Advantages of delaying the onset of rehabilitative reaching training in rats with incomplete spinal cord injury

We have previously reported that rehabilitative reaching training initiated 4 days following an incomplete cervical spinal cord injury (SCI) in adult rats promotes plasticity and task-specific recovery. This training, however, also resulted in impairments in an untrained task. Here we examined whether delaying the rehabilitative training following cervical SCI is still effective in promoting task-specific recovery, but circumvents impairments in an untrained task, comparable to what has been reported in stroke models. Therefore, reaching training for a period of 6 weeks was initiated at Day 12 following a cervical dorso-lateral quadrant lesion. Thereupon the rats' ability to reach and to walk on a horizontal ladder (i.e. the untrained task) was assessed, and 8 weeks post-injury cortical map changes were investigated through microstimulation. Further, we examined changes in phospho protein kinase A (pPKA) levels following an immediate and a delayed onset of reaching training in rats with cervical SCI. We found that delayed rehabilitative training was comparably effective as immediate training in promoting task-specific recovery and sprouting of injured axons. Importantly, delayed training did not impair the performance on horizontal ladder walking. Strikingly, only delayed reaching training restored cortical PKA levels that had dropped significantly over 2 weeks post-injury. Additionally, delayed training did not influence cortical map changes following injury, but decreased white matter damage. In conclusion, our results show that a short delay in the onset of training in a forelimb task significantly alters our outcome measures, which should be considered in future rehabilitative approaches

Characterization of A11 neurons projecting to the spinal cord of mice.

The hypothalamic A11 region has been identified in several species including rats, mice, cats, monkeys, zebrafish, and humans as the primary source of descending dopamine (DA) to the spinal cord. It has been implicated in the control of pain, modulation of the spinal locomotor network, restless leg syndrome, and cataplexy, yet the A11 cell group remains an understudied dopaminergic (DAergic) nucleus within the brain. It is unclear whether A11 neurons in the mouse contain the full complement of enzymes consistent with traditional DA neuronal phenotypes. Given the abundance of mouse genetic models and tools available to interrogate specific neural circuits and behavior, it is critical first to fully understand the phenotype of A11 cells. We provide evidence that, in addition to tyrosine hydroxylase (TH) that synthesizes L-DOPA, neurons within the A11 region of the mouse contain aromatic L-amino acid decarboxylase (AADC), the enzyme that converts L-DOPA to dopamine. Furthermore, we show that the A11 neurons contain vesicular monoamine transporter 2 (VMAT2), which is necessary for packaging DA into vesicles. On the contrary, A11 neurons in the mouse lack the dopamine transporter (DAT). In conclusion, our data suggest that A11 neurons are DAergic. The lack of DAT, and therefore the lack of a DA reuptake mechanism, points to a longer time of action compared to typical DA neurons

A11 TH positive neurons are also expressing aromatic amino acid decarboxylase (AADC).

<p>Immunohistochemistry targeted against TH (green) and AADC (magenta) shown in a representative middle region section of A11 (A, B) and the locus coeruleus (C). Note the co-localization between TH and AADC positive neurons (arrows) in A11 (B). Locus coeruleus served as a positive control (C). Scale bar 50 µm. A and B are derived from data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109636#pone-0109636-g001" target="_blank">Figure 1</a>. B: Higher magnification of boxed area in A.</p

YFP positive fibers in the lumbar spinal cord originating from A11.

<p>Schematic of fiber localization in the lumbar spinal cord. Representative micrographs of YFP-labeled fibers in transverse (A, B) and parasagittal (C, D) lumbar spinal cord sections. Fibers were found in the dorsal (A) and ventral horn (B) as well as in the grey (C) and the white matter (D). Scale bar: 10 µm.</p

Characterization of tyrosine hydroxylase (TH) positive cells in A11.

<p>A: Diagram showing the middle region of the A11 area in the mouse. The red frame represents the area where representative micrographs were taken. The diagram was adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109636#pone.0109636-Paxinos1" target="_blank">[24]</a> with permission. B: TH immunohistochemistry (green) of the middle region of A11. Scale bar 100 µm. 3 V = third ventricle, PF = parafascicular thalamic nucleus, fr = fasciculus retroflexus, PH = posterior hypothalamic nucleus, mt = mammillothalamic tract. C: Diameter of TH positive cells in three regions - rostral (AP −2.0 mm), middle (AP −2.3 mm) and caudal region (AP −2.5 mm). The mean cell diameter was 16.7±0.3 µm and the cell diameters between sections were similar.</p

A11 TH positive neurons project to the spinal cord.

<p>Example of retrograde labelling in the A11 caudal area following FluoroGold (FG) injections into the lumbar spinal cord between lumbar vertebral segments L4 and L5. Representative double-fluorescent immunostaining of tyrosine hydroxylase (TH; green) and FluoroGold (FG; magenta). The white arrows point to double-labeled cells. Most of the TH positive cells also contain FG (arrows), one cell shows labelling for TH but not FG. Several cells that contain FG but not TH are also seen (not co-localizing). Scale bar: 50 µm.</p

YFP expression in A11 neurons following Cre-dependent viral expression and retrograde tracing with FG from the spinal cord.

<p>Rostral region section of A11 from a TH-Cre mouse transduced with a Cre-dependent YFP AAV (green) and immunohistochemistry against FG (magenta) (A–D). Boxed inset in (B (b)) shows FG labeled cells in the motor cortex. Only cells in A11 were co-labeled with YFP and FG (C, D). Scale bars: 50 µm. D: Higher magnification of boxed area in A. Scale bar: 20 µm.</p