Mechanisms underlying the early establishment of thalamocortical connections in the rat

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.

Fig. 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.

Fig. 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 arrowin E) head to a more posterior region then those labeled with DiA (filled arrows) from the more medial crystal placement (filled black arrow inE). Scale bar, 500 μm.

Fig. 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). Theright 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 inD. Scale bar, 500 μm for all panels.

Fig. 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 inA) 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.

Fig. 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), andP2 (bottom row). Left panels, DiI labeling; right panels, bisbenzimide labeling of the same section. The examples shown for E16and E19 are higher-power fluorescence photomicrographs from Figure 3, G and H andK 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 andP2. Scale bar, 100 μm for all panels.

Fig. 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 thetop continuous line marks the pial surface, whereas theinterrupted 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.

Fig. 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 ofB (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 inC). 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.

Fig. 8.

Outgrowth of the first corticofugal projections. The photomicrographs were taken from the regions indicated in the schematic drawing on the left: A andB at E13; C at E13.5; andD–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.

Fig. 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 themiddle), 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.

Fig. 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 anasterisk), 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 thearrow. 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 andgreen 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.

Fig. 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 inA 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.

Fig. 12.

The trajectories of individual thalamic and cortical axons in the region shown in Figure 10Dwere 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.

Fig. 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 theleft, 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 toB 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 anarrow. 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.

Fig. 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 theleft, 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.

Fig. 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.