Thoracic Hemisection in Rats Results in Initial Recovery Followed by a Late Decrement in Locomotor Movements, with Changes in Coordination Correlated with Serotonergic Innervation of the Ventral Horn

<div><p>Lateral thoracic hemisection of the rodent spinal cord is a popular model of spinal cord injury, in which the effects of various treatments, designed to encourage locomotor recovery, are tested. Nevertheless, there are still inconsistencies in the literature concerning the details of spontaneous locomotor recovery after such lesions, and there is a lack of data concerning the quality of locomotion over a long time span after the lesion. In this study, we aimed to address some of these issues. In our experiments, locomotor recovery was assessed using EMG and CatWalk recordings and analysis. Our results showed that after hemisection there was paralysis in both hindlimbs, followed by a substantial recovery of locomotor movements, but even at the peak of recovery, which occurred about 4 weeks after the lesion, some deficits of locomotion remained present. The parameters that were abnormal included abduction, interlimb coordination and speed of locomotion. Locomotor performance was stable for several weeks, but about 3–4 months after hemisection secondary locomotor impairment was observed with changes in parameters, such as speed of locomotion, interlimb coordination, base of hindlimb support, hindlimb abduction and relative foot print distance. Histological analysis of serotonergic innervation at the lumbar ventral horn below hemisection revealed a limited restoration of serotonergic fibers on the ipsilateral side of the spinal cord, while on the contralateral side of the spinal cord it returned to normal. In addition, the length of these fibers on both sides of the spinal cord correlated with inter- and intralimb coordination. In contrast to data reported in the literature, our results show there is not full locomotor recovery after spinal cord hemisection. Secondary deterioration of certain locomotor functions occurs with time in hemisected rats, and locomotor recovery appears partly associated with reinnervation of spinal circuitry by serotonergic fibers.</p></div

Comparison of EMG activity of the forelimb and hindlimb muscles.

<p>EMG activity during locomotion of a representative rat before (A), 7 days (B), 14 days (C) and 42 days (D) after spinal cord hemisection. Interlimb coordination (onset of EMG activity in the left TA (l TA) with respect to the contralateral right TA (r TA)) and intralimb coordination (onset of EMG activity in the left extensor (l Sol) with respect to the left TA (l TA)) for these time points are shown in the left and right polar plots, respectively. Zero corresponds to the onset of activity in the right TA muscle, and the positions of the filled black circles indicate the times of onset of activity in the left TA<i>—</i>interlimb coordination (left panel) or the onset of activity in the left Sol <i>-</i>intralimb coordination (right panel) in the relation to the onset of activity in the left TA muscle. The black lines each represent data from one animal, and the red line represents the global mean for the group. l Tri—left <i>Triceps</i>; l/r Sol—left and right <i>Soleus</i> and l/r TA—left and right <i>Tibialis Anterior</i>.</p

Immunolabeling of transverse spinal cord sections showing 5-HT-positive staining in intact rats and at different time points after hemisection.

<p>The representative images show the ventral horns below the lesion (L3-L5 level) in the ipsilateral (left) and contralateral (right) side to hemisection. (A) intact rat, (B) 1 week, (C) 2 weeks, (D) 3 months, (E) 6 months after the lesion. 1 and 3 –real images, 2 and 4 –images after skeletonization.</p

Unusual Quadrupedal Locomotion in Rat during Recovery from Lumbar Spinal Blockade of 5-HT7 Receptors

Coordination of four-limb movements during quadrupedal locomotion is controlled by supraspinal monoaminergic descending pathways, among which serotoninergic ones play a crucial role. Here we investigated the locomotor pattern during recovery from blockade of 5-HT7 or 5-HT2A receptors after intrathecal application of SB269970 or cyproheptadine in adult rats with chronic intrathecal cannula implanted in the lumbar spinal cord. The interlimb coordination was investigated based on electromyographic activity recorded from selected fore- and hindlimb muscles during rat locomotion on a treadmill. In the time of recovery after hindlimb transient paralysis, we noticed a presence of an unusual pattern of quadrupedal locomotion characterized by a doubling of forelimb stepping in relation to unaffected hindlimb stepping (2FL-1HL) after blockade of 5-HT7 receptors but not after blockade of 5-HT2A receptors. The 2FL-1HL pattern, although transient, was observed as a stable form of fore-hindlimb coupling during quadrupedal locomotion. We suggest that modulation of the 5-HT7 receptors on interneurons located in lamina VII with ascending projections to the forelimb spinal network can be responsible for the 2FL-1HL locomotor pattern. In support, our immunohistochemical analysis of the lumbar spinal cord demonstrated the presence of the 5-HT7 immunoreactive cells in the lamina VII, which were rarely 5-HT2A immunoreactive

Serotonin fiber length.

<p>(A) Serotonin fiber length (in pixels) at the lumbar spinal cord level (below the area of hemisection) in the ipsi- and contralateral sides before and at consecutive time points after injury (mean±SEM). (B) Expanded scale of the diagram in (A) to show changes in serotonin fiber length (in pixels) in the lumbar spinal cord level (below the area of hemisection) in the ipsilateral side at consecutive time points after the injury (mean±SEM). Abbreviations: w—weeks post operation, m—months post operation. * − <i>p</i><0.05, ** − <i>p</i><0.01, *** − <i>p</i><0.001.</p

Changes in speed and spatial parameters induced by spinal cord hemisection.

<p>(A) Changes in the hindlimb base of support (BOS) before and at consecutive time points after hemisection (mean±SEM), (B) changes in hindlimb abduction (HA) before and at consecutive time points after hemisection (mean±SEM), (C) changes in the relative print distance (RPD) before and in the consecutive time points after spinal cord hemisection (mean±SEM), (D) changes in locomotor speed (V) before and at consecutive time points after hemisection (mean±SEM). Abbreviations: w—weeks post operation, m—months post operation, * − <i>p</i><0.05, ** − <i>p</i><0.01, *** − <i>p</i><0.001.</p

LFP Oscillations in the Mesencephalic Locomotor Region during Voluntary Locomotion

Oscillatory rhythms in local field potentials (LFPs) are thought to coherently bind cooperating neuronal ensembles to produce behaviors, including locomotion. LFPs recorded from sites that trigger locomotion have been used as a basis for identification of appropriate targets for deep brain stimulation (DBS) to enhance locomotor recovery in patients with gait disorders. Theta band activity (6–12 Hz) is associated with locomotor activity in locomotion-inducing sites in the hypothalamus and in the hippocampus, but the LFPs that occur in the functionally defined mesencephalic locomotor region (MLR) during locomotion have not been determined. Here we record the oscillatory activity during treadmill locomotion in MLR sites effective for inducing locomotion with electrical stimulation in rats. The results show the presence of oscillatory theta rhythms in the LFPs recorded from the most effective MLR stimulus sites (at threshold ≤60 μA). Theta activity increased at the onset of locomotion, and its power was correlated with the speed of locomotion. In animals with higher thresholds (&gt;60 μA), the correlation between locomotor speed and theta LFP oscillations was less robust. Changes in the gamma band (previously recorded in vitro in the pedunculopontine nucleus (PPN), thought to be a part of the MLR) were relatively small. Controlled locomotion was best achieved at 10–20 Hz frequencies of MLR stimulation. Our results indicate that theta and not delta or gamma band oscillation is a suitable biomarker for identifying the functional MLR sites