Up-regulation of 5-HT₂ receptors is involved in the increased H-reflex amplitude after contusive spinal cord injury

Abstract

The amplitude of the H-reflex increases chronically after incomplete SCI and is associated with the development of exaggerated hindlimb reflexes. Although the mechanism for this increased H-reflex is not clear, previous studies have shown that pharmacological activation of the 5-HT₂ receptors (5-HT₂R) can potentiate the monosynaptic reflex. This study tested the hypothesis that increased expression of 5-HT₂R on motoneurons is involved in increased H-reflex amplitude after a standardized clinically relevant contusive SCI. Adult female rats were subjected to contusion, complete surgical transection, or a T8 laminectomy only. At 4 weeks after surgery, H-reflex recordings from the hindpaw plantar muscles of contused rats showed twice the amplitude of that in laminectomy controls or transected rats. To probe the role of 5-HT₂R in this increased amplitude, dose–response studies were done with the selective antagonists mianserin or LY53857 and the 5-HT₂R agonist (±)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI). The drugs were intrathecally infused into the lumbar cord while recording the H-reflex. Mianserin did not have any significant effects on the H-reflex after transection, consistent with the loss of distal serotonergic innervation. After contusion, both 5-HT₂R antagonists reduced the H-reflex reflex amplitude with a significantly higher ID₅₀ compared to the uninjured controls. The 5-HT₂R agonist DOI significantly increased reflex amplitude in contused but not control rats. Furthermore, while 5-HT immunoreactivity was similar, contused rats displayed increased 5-HT_2AR immunoreactivity in plantar muscle motoneurons compared to uninjured controls. We conclude that increased expression of 5-HT₂R is likely to be involved in the enhanced H-reflex that develops after contusive SCI.

Introduction

Segmental hindlimb reflexes located in the lumbar spinal cord are heavily influenced by descending and ascending pathways (Carpenter et al., 1966, Chen et al., 2001). When these pathways are disrupted after spinal cord injury (SCI), abnormalities of the local lower limb reflexes often develop (Hornby et al., 2004, Thompson et al., 1992). One of the most commonly studied local reflexes after SCI is the monosynaptic connection between the primary muscle afferents and the alpha motoneurons, which is often studied in patients and animals using the Hoffman (H)-reflex. The H-reflex is elicited by an electrical stimulation of a peripheral nerve that innervates the muscle from which the reflex is recorded. A single stimulus generates a short-latency M-wave, resulting from direct stimulation of the motor axons innervating the muscle, and a long-latency H-wave, which is a measure of the alpha motoneurons activated by Ia afferents (Gozariu et al., 1998).

Properties of the H-reflex are known to be affected in both humans and rodents after SCI (Lee et al., 2005, Schindler-Ivens and Shields, 2000, Thompson et al., 1998, Valero-Cabre and Navarro, 2001). One of these alterations is elevated reflex amplitude that develops over time after SCI (Lee et al., 2005, Thompson et al., 1998). The mechanism for this elevated reflex is still unclear. Our previous study demonstrated that the increased H-reflex amplitude observed at 4 weeks after different severities of contusive SCI in rats is positively correlated with serotonin immunoreactivity around motoneurons involved in the reflex (Lee et al., 2005). That is, the greater their apparent chronic serotonergic (re)innervation, the more abnormally elevated their reflex activation. Pharmacological activation of 5-HT₂ receptors (5-HT₂R) has been shown to potentiate the monosynaptic reflex and restore the excitability of the extensor stretch reflex in spinalized rat and cat preparations, respectively (Hasegawa and Ono, 1996, Miller et al., 1996), in which serotonergic innervation has been completely lost. However, whether alterations in 5-HT₂ receptors are involved in the elevated H-reflex after SCI, particularly after clinically relevant incomplete contusive SCI, has not previously been examined.

We therefore investigated the role of 5-HT₂R in the increased H-reflex amplitude observed after a standardized incomplete contusive SCI using a well-characterized rat model of injury (Basso et al., 1996, Lee et al., 2005, Pikov and Wrathall, 2001). We employed local intrathecal administration of the 5-HT₂R antagonists mianserin and LY53857 and the agonist DOI as well as immunohistochemistry of 5-HT₂R expression on plantar motor neurons and found evidence in support of the hypothesis that increased 5-HT₂R expression by plantar muscle motoneurons is causally related to the increased H-reflex amplitude of the plantar muscle after contusive SCI.