Characterization of dendritic morphology and neurotransmitter phenotype of thoracic descending propriospinal neurons after complete spinal cord transection and GDNF treatment

Abstract

After spinal cord injury (SCI), poor regeneration of damaged axons of the central nervous system (CNS) causes limited functional recovery. This limited spontaneous functional recovery has been attributed, to a large extent, to the plasticity of propriospinal neurons, especially the descending propriospinal neurons (dPSNs). Compared with the supraspinal counterparts, dPSNs have displayed significantly greater regenerative capacity, which can be further enhanced by glial cell line-derived neurotrophic factor (GDNF). In the present study, we applied a G-mutated rabies virus (G-Rabies) co-expressing green fluorescence protein (GFP) to reveal Golgi-like dendritic morphology of dPSNs. We also investigated the neurotransmitters expressed by dPSNs after labeling with a retrograde tracer Fluoro-Gold (FG). dPSNs were examined in animals with sham injuries or complete spinal transections with or without GDNF treatment. Bilateral injections of G-Rabies and FG were made into the 2nd lumbar (L2) spinal cord at 3 days prior to a spinal cord transection performed at the 11th thoracic level (T11). The lesion gap was filled with Gelfoam containing either saline or GDNF in the injury groups. Four days post-injury, the rats were sacrificed for analysis. For those animals receiving G-rabies injection, the GFP signal in the T7-9 spinal cord was visualized via 2-photon microscopy. Dendritic morphology from stack images was traced and analyzed using a Neurolucida software. We found that dPSNs in sham injured animals had a predominantly dorsal-ventral distribution of dendrites. Transection injury resulted in alterations in the dendritic distribution with dorsal-ventral retraction and lateral-medial extension. Treatment with GDNF significantly increased the terminal dendritic length of dPSNs. The density of spine-like structures was increased after injury, and treatment with GDNF enhanced this effect. For the group receiving FG injections, immunohistochemistry for glutamate, choline acetyltransferase (ChAT), glycine, and GABA was performed in the T7-9 spinal cord. We show that the majority of FG retrogradely-labeled dPSNs were located in the Rexed Lamina VII. Over 90% of FG-labeled neurons were glutamatergic, with the other three neurotransmitters contributing less than 10% of the total. To our knowledge this is the first report describing the morphologic characteristics of dPSNs and their neurotransmitter expressions, as well as the dendritic response of dPSNs after transection injury and GDNF treatment.

Keywords: Dendrite; Descending propriospinal neuron; GDNF; Rabies virus; Spinal cord injury; Spine.

Figure 1

(A) Schematic diagram of the descending propriospinal tract system (dPST). dPST pathways descending several spinal segments are located in both the ventral and ventral lateral funiculi (VF and VLF, respectively). The dPST projects through either contralateral or ipsilateral VF or VLF and innervates motoneuron pools directly or indirectly through interneurons. dPST neurons receive convergent supraspinal innervation, including those from the corticospinal (CST) and rubrospinal (RST) tracts. Descending propriospinal neurons are indicated in green, interneurons in brown and motoneurons in purple. (B, C) Schematic diagrams of the experimental designs for the dendritic morphology study (B) and the neurotransmitter study (C). The three figures in each panel, from left to right, show propriospinal neurons were first retrogradely infected by a G-mutated rabies virus (green particles) that expressed green fluorescence protein which filled the dendritic compartments of dPST neurons (B) or retrogradely labeled by FluoroGold (FG) (blue particles) (C). Spinal cords then received either a transection injury or a transection + glial cell line-derived neurotrophic factor (GDNF) (red dots) applied to the lesion site to be retrogradely transported to the soma.

Figure 2

Camera lucida reconstructions of three propriospinal neurons (rows A, B, C) with different dendritic patterns as if viewed in the transverse (D), parasagittal (E), and horizontal (F) planes. Row A shows a dPSN that extended its dendritic branches in medial, lateral, ventral and dorsal directions. Row B shows a dPSN that extended its dendritic branches predominantly in medial and lateral directions. Row C shows a dPSN that has more dendritic branches extending in ventral and dorsal directions. Scale bar: 100μm.

Figure 3

Percentage of dendritic distribution of dPSNs in four quadrant areas. (A) Schematic drawing of a spinal cord transverse section, light grey area corresponds to lamina VII in which all of the dPSNs in this study were located. A representative neuron is indicated at right in lamina VII. (B, C) The area surrounding the soma was separated into four quadrants. We defined these four quadrants as lateral: 320°~50°; dorsal: 50°~140°; medial: 140°~230°; ventral: 230°~320°. (D) Comparison of percentage distribution of dendrites among the sham, transected (TX) and TX + GDNF groups. * p < 0.05 TX + GDNF lateral versus sham lateral; ** p < 0.01 TX lateral versus sham lateral; & p < 0.05 TX dorsal or TX + GDNF dorsal versus sham dorsal; ## p < 0.01 sham medial versus sham dorsal; ### p < 0.001 sham lateral versus sham dorsal; $ p < 0.05 sham ventral versus sham medial; $$$ p < 0.001 sham ventral versus sham lateral..

Figure 4

Comparison of the dendritic features of the 36 GFP-labeled and fully reconstructed dPSN neurons from sham (n=15), transection injury (TX, n=10) and TX + GDNF treatment groups (n=11). (A) total dendritic length; (B) dendritic interval length; (C) terminal dendritic length; (D) number of dendritic end branches/cell; (E) number of total dendritic branches; (F) number of dendritic nodes; (G) number of terminal dendritic branches; and (H) maximal order of dendritic branches. *: p < 0.05; **: p < 0.01, Compared to sham group. Bar heights represent means ± SD.

Figure 5

Morphological plasticity of dendritic spine-like structures of dPSNs after axotomy injury treated with or without GDNF. (A) Schematic drawings and representative images of the five different types of spine-like structures. Comparisons of the number (B), density (C), number by branch order, (D) percentage of types of spine-like structures overall and by the different types across the sham, TX, and TX + GDNF groups. * p < 0.05 compared with sham group. ** p < 0.01 compared with sham group. Bar heights represent means ± SD.

Figure 6

Co-localizations of retrograde tracer FluoroGold (FG) with neurotransmitter markers in sub-populations of dPSNs. (A-C) The majority of neurons labeled by FG were glutamatergic (white arrow, red glutamate, blue FG), (D-F) a small percentage of neurons labeled by FG were ChAT-positive neurons (white arrow, red ChAT, blue FG). (G-I) a small percentage of neurons labeled by FG were GABAergic neurons (white arrow, green GABA, blue FG). (J-L) a small percentage of neurons labeled by FG were glycinergic neurons (white arrow, green glycine, blue FG). Scale bar: 10μm.

Figure 7

(A) Percentage distribution of subpopulations of four different neurotransmitters in the sham group. The majority of FG-labeled dPSNs are glutamatergic neurons. *** p <0.001 compared with glutamatergic neurons. (B) Total number of FG-labeled neurons in the T7 and T9 spinal segments in sham animals and after spinal transection (TX) with or without GDNF treatment. The number of FG-labeled cells is decreased in T9 spinal cord after transection injury regardless of treatment with GDNF. *** p < 0.001 compared with sham group.