Distribution and localization of 5-HT(1A) receptors in the rat lumbar spinal cord after transection and deafferentation

Abstract

The serotonergic system is highly plastic, capable of adapting to changing afferent information in diverse mammalian systems. We hypothesized that removing supraspinal and/or peripheral input would play an important role in defining the distribution of one of the most prevalent serotonergic receptors, the 5-HT(1A) receptor (R), in the spinal cord. We investigated the distribution of this receptor in response to a complete thoracic (T7-T8) spinal cord transection (eliminating supraspinal input), or to spinal cord isolation (eliminating both supraspinal and peripheral input) in adult rats. Using two antibodies raised against either the second extracellular region (ECL(2)) or the third intracellular region (ICL(3)) of the 5-HT(1A)R, we compared the 5-HT(1A)R levels and distributions in specific laminae of the L3-L5 segments among the control, spinal cord-transected, and spinal cord-isolated groups. Each antibody labeled different populations of 5-HT(1A)R: ECL(2) labeled receptors in the axon hillock, whereas ICL(3) labeled receptors predominantly throughout the soma and proximal dendrites. Spinal cord transection increased the number of ECL(2)-positive cells in the medial region of laminae III-IV and lamina VII, and the mean length of the labeled axon hillocks in lamina IX. The number of ICL(3)-labeled cells was higher in lamina VII and in both the medial and lateral regions of lamina IX in the spinal cord-transected compared to the control group. In contrast, the length and number of ECL(2)-immunolabeled processes and ICL(3)-immunolabeled cells were similar in the spinal cord-isolated and control groups. Combined, these data demonstrate that the upregulation in 5-HT(1A)R that occurs with spinal cord transection alone is dependent on the presence of sensory input.

FIG. 1.

Differential topography of 5-HT_1AR detected with an intracellular (ICL₃) and an extracellular (ECL₂) epitope-recognizing antibody. Panels a–c are images projected from 30 single plane 1 μm confocal images of L4 lamina IX neurons in a control rat labeled for both ECL₂ and ICL₃ antibodies, whereas d–g are single plane (1 μm) images of an L4 control motoneuron. The ECL₂ antibody stained primarily the axon hillock and initial segment (arrows in b, c, e, f, and g) of the axon, while the cell body (double arrows in b) showed weak, diffuse staining. The ICL₃ antibody detected 5-HT_1AR proteins primarily on proximal dendrites (asterisks in a, c, d, f, and g), and on the cell body (arrowheads in a, c, d, f, and g). Motoneurons were identified by HSP27 (f and g) (scale bars for a–c = 20 μm, and d–g = 10 μm). (Color image is available online at www.liebertpub.com/jon.)

FIG. 2.

Laminae distribution of 5-HT_1AR immunoreactivity. Regions 1–5 on the schematic spinal cord cross-section show areas of quantification for 5-HT_1AR staining: the lateral region of laminae III–IV (1), the medial region of laminae III–IV (2), the medial cell column of lamina VII (3), the lateral region of lamina IX (4), and the medial region of lamina IX (5). Fluorescence microscopy images of ECL₂ immunostaining (a–e) and ICL₃ immunostaining (f–j) are from a control L4 spinal cord section. Scale bar in e = 20 μm and applies to a–e; scale bar in g = 10 μm and applies to f–h; scale bar in i = 25 μm and applies to i and j.

FIG. 3.

The number of 5-HT_1AR-positive cell bodies and axon hillocks in spinal cord segments L3–L5 are reduced by the elimination of supraspinal and/or afferent input. Graphs a and b show quantitative changes of ECL₂ (a) or ICL₃ (b) 5-HT_1AR immunoreactivity in the same regions shown in Figure 2 in control (Con; n = 6), spinal cord–transected (ST, supraspinal input eliminated; n = 6), and spinal cord–isolated (SI, supraspinal and afferent inputs eliminated; n = 6) rats. Values are mean ± SEM (*, ^† indicate significantly different from Con and ST rats, respectively, at p < 0.05).

FIG. 4.

Elimination of supraspinal and afferent inputs affects the distribution of 5-HT_1AR proteins in the axon hillock. The mean length of ECL₂-positive axon hillocks from confocal projected images in control (Con), spinal cord–transected (ST), and spinal cord–isolated (SI) rats in the lateral and medial regions of lamina IX is shown. The 5-HT_1AR protein is distributed over a longer length of axon hillocks in ST compared to Con and SI rats in both regions. Values are mean ± SEM, n = 6 rats/group, using the mean of 50 neurons/rat examined in each region (* and ^† indicate significantly different from Con and ST, respectively, at p < 0.01).

FIG. 5.

Summary of 5-HT_1AR distribution in the spinal cord in response to elimination of supraspinal and/or afferent inputs. Significant changes are shown for the number of 5-HT_1AR ECL₂-positive processes (black), ICL₃-positive cell bodies (white) for all five regions, and the length of ECL₂-positive processes (gray) for lamina IX. Arrows and equal signs signify significant increases (up arrows), or the absence (=) of significant changes (p < 0.05). Changes of 5-HT_1AR immunoreactivity in spinal cord–transected (ST) relative to control (Con) (a), spinal cord–isolated (SI) relative to Con (b), and ST relative to SI (c) are shown schematically on a spinal cord cross-section.