Increasing the frequency of spontaneous rhythmic activity disrupts pool-specific axon fasciculation and pathfinding of embryonic spinal motoneurons

Abstract

Rhythmic spontaneous bursting activity, which occurs in many developing neural circuits, has been considered to be important for the refinement of neural projections but not for early pathfinding decisions. However, the precise frequency of bursting activity differentially affects the two major pathfinding decisions made by chick lumbosacral motoneurons. Moderate slowing of burst frequency was shown previously to cause motoneurons to make dorsoventral (D-V) pathfinding errors and to alter the expression of molecules involved in that decision. Moderate speeding up of activity is shown here not to affect these molecules or D-V pathfinding but to strongly perturb the anteroposterior (A-P) pathfinding process by which motoneurons fasciculate into pool-specific fascicles at the limb base and then selectively grow to muscle targets. Resumption of normal frequency allowed axons to correct the A-P pathfinding errors by altering their trajectories distally, indicating the dynamic nature of this process and its continued sensitivity to patterned activity.

Figure 1.

Intervals between episodes of spontaneous rhythmic activity in sarcosine-treated and control embryos. A, Bar graph of the intervals between electrically recorded episodes of activity in isolated cord preparations from control and chronic sarcosine-treated embryos at different developmental stages. The sarcosine-treated cords were dissected and recorded in Tyrode's containing 100 μm sarcosine. B, Bar graph of the intervals between in ovo movements quantified at St 23 in control embryos and those treated acutely or chronically (from St 20) with sarcosine (Sarc). The 1.0X acute and chronic sarcosine values were significantly different from control (p < 0.05). C, When sarcosine treatment was stopped at St 24, the intermovement intervals had returned to control values by St 26. D, The recovery to control values of interepisode intervals was also assessed with electrical recordings from isolated spinal cords at different developmental stages.

Figure 2.

The sorting and pathfinding of axons that project to dorsal versus ventral limb muscles in control and sarcosine-treated embryos. A, Diagram showing the reorganization and D-V pathfinding of two dorsal motoneuron pools (sartorius, green; femorotibialis, red). Cross sections at the right show the positions of these and ventrally projecting axons (blue) to the adductor (Adduct) muscle in the proximal spinal nerve where they are extensively intermingled (top), just before the convergence of spinal nerves into the crural plexus, where dorsal and ventral projecting axons have segregated along the D-V axis (middle) and within the dorsal (crural) nerve trunk after the ventral (obturator) trunk has diverged and sartorius (Sart) and femorotibialis (Fem) axons have formed discrete fascicles (bottom). The dorsal nerve trunk diverges to innervate multiple muscles (ITR, iliotrochanterici; Femoro, femorotibialis; Ailtib, anterior iliotibialis). The ventral trunk projects to the adductor muscle. B, C, Whole mounts as seen from the ventral surface of control and sarcosine-treated embryos, respectively, of axons retrogradely labeled with dextrans from the sartorius (green) and the adductor (white). Labeled axons are seen in spinal nerves LS1 and LS2. D, E, Cross sections showing the ventral spinal cord on one side from control and sarcosine-treated embryos, respectively, at the level of LS1 (ventral, down; medial, left). In both cases, the sartorius motoneuron somas (green) are located lateral to those projecting to the adductor (white), in appropriate LMCl and LMCm locations, showing that D-V pathfinding has not been affected. The LMCl is encircled with a dashed line. Scale bar, 100 μm.

Figure 3.

Motoneuron pool-specific pathfinding in control embryos, those treated with sarcosine, and those in which sarcosine treatment was stopped at St 24 (sarcosine recovery) and activity was allowed to recover to control values. A–C, Whole mounts of control, sarcosine, and sarcosine recovery embryos in which axons projecting to the sartorius (Sart; green) and femorotibialis (Fem; red) were retrogradely labeled at St 30 by fluorescent dextrans. White arrows show green labeled axons in spinal nerve LS2 projecting to the sartorius in control (A) and sarcosine recovery (C) but not in embryos treated with sarcosine throughout the growth of axons from the spinal cord to their entry into the muscle (B). The plexus region, where spinal nerves LS2 and LS3 converge, is marked by a white arrowhead. In the control embryo (A), the sartorius labeled axons in LS2 diverge at this point to join the sartorius axons in LS1 (red labeled axons projecting to the femorotibialis were also present in LS1 but cannot be distinguished in this whole mount at this level of magnification). With chronic sarcosine treatment, no axons from LS2 project to the sartorius (B). When sarcosine treatment was stopped after plexus formation (C), the sartorius-labeled axons fail to diverge at this point and continue to grow with the femorotibialis axons (red) toward the femorotibialis muscle. However, more distally, they diverge and grow to the sartorius muscle. D–F, Histograms of the rostrocaudal location of motoneuron somas retrogradely labeled from the sartorius (top) and femorotibialis (bottom) quantified from serial frozen sections and expressed as the percentage of the total number of neurons in each pool (3 embryos/histogram). The sartorius pool extends into LS2 in the control and sarcosine recovery but not with chronic sarcosine. Similarly, the femorotibialis pool extends into LS1 and LS3 in control and sarcosine recovery but not sarcosine. G, H, The location of motoneuron somas retrogradely labeled from the sartorius (green, top) at the level of LS1 or from the femorotibialis (red, bottom) at the level of LS2. The sections were also immunostained with antibody for islet 1/2, which labels the nuclei of all LMCm motoneurons. In the sarcosine-treated embryos, all sartorius and femorotibialis somas were located lateral to the LMCm, consistent with a lack of D-V pathfinding errors. However, in general, the somas of each pool were distributed throughout the LMCl, rather than being clustered in distinct locations.

Figure 4.

EMG recordings reveal the functional innervation of sartorius (Sart) and femorotibialis (Femoro) muscles by different spinal cord segments in control, sarcosine, and sarcosine recovery embryos. The amplitude of EMG responses, in response to a single maximal stimulus (Stim) to each spinal nerve, shows that the functional segmental innervation by the three spinal nerves that compose the crural plexus is altered in the chronic (Chr) sarcosine (Sarc) treatment (B) compared with control (A), but that recovery of activity after cessation of sarcosine treatment at St 24 restores the segmental innervation pattern toward control values. Calibration: 30 ms, 5 mV.

Figure 5.

Muscle nerve recordings reveal that motoneurons that have innervated foreign muscles retain their original pool-specific bursting patterns. A, Recordings of an episode of activity induced by a single stimulus to the thoracic cord from control sartorius (Sart; top) and femorotibialis (Fem; bottom) nerves show that these are activated out of phase in the three “step” cycles that comprise an episode of activity at St 30. The bracket delineates one cycle in the top trace. After a brief activation, the sartorius exhibits a period of inhibition during which the femorotibialis fires. Subsequently, the sartorius resumes bursting until the next cycle. B, C, Expanded time base traces of a single cycle in control (B) and sarcosine (C)-treated embryos. The arrowhead in C denotes units in the femorotibialis nerve firing during the normal sartorius inhibitory period (shown by a bar in the top trace of B). The arrowhead in C shows units firing in the sarcosine-treated femorotibialis muscle during the prolonged period of the sartorius burst in the control (B, asterisk), whereas no units fire during this time in the control femorotibialis (B, arrowhead).

Figure 6.

Sartorius axons that made pathfinding errors in the plexus after sarcosine treatment are able to correct these errors via the formation of novel distal nerves when sarcosine treatment is stopped and activity returns to normal levels. A, Orthograde labeling of LS2 with a fluorescent dextran shows that axons from this segment project out the sartorius nerve (shown by the dashed green line) in the control (left). When the frequency of activity was altered by sarcosine treatment until St 24 (right), LS2 axons failed to project into the sartorius nerve (location shown by dashed green line), but after resumption of activity axons from LS2 formed novel nerves (arrow) that diverged more distally to project to the sartorius muscle. B, After transection of spinal nerve LS1 and injection of the sartorius muscle with a dextan dye, the motoneuron cell bodies of axons that projected to the sartorius muscle via the novel aberrant nerves were found to extend throughout spinal segment LS2, similar to the control sartorius pool. Histograms show the number of motoneurons at different rostrocaudal levels from serial frozen cross sections of cord, expressed as the percentage of the total labeled pool. C, A diagram to show the formation of novel aberrant nerves projecting to the sartorius (sart axons green, femoro axons red). D–G, Nerve recordings from the sartorius nerve (D), an aberrant nerve (E), and at different proximodistal levels of the femorotibialis nerve (F, G). Axons projecting via the aberrant nerve (E) had typical sartorius bursting patters with a clear inhibitory period (bracket) and prolonged unit activity (asterisk) until the next cycle. Recordings from the femorotibialis nerve proximal to the divergence of the aberrant nerve (G) showed a composite sartorius/femorotibialis bursting pattern, whereas recordings distal to this point showed a cleaner femorotibialis pattern (F), with units no longer firing during the prolonged sartorius bursting period (brackets, compare F and G). H, The location of cell bodies in LS2 retrogradely labeled via aberrant nerves to the sartorius (green) and those cell bodies that project to the femorotibialis (red). The yellow dashed line encircles the LMCl, whereas the white dashed line indicates the lateral boundary of the ventral cord. Scale bar, 100 μm. I, The location of all LS2 cell bodies retrogradely labeled from the sartorius from one sarcosine recovery embryo and projecting to the sartorius via aberrant nerves in different frozen sections have been marked with Xs. Although many cell bodies in the sarcosine recovery embryo are in the location of the control pool (green line), some appear to be displaced and in the location of the femorotibialis pool.

Figure 7.

The effect of sarcosine on the frequency of spontaneous episodes and on pool-specific pathfinding errors is prevented by simultaneous application of the glycine receptor antagonist strychnine. A, In ovo intermovement intervals in control, sarcosine- and combined sarcosine/strychnine-treated embryos. B, EMG recordings in isolated St 30 cord preparations after washout of drugs reveal control-like bursting patterns in the strychnine-treated (top) and combined sarcosine/strychnine (bottom)-treated embryos. In each pair of traces, the sartorius is on the top, and the femorotibialis is on the bottom. C, After the combined strychnine/sarcosine treatment, the location of sartorius and femorotibialis pools are unaltered from control levels showing that A-P pathfinding errors produced by sarcosine alone have been prevented.