Morphology and growth patterns of developing thalamocortical axons

Abstract

It is increasingly evident that the actions of guidance factors depend critically on the cellular and molecular context in which they operate. For this reason we examined the growth cone morphology and behavior of thalamic fibers in the relatively natural environment of a slice preparation containing the entire pathway from thalamus to cortex. Axons were labeled with DiI crystals and imaged with a laser-scanning confocal microscope for up to 8 hr. Their behavior was analyzed in terms of morphology, extension rates, shape of trajectory, frequency of branching, and percentage of time spent in advance, pause, and retraction. Thalamic fibers had distinct and stereotyped growth patterns that related closely to their position; within the striatum growth cones were small and elongated, rarely extending filopodia or side branches. Axons grew quickly, in straight trajectories, with minimal pauses or retractions. When they reached the ventral intermediate zone, axons slowed down, often coming to a complete stop for up to several hours, and their growth cones became larger and more complex. During pauses there were continuous extensions and retractions of filopodia and/or side branches. When advance resumed, it was often to a different direction. These results demonstrate consistent regional variations in growth patterns that identify an unexpected decision region for thalamic axons. They provide the basis for examining the roles of guidance cues in an accessible yet intact preparation of the thalamocortical pathway and allow for an evaluation of previously suggested pathfinding mechanisms.

Fig. 1.

Photomicrograph, taken under transillumination, of a 400 μm whole forebrain slice from the left hemisphere of an E17 rat brain cut at 45° to the coronal and sagittal planes. Broken lines enclose the two locations in which growing axons were monitored: a, the region of the internal capsule;b, the ventral intermediate zone at the border between dorsal and ventral telencephalon. CX, Cortex;T, thalamus, S, corpus striatum.

Fig. 2.

Thalamic fibers growing through the internal capsule (IC) in a slice from an E18 embryo. A, Thetop graph plots the growth of two axons (axons 4 and 6) for the duration of the recording sessions (3 and 4.5 hr, respectively). Measurements were made from extended-focus images collected at regular intervals (as indicated by the redand blue data points) and were normalized with respect to the measurements taken at the beginning of the sequence.B, Closer observation of the behavior of axon 4 over the first 60 min of the recording is shown on an expanded time scale in thebottom graph, each dot indicating the position of the leading tip of the axon relative to a distinctive and fixed reference point on the axon shaft. Single-frame confocal images were obtained at 90 sec intervals (gaps reflect loss of the focal plane). The sidepanels are single-frame images of axon 4 at selected times during the first hour of the recording session. The times at which the images were obtained are indicated at the topright of each frame. Both axons are advancing most of the time, with brief pauses (e.g., 20–30 min, in B) and occasional retractions (e.g., from 47 to 56 min, in B). Note the morphology of the growth cone, which is small and streamlined with no side branches. Scale bar, 10 μm.

Fig. 3.

E16 thalamic fiber growing through the IC.Side panels are extended-focus images of the distal segment of the axon and its growth cone, collected at various times throughout a 3 hr imaging session. The axon has beenoutlined for clarity. The times at which the stacks were collected are indicated at the top left of each image, and the star indicates a stable point along the axon for clearer illustration of axon extension. The top graphshows that it advanced at a relatively constant growth rate. Thebottom graph, at an expanded time scale, provides a better view of the momentary pauses and retractions. Scale bar, 10 μm.

Fig. 4.

Thalamic axon with its growth cone located in the ventral intermediate zone (VIZ) of an E17 forebrain slice.Panels at the top andbottom are single confocal frames obtained at the times indicated. These images are framed to illustrate growth cone morphology and do not necessarily reflect the absolute position of the growth cone with respect to the slice. The graph illustrates the behavior of this axon for the 8 hr of the imaging session. Measurements were made from a stable inflection point further down along the axon shaft and were normalized to the beginning of the sequence.Dots indicate the principal axon length, i.e., the distance from the measuring point to the leading edge of the body of the growth cone (excluding filopodia and side branches, examples of which are indicated by the arrows of the leading segment of the axon). Periods lacking data points reflect either loss of the focal plane or deliberate changes in the area that was imaged. Although the out-of-focus images were not sharp enough to allow for accurate measurements, they confirmed that the growth cone was still in the field of view and had not manifested massive extensions or retractions. Note that the net forward growth rate is at its highest at the end of the 8 hr imaging session, indicating the continuing good health of the slice. Scale bar, 10 μm.

Fig. 5.

“Exploratory” behavior. The graphs illustrate on expanded time scales the behavior of the axon shown in the previous figure. The red symbols and line indicate the changes in total side branch length—i.e., the summed length of all visible filopodia and side branches of the leading tip of the axon. Theblue symbols and trace indicate the principal axon length, i.e., the distance from a stable measuring point to the most distal tip of the body of the growth cone. Both measurements are normalized to the respective measurements made at the beginning of the sequence. Note the lack of net advance (blue trace) and the considerable “stationary” movement (redtrace). Negative numbers in either case indicate retractions with respect to the position at the onset of the observation period.

Fig. 6.

Side branch exploration of an axon for which the leading segment was in the VIZ, in a slice that was obtained from an E18 rat embryo. The four side panels are single-frame confocal images at selected times during a 30 min imaging session (times indicated at top left of each panel). Thegraph illustrates the behavior of each branch on this axon separately. Measurements for the principal axon and the main side branch were made from the point of bifurcation of the main side branch (star in topleft panel) and normalized to the beginning of the recording session, The principal axon (green) as well as the main side branch (light blue) are primarily pausing, whereas side branches 2 (red), 3 (yellow), and 4 (black) along the axon shaft are being extended and retracted continually. The axon and branches in the four panels have been outlined for clarity. Scale bar, 10 μm.

Fig. 7.

Growth dynamics. The graphillustrates the contrasting behaviors of the main branch (blue, filled circles) and the side branch (red, open circles) of a thalamic axon in the VIZ. Thepanels at the bottom are single-frame confocal images collected at the times indicated by the solid lines. The lengths of the main axon (open arrow) and of the side branch (filled arrow) are measured from the stable bifurcation point at different times during a 70 min imaging session. Initially, most of the activity occurs in the side branch, which has branches of its own and appears to explore the surrounding territory. Gradually, the side branch becomes more quiescent and eventually retracts into the axon shaft; at the same time the main axon becomes “activated”: its growth cone becomes enlarged and it seems to take over the exploration. Scale bar, 10 μm.

Fig. 8.

Example of “transition” behavior in an axon for which the leading segment is at the border between IC and VIZ, in a slice obtained from an E17 embryo. The panels at thetop are single-frame confocal images of the growth cone. The data points in the graph illustrate the net advance of the axon during the period of observation. Scale bar, 10 μm.