Parvalbumin-positive primary afferent projections to motoneurons increase after complete spinal transection in neonatal and juvenile rats

Abstract

Hindlimb locomotor activity spontaneously recovers after complete spinal cord transection (CST) in neonatal rats, but not in juvenile rats. A previous study in neonatal rats that underwent CST at the thoracic level demonstrated that primary afferent projections increase in the ventral horn and intermediate zone at the lumbar level. It remains unclear whether primary afferent terminals of motoneurons increase and whether primary afferent projections to the spinal cord are altered after CST in juvenile rats. Here, we used biotinylated dextran amine as a tracer to demonstrate that primary afferent projections to the ventral horn and intermediate zone were significantly increased in rats that underwent CST in the juvenile period compared to intact rats of the same age. We then examined Ⅰa afferents using immunohistochemistry for parvalbumin. Our findings revealed an increase in parvalbumin-immunoreactive terminals on motoneurons in both neonatal and juvenile rats after CST compared to intact rats of the same age. These results suggest that proprioceptive afferent terminals on motoneurons are increased after CST in both neonatal and juvenile rats. In neonatal rats, this increase might contribute to the spontaneous recovery of hindlimb motor activity after CST, whereas in juvenile rats, the increase in proprioceptive afferent terminals on motoneurons does not contribute to recovery following CST.

Keywords: Juvenile; Motoneurons; Neonate; Parvalbumin; Primary afferents; Spinal cord injury.

Fig. 1

Localization of BDA-labeled primary afferent fibers in the intermediate zone (IZ), lateral part of the ventral horn (VHl) and medial part of the ventral horn (VHm) of the L5 spinal cord. A-D: Images of intact rats at P40 (A, B) and P20ST rats at P40 (C, D). BDA was administered into the left DRG 1 week prior (P33) to perfusion. Arrowheads in B and D indicate terminal buttons. Asterisks: central canal. Scale bars: 100 μm in C (apply also to A), and 50 μm in D (apply also to B). E, F: Total axonal length (E) and the number of terminal buttons (F) of BDA-labeled fibers in the IZ, VHl, and VHm were significantly higher in P20ST rats than in intact rats. Data are expressed as mean ± SEM. Significant differences are indicated by * * (p < 0.01) or * (p < 0.05).

Fig. 2

Representative parvalbumin-immunoreactivity in the L5 spinal cord of a P5ST rat. Parvalbumin-immunoreactive fibers (green) were observed in the IZ, and around ChAT-positive motoneurons (red) in the ventral horn. Inset: a higher magnification image of parvalbumin-immunoreactive terminals (arrowheads) on ChAT-positive motoneurons. CC: central canal. Scale bars: 100 μm, and 50 μm in the inset.

Fig. 3

Parvalbumin-immunoreactive nerve terminals on ChAT-immunoreactive neurons in the L5 ventral horn. A, B: Images of an intact rat at P19 (A), and P34 rats (B). C, D: Images of a rat with CST at P5 (P5ST rats, C), and P20 (P20ST rats, D). Arrowheads indicate parvalbumin-nerve terminals on ChAT-immunoreactive neurons. E, F: Triple labeling of parvalbumin (red), ChAT (blue), and synapsin Ⅰ (green). The parvalbumin-immunoreactive nerve terminals (arrowheads in E) on ChAT-positive neurons were also positive for synapsin Ⅰ (arrowheads in F). Scale bars: 50 μm in A (applies also to B-D), and 50 μm in E (apply also to F). G, H: Number of parvalbumin-immunoreactive terminals on ChAT-immunoreactive neurons per section (G) and per neuron (H). Data are expressed as mean ± SEM. Dots indicate individual data points. Significant differences are indicated by * * (p < 0.01) or * (p < 0.05).