons does not prove that the former actually guide the latter. But, at the very least, the early corticofugal scaffold and
the subplate layer appear to provide attractive compartments within
which thalamic axons grow. The subplate is rich in chondroitin sulfate
proteoglycans (Bicknese et al., 1994), especially neurocan (Fukuda et
al., 1997), and it is possible that proteoglycan expression in the
intermediate zone, although much weaker (Bicknese and Pearlman, 1992;
Fukuda et al., 1997), is associated with the axons of subplate cells.
Indeed, Emerling and Lander (1994) found that dissociated embryonic
thalamic neurons selectively adhere to and extend neurites on the
intermediate zone as well as the subplate of embryonic cortical slices
(E15-E16), and this behavior is eliminated by the enzymatic removal of
chondroitin sulfate (Emerling and Lander, 1996).
Is there a "waiting period" in rodents?
Although the accumulation of thalamic fibers below the cortical
plate, which is such a striking feature of development in carnivores
(Shatz and Luskin, 1986; Ghosh and Shatz, 1992) and primates (Rakic,
1977, Kostovic and Rakic, 1990), was first described by Lund and
Mustari (1977) in the rat, the existence of a waiting period in rodents
has recently been questioned. Catalano et al. (1991, 1996) and Kageyama
and Robertson (1993) observed thalamic fibers simply advancing steadily
into the cortex as it matures. We saw some axons entering the lower
layers of the occipital cortex as early as E16 (Fig. 5), but they were
rare and trivial in density compared with the massive invasion that
starts at E19-E20, 2-3 d after their arrival (Figs. 3, 5). This is
compatible with the results of co-culture experiments, which suggest
that the occipital cortex does not express membrane-bound
growth-permissive properties until approximately E19-E20 (Götz
et al., 1992; Molnár and Blakemore, 1995).
Chemospecificity and chronotopy
Molecular signals, perhaps expressed in gradients across the
developing cortex, might play a part in guiding, or at least refining,
the distribution of thalamic fibers. Experiments involving co-culture
of thalamic and cortical explants (Yamamoto et al., 1992) suggest that
the cortex produces a diffusible factor that stimulates the outgrowth
of thalamic neurites from as early as E14 (Blakemore and Molnár,
1990; Molnár and Blakemore, 1991, 1995; Rennie et al., 1994;
Lotto and Price 1995; Molnár, 1998). This remote influence may
play a part in initiating outgrowth of thalamic axons in
vivo, but it seems inconceivable that it could impose the
topographic order that they exhibit throughout their course.
Bolz and Götz (1992) have shown that a thalamic explant extends
neurites somewhat more profusely over a surface coated with membranes
prepared from the corresponding region of cortex than over a membrane
preparation from the opposite end of the hemisphere. However, the
cortex does not express membrane-bound growth-permissive properties
until after thalamic axons have already arrived, in topographic order,
below the cortical plate; it therefore seems unlikely that such
properties control their overall distribution. However, chemical cues
expressed at the growing tips of preplate axons might play a role in
establishing their relationships with thalamic axons during the
handshake.
In the rat, like all mammalian species so far examined, there is a
pronounced gradient of maturation across the cerebral hemispheres (Bayer and Altman, 1991). There is also a temporal pattern of maturation within the thalamus (Jones, 1985), the first thalamic axons
to reach the cortex being from the ventrobasal complex (Braisted and
O'Leary, 1995). If outgrowing axons deposit themselves on the carpet
of axons that have already extended, and if axons tend to maintain
neighborly order and not to cross each other, the timing of fiber
outgrowth could potentially determine one axis of the initial mapping
of thalamus to cortex (Molnár and Blakemore, 1995).
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FOOTNOTES |
Received March 3, 1998; revised May 8, 1998; accepted May 14, 1998.
This study was supported by grants from the Medical Research Council
(MRC), the Wellcome Trust, the Soros-Hungarian Academy of Sciences
Foundation, the Human Frontier Science Program, and Merton College
(Oxford, UK). It forms part of the work of the Oxford McDonnell-Pew
Centre and the MRC Interdisciplinary Research Centre for Cognitive
Neuroscience, for which R.A. is a Research Scientist. Z.M. held an MRC
Training Fellowship. We are indebted to Laurence Waters, Lorraine
Chapell, and William Hinkes for help with photography and to Pat Owens
and Duncan Fleming for excellent animal care.
Correspondence should be addressed to Zoltán Molnár,
University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK.
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Top
Abstract
Introduction
