Activation of locomotion in adult chronic spinal rats is achieved by transplantation of embryonic raphe cells reinnervating a precise lumbar level - PubMed (original) (raw)

Activation of locomotion in adult chronic spinal rats is achieved by transplantation of embryonic raphe cells reinnervating a precise lumbar level

M G Ribotta et al. J Neurosci. 2000.

Abstract

Traumatic lesions of the spinal cord yield a loss of supraspinal control of voluntary locomotor activity, although the spinal cord contains the necessary circuitry to generate the basic locomotor pattern. In spinal rats, this network, known as central pattern generator (CPG), was shown to be sensitive to serotonergic pharmacological stimulation. In previous works we have shown that embryonic raphe cells transplanted into the sublesional cord of adult rats can reinnervate specific targets, restore the lesion-induced increase in receptor densities of neurotransmitters, promote hindlimb weight support, and trigger a locomotor activity on a treadmill without any other pharmacological treatment or training. With the aim of discriminating whether the action of serotonin on CPG is associated to a specific level of the cord, we have transplanted embryonic raphe cells at two different levels of the sublesional cord (T9 and T11) and then performed analysis of the kinematic and EMG activity synchronously recorded during locomotion. Locomotor performances were correlated to the reinnervated level of the cord and compared to that of intact and transected nontransplanted animals. The movements expressed by T11 transplanted animals correspond to a well defined locomotor pattern comparable to that of the intact animals. On the contrary, T9 transplanted animals developed limited and disorganized movements as those of nontransplanted animals. The correlation of the locomotor performances with the level of reinnervation of the spinal cord suggests that serotonergic reinnervation of the L1-L2 level constitutes a key element in the genesis of this locomotor rhythmic activity. This is the first in vivo demonstration that transplanted embryonic raphe cells reinnervating a specific level of the cord activate a locomotor behavior.

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Figures

Fig. 1.

Fig. 1.

Sequences of selected video frames in five different conditions. Frames were grabbed using a video grabber at approximately every 50 msec starting at foot lift of one step sequence (beginning of swing) on the left side. The spots are the light reflecting markers placed on the left leg. The white lines were added post hoc to facilitate the visualization of angular movements as shown in figures such as Figure2_A_. Times are approximations to the closest 50 msec. Upward and _downward facing arrows_indicating foot lifts and foot contacts have been added when appropriate. A, An intact adult rat; B, A spinal adult rat 10 weeks after transection; C, A T11-transplanted rat (9 weeks after grafting); D, The same animal as in C (10 weeks after grafting) after a new transection at T8 spinal cord level. E, A T9-transplanted rat (9 weeks after grafting).

Fig. 2.

Fig. 2.

Comparison of rhythmic locomotor activity in an intact rat (A–C) and a T11 animal 9 weeks after transplantation of embryonic raphe cells from E14 embryos (D–F). A, _D,_Reconstruction, as stick diagrams, of treadmill locomotor movements during swing and stance phases. Each stick figure is displaced from the previous by an amount equivalent to the foot displacement to avoid overlap of all the figures. B, _E,Variations of mean angle joints (thick lines) and their SDs (thin lines) from six consecutive step cycles, in (from top to bottom) hip, knee, ankle, and metatarsophalangeal (MTP) joints. The same normalized step cycle is displayed twice to facilitate viewing the events at around the trigger point (foot contact of the limb facing the camera, downward facing arrow). The foot lift of the same limb is also indicated at the bottom of the figure by_upward facing arrows. Angular excursion of various joints are averaged for six cycles and synchronized on foot contact.C, F, Corresponding synchronized EMG activity in various muscles of ipsi (i) or contralateral (co) hindlimbs. _Ip,_Iliopsoas (hip flexor); St, semitendinosus (knee flexor and hip extensor); VL, vastus lateralis (knee extensor);TA, tibialis anterior (ankle flexor); _GM,_gastrocnemius medialis (ankle extensor). Note the discharges of iIp, iSt, and iTA during the swing phase and that of iVL and iGM during stance. The contralateral St (coSt) also discharges during the ipsilateral stance, indicating a good alternation between limbs.

Fig. 3.

Fig. 3.

Relationships between duration of extensor (GM for gastrocnemius medialis) or flexor (TA for tibialis anterior) EMG bursts in intact (A) or transplanted (B) adult rats 10 weeks after spinal cord transection. A, Intact rat. n = 21;_r_2 = 0.035; slope = −0.30 ± 0.036 for TA muscle and n = 14,_r_2 = 0.87; slope = 0.98 ± 0.30 for GM muscle. B, Spinal rat (T8) transplanted (T11) for 10 weeks. n = 12;_r_2 = 0,091; slope = −0.051 ± 0.051 for TA and n = 12;_r_2 = 0.83; slope = 0.75 ± 0.11 for GM.

Fig. 4.

Fig. 4.

Diagrams and immunocytochemical detection of serotonin (5-HT) in vibratome sections of the spinal cord.A, Diagram illustrating the spinal cord in a T11 animal and the segments taken for immunocytochemical detection of 5-HT as seen in micrographs B and C. _B,_A longitudinal rostrocaudal section through the transplant area showing ovoid and multipolar 5-HT-immunoreactive neurons with thin and varicose processes distributed in the gray as well as white matter. Scale bar, 100 μm. C, A transverse section at L2 level that shows very varicose 5-HT fibers distributed in the ventral horn. Scale bar, 25 μm. D, Diagram illustrating the spinal cord in a T9 animal and segments taken for immunocytochemical detection of 5-HT as seen in micrographs E and F.E, A longitudinal rostrocaudal section through the transplant area showing, as in T11 animals, 5-HT-immunoreactive neurons with a dense immunoreactive neuropil. Scale bar, 100 μm.F, A transverse section at L2 level where no 5-HT-immunoreactive fibers can be detected. Scale bar, 25 μm.

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