Local neurotrophin effects on central trigeminal axon growth patterns

Abstract

In dissociated cell and wholemount explant cultures of the embryonic trigeminal pathway NGF promotes exuberant elongation of trigeminal ganglion (TG) axons, whereas NT-3 leads to precocious arborization [J. Comp. Neurol. 425 (2000) 202]. In the present study, we investigated the axonal effects of local applications of NGF and NT-3. We placed small sepharose beads loaded with either NGF or NT-3 along the lateral edge of the central trigeminal tract in TG-brainstem intact wholemount explant cultures prepared from embryonic day 15 rats. Labeling of the TG with carbocyanine dye, DiI, revealed that NGF induces local defasciculation and diversion of trigeminal axons. Numerous axons leave the tract, grow towards the bead and engulf it, while some axons grow away from the neurotrophin source. NT-3, on the other hand, induced localized interstitial branching and formation of neuritic tangles in the vicinity of the neurotrophin source. Double immunocytochemistry showed that axons responding to NGF were predominantly TrkA-positive, whereas both TrkA and TrkC-positive axons responded to NT-3. Our results indicate that localized neurotrophin sources along the routes of embryonic sensory axons in the central nervous system, far away from their parent cell bodies, can alter restricted axonal pathways and induce elongation, arborization responses.

Fig. 1

(A) Schematic illustration of the whisker pad-TG-brainstem wholemount cultures and the experimental setup. The whisker pads are sectioned off from dashed lines. Small diagram at the bottom left illustrates the preparation of the “open book” brainstem explants and inset diagram illustrates the relative positions of the tract and the bead. In a few cases the bead was located directly across from the tract (gray bead) on the ventricular surface. ATr: ascending trigeminal tract; DTr: descending trigeminal tract; BSTC: brainstem trigeminal complex, TG: trigeminal ganglion, WP: whisker pad. (B) DiI labeled DTr axons in the presence of a control (BSA-loaded) bead. Note that central trigeminal axons are restricted to the tract and do not show any response to control bead. (C) NGF-loaded sepharose bead induces extensive axon outgrowth both towards and away (arrow) from the source of neurotrophin. Many axons leave the tract and encircle the bead. (D) NT-3-loaded beads, on the other hand, induce attraction, dense arborization and knot-like structures in the vicinity of the bead. Asterisks indicate the beads in (B–D). Scale bar=200 μm for (B–D).

Fig. 2

Effects of NGF and NT-3 on descending trigeminal tract axons and quantitative analysis of axon growth. (A) Note that with the exception of a few stray axons laterally, all other axons grow tightly fasciculated and none turn towards the control (BSA) bead. (B) DiI-labeled axons (arrows) growing medially and away from an NGF bead. (C) The lateral side of the same tract showing axons that grow and encapsulate the NGF-loaded bead. (D) Another NGF bead case with DiI labeling photoconverted. Note the axons that grow both towards and away (arrows) from the bead. (E, F), Attraction and axon branching towards NT-3 beads. Note that a few axons are also growing away from the bead in F (arrows). (G, H), camera lucida drawings of axons from single images through z-series obtained with confocal microscopic images illustrating single axons from the parent tract which are turning laterally or medially and growing unbranched, in response to NGF (G) and the nodal branching and some turning of parent trigeminal axons in the tract in response to NT-3 (H). In (B–H) lateral is to the right and rostral is to the top, and in the control case (A) lateral is to the left. Asterisks mark the beads. Scale bar=100 μm for (A–F), 50 μm for (G, H). (I) Bar graph representation of axon outgrowth analysis both towards (lateral) and away (medial) from the neurotrophin source. The thickness of the tract between the medial and lateral edges tract was designated as zero reference point for each case. *p<0.01, **p<0.001.

Fig. 3

The effects of NGF bead over a three-day culture period. (A) After 24 h most cases did not show any axonal response to the NGF bead. (B) When NGF bead was placed over the trigeminal tract on the ventricular side of the explant, a distinct defasciculation and turning response of axons that have reached this level is seen. Some axons tipped with growth cones (arrows) leave the tract and grow away from the bead. After 48 h in culture, many axons show attraction response to the NGF bead (C). (D) A case with an NGF bead over the tract. A clear turning of the leading edge of immature axons is evident (arrows). After 3 days in culture, central trigeminal axons extended all the way to the most laterally placed beads, forming spiraling funnels (E, F). In these two exemplary cases the central trigeminal tract is located to the left of the micrographs. Dashed circles outline the beads and asterisks mark the center of the beads. Scale bar=150 μm.

Fig. 4

The effects of NT-3 beads over a three-day culture period. (A) After 24 days most cases did not show any axonal response to the NT-3 bead as in NGF cases. (B) In a couple cases axonal responses to the NT-3 bead became apparent by 24 h. Axonal effects were pronounced by the end of the second day (C, D) and not much different from that seen by the end of the third day (E, F). Many axons within the tract formed interstitial branches that extended towards the bead and arborized extensively (arrows D, F) or formed “knots” (arrows, C, E). Dashed circles outline the beads and asterisks mark the center of the beads. Scale bar=150 μm.

Fig. 5

TrkA and TrkC double immunocytochemistry. (A) TrkA and (B) TrkC expression in the same TG from a TG-brainstem wholemount culture with an NGF-bead. Merged images are shown in (C), (F) and (I). Different populations of cells are labeled with either antibody, and there are hardly any cells that express both receptors. (D) and (E) illustrate the initial (rostral) part of the central trigeminal tract, and (G) and (H) at the level of an NGF-loaded bead. Note that at all levels of the tract different populations of trigeminal axons express TrkA and TrkC. Scale bar=100 μm.

Fig. 6

Examples of TrkA and TrkC-labeled axons with respect to neurotrophin bead. (A–I) show TrkA and TrkC-labeled axons with respect to an NGF bead from three different cases. Note that mostly TrkA-labeled axons are around the NGF bead (marked with +). While in some cases there are no TrkC-positive axons (A–C), in others, few axons are seen in the vicinity of the bead (D–I, arrow heads). (J–R), show the presence of both TrkA and TrkC-positive axons near NT-3-loaded beads (marked with *). Since these are cryostat sections through the explants, the size of the bead, and the density of the labeled fibers appear different for each case due to the level of section through the explant. Scale bar=100 μm.