Chronic Spinal Cord Injury Reduces Gastrin-Releasing Peptide in the Spinal Ejaculation Generator in Male Rats

Abstract

Spinal cord injury (SCI) causes sexual dysfunction, including anejaculation in men. Likewise, chronic mid-thoracic contusion injury impairs ejaculatory reflexes in male rats. Ejaculation is controlled by a spinal ejaculation generator (SEG) comprised of a population of lumbar spinothalamic (LSt) neurons. LSt neurons co-express four neuropeptides, including gastrin-releasing peptide (GRP) and galanin and control ejaculation via release of these peptides in lumbar and sacral autonomic and motor nuclei. Here, we tested the hypothesis that contusion injury causes a disruption of the neuropeptides that are expressed in LSt cell bodies and axon terminals, thereby causing ejaculatory dysfunction. Male Sprague Dawley rats received contusion or sham surgery at spinal levels T6-7. Five to six weeks later, animals were perfused and spinal cords were immunoprocessed for galanin and GRP. Results showed that numbers of cells immunoreactive for galanin were not altered by SCI, suggesting that LSt cells are not ablated by SCI. In contrast, GRP immunoreactivity was decreased in LSt cells following SCI, evidenced by fewer GRP and galanin/GRP dual labeled cells. However, SCI did not affect efferent connections of LSt, cells as axon terminals containing galanin or GRP in contact with autonomic cells were not reduced following SCI. Finally, no changes in testosterone plasma levels or androgen receptor expression were noted after SCI. In conclusion, chronic contusion injury decreased immunoreactivity for GRP in LSt cell soma, but did not affect LSt neurons per se or LSt connections within the SEG. Since GRP is essential for triggering ejaculation, such loss may contribute to ejaculatory dysfunction following SCI.

Keywords: anejaculation; contusion injury; lumbar spinal cord; sexual dysfunction; urogenital.

FIG. 1.

Galanin and gastrin-releasing peptide (GRP) immunoreactivity in sham and spinal cord injury (SCI) animals in L3 and L4 spinal levels. Representative fluorescent microscope images demonstrating immunofluorescent labeling for galanin (red; A, D, G, J, M), GRP (green; B, E, H, K, N), and merged channels (C, F, I, L, O) in a sham (A-C and G-I) and chronic spinal contusion injury (SCI; D-F, J-L, M-O) animal showing lumbar spinothalamic cells in the L3 (A-I) and L4 (G-O) levels of the spinal cord. Images in J-O demonstrate the reduced GRP-immunofluorescent signal, with M-O showing cells at a higher magnification. cc, central canal. Scale bar indicates 50 (A-L) or 25 (M-O) μm. Color image is available online.

FIG. 2.

Lumbar spinothalamic (LSt) axon terminal distribution in spinal autonomic regions. Representative fluorescent microscope images showing immunofluorescent labeling of LSt axons for galanin (red; A, D, G, J), gastrin-releasing peptide (GRP; green; B, E, H, K), and merged channels (with NeuN immunoreactivity in blue; C, F, I, L) innervating the intermediolateral cell column (IML; A-C), central autonomic nucleus (CAN; D-F), sacral parasympathetic nucleus (SPN; G-I), and central gray area (Central Gray; J-K) in a representative sham animal. Scale bar indicates 50 μm. Color image is available online.

FIG. 3.

Spinal cord injury (SCI) reduces gastrin-releasing peptide (GRP) immunoreactivity in lumbar spinothalamic neurons. Quantitative analysis showing numbers of cells immunoreactive for galanin and GRP (Dual), or only for galanin (Galanin Only) or GRP (GRP Only) in the sham (Sham) or contusion spinal injury (SCI) groups. Data are shown as total numbers of cells that were counted (A) or as cells per analyzed section (B). Data are expressed as mean ± standard error of the mean. * indicates significant difference from Sham group.

FIG. 4.

Lumbar spinothalamic (LSt) axon terminals innervating autonomic preganglionic neurons. Representative confocal images (1-μm optical sections) in a sham animal showing galanin-immunoreactive (red) and synaptophysin-immunoreactive (blue) axon terminals in close proximity to ChAT-immunoreactive neurons (green) in intermediolateral cell column (IML; A), central autonomic nucleus (CAN; B), and sacral parasympathetic nucleus (SPN; C). An enlarged view at higher magnification (D) demonstrates axon terminals in close apposition to ChAT-ir soma membrane that are either immunoreactive for galanin and synaptophysin (magenta; red arrows), or synaptophysin only (blue; white arrows). Scare bars indicates 50 μm (A-C) or 5 μm (D). Schematic drawings (based on Paxinos Atlas80) of L1 (E), L4 (F), and L6 (G) demonstrate the anatomical locations of LSt cells and the areas of analysis of LSt axon terminal inputs to cells in IML, CAN, SPN, and central gray (CG). Color image is available online.

FIG. 5.

Spinal cord injury (SCI) did not affect lumbar spinothalamic axon terminal innervation of intermediolateral cell column (IML). Quantitative analysis of galanin- (A-G) or gastrin-releasing peptide (GRP)-immunoreactive (B-H) axon terminals in close apposition (Contacts) to ChAT-immunoreactive cells in IML in Sham (white bars and circles) and SCI (black bars and circles) groups. (A and B) Numbers of contacts are expressed per 100 μm of ChAT-ir soma membrane, for axon terminals co-labeled for synaptophysin (Syn) and galanin (A) or GRP (B), single labeled for Syn, and the sum of these synaptophysin and galanin (A) or GRP (B) and synaptophysin-only contacts (Total). C and D demonstrate a positive correlation of numbers of galanin- (C) or GRP-immunoreactive (D) contacts and the size of ChAT-ir cells using the Pearson Product-Movement Correlation (see Table 2 for statistical data). Line represents the slopes in Sham (black: Galanin - y = 0.7336x - 27.216, R² = 0.6215; GRP - y = 0.4116x - 16.013, R² = 0.787) and SCI (gray: Galanin - y = 0.8943x - 33.732, R² = 0.6949; GRP - y = 0.4533x - 17.593, R² = 0.6376) groups. E-H). Numbers of contacts immunoreactive for galanin (E) or GRP (F) on ChAT-ir cells with soma perimeters equal to or larger (E,F), or smaller than 60 μm (G,H). Data are expressed as ± standard error of the mean. * indicates significant difference from Sham group.

FIG. 6.

Spinal cord injury (SCI) did not affect lumbar spinothalamic axon terminal innervation of central autonomic nucleus (CAN) or sacral parasympathetic nucleus (SPN). Quantitative analysis of galanin- (A-C) or gastrin-releasing peptide (GRP)-immunoreactive (B-D) axon terminals in close apposition (Contacts) to ChAT-immunoreactive cells in Sham (white bars) and SCI (black bars) groups. Numbers of contacts are expressed per 100 μm of ChAT-ir soma membrane, for axon terminals co-labeled for synaptophysin and galanin (A, C) or GRP (B, D), single labeled for synaptophysin (Syn), and the sum of these dual labeled synaptophysin and galanin (A, C) or GRP (B, D) plus synaptophysin-only contacts (Total). Data are expressed as mean ± SEM.

FIG. 7.

Spinal cord injury (SCI) did not affect lumbar spinothalamic axon terminal innervation of CG neurons. Representative confocal image (1-μm optical sections) in a sham animal showing galanin- (A; red) and gastrin-releasing peptide (GRP)-immunoreactive (B; green) axon terminals in close proximity to a NeuN-immunoreactive neuron (blue) in Central Gray with merged channels shown in (C). White arrows indicate galanin and GRP dual labeled-axon terminals in close apposition to the soma; green arrow indicates a putative contact labeled for GRP only. Scale bar indicates 5 μm. (D) Quantitative analysis of axon terminals labeled for galanin-only (Galanin), GRP-only (GRP) or both galanin and GRP (Dual) in close apposition to NeuN-labeled soma of CG cells (Contacts) expressed per 100 μm of soma membrane. Data are expressed as mean ± standard error of the mean. * indicates significant difference from Sham group. Color image is available online.

FIG. 8.

Spinal cord injury (SCI) did not affect measures of testosterone release or action. Testes weights (A; in grams) and testosterone plasma concentrations (in ng/dL; B) in Sham (white bars) and SCI (black bars) groups. Data are expressed as mean ± standard error of the mean. Representative images from a sham (C, E, G) and SCI (D, F, H) animal showing chromogen labeling for androgen receptors (black nuclei) and galanin (brown) in lumbar spinothalamic cells (C, D), sacral nucleus of the bulbocavernosus (SNB; E-F), sacral parasympathetic nucleus (SPN), and central gray (CG; G-H). Arrows indicate general locations of SNB, SPN, and CG cells immunoreactive for androgen receptors. cc, central canal. Scale bars indicate 50 μm. Color image is available online.