Recovery of motoneuron and locomotor function after spinal cord injury depends on constitutive activity in 5-HT2C receptors

Abstract

Muscle paralysis after spinal cord injury is partly caused by a loss of brainstem-derived serotonin (5-HT), which normally maintains motoneuron excitability by regulating crucial persistent calcium currents. Here we examine how over time motoneurons compensate for lost 5-HT to regain excitability. We find that, months after a spinal transection in rats, changes in post-transcriptional editing of 5-HT2C receptor mRNA lead to increased expression of 5-HT2C receptor isoforms that are spontaneously active (constitutively active) without 5-HT. Such constitutive receptor activity restores large persistent calcium currents in motoneurons in the absence of 5-HT. We show that this helps motoneurons recover their ability to produce sustained muscle contractions and ultimately enables recovery of motor functions such as locomotion. However, without regulation from the brain, these sustained contractions can also cause debilitating muscle spasms. Accordingly, blocking constitutively active 5-HT2C receptors with SB206553 or cyproheptadine, in both rats and humans, largely eliminates these calcium currents and muscle spasms, providing a new rationale for antispastic drug therapy.

Figure 1

Constitutive 5-HT₂ receptor activity, but not residual 5-HT, causes spasms. (a) Schematic of tail spasm in an awake chronic spinal rat with S2 sacral transection. (b) Representative immunofluorescence images of 5-HT fibers (beaded) in the S4 ventral horn of normal rats (top; mn, motoneuron, n = 5 rats) and chronic spinal rats (bottom; the arrow indicates a residual fiber, n = 5; scale bar, 50 μm). (c,d) Spasms in chronic spinal rat evoked by cutaneous electrical stimulation of the tail (pulse three times the threshold (3×T)) and recorded with EMG (quantified during the length of time indicated by the bar, LLR) before and after blocking effects of residual 5-HT with i.t. injection of the neutral antagonist SB242084 (3 mM in 30 μl saline). (d) Lack of spasm (LLR) after blocking constitutive receptor activity with the inverse agonist SB206553 (i.t., 3 mM in 30 μl saline). (e) Tail flexion angle during spasms before and after SB206553 injection, quantified during the length of time indicated by the bar. (f) Group means of spasms (normalized to predrug control) with SB242084 (abbreviated SB242; LLR), SB206553 (SB206 for LLR EMG recording; and SB206+ for tail-angle spasms) and cyproheptadine (cypro; LLR; 10 mg per kg body weight, orally), and after depletion of residual 5-HT with para-chlorophenylalanine-methylester (pCPA) (two 300 mg per kg body weight intraperitoneal injections over 48 h; tail-angle), with n = 5 rats per drug. (g,h) Normalized group means of SLR and background EMG with SB242084 and SB206553. **P < 0.01 relative to predrug control, 100%. Error bars indicate s.e.m.

Figure 2

Constitutive 5-HT₂ receptor activity contributes to LLRs in the isolated spinal cord in vitro. (a) Whole sacrocaudal spinal cord below chronic S2 transection maintained in vitro. (b) Long-lasting reflex triggered by dorsal root stimulation (single pulse, 3×T) and recorded from the ventral roots (LLR, quantified during the length of time indicated by the horizontal bar; counterpart of spasms in Figure 1) before and after blocking effects of residual 5-HT with the neutral 5-HT₂ receptor antagonist SB242084 (3–5 μM). (c) Elimination of LLR, but not SLR, after blocking constitutive 5-HT₂ receptor activity with the inverse agonist SB206553 (3–5 μM). Inset, SLR (expanded time scale). (d) Group means of LLRs (normalized to predrug LLRs) with SB242084 (abbreviated SB242, n = 11), methysergide (Methys, 10 μM, neutral antagonist, n = 12), SB206553 (SB206, n = 24), cyproheptadine (Cypro, 20 μM; n = 6), and SB206553 after prior application of methysergide (30 μM; white bar; Methy+SB206; n = 8). (e,f) Normalized group means of the SLR and background ventral root activity with SB206553 and SB242084. *P < 0.05, **P < 0.01 relative to control, 100%. Error bars indicate s.e.m.

Figure 3

Constitutively active 5-HT₂ receptors on motoneurons contribute to Ca²⁺ PICs underlying spasms. (a,b) Intracellular recording from motoneuron (mn) in whole spinal cord, in vitro. (b) Top, Ca²⁺ PIC in motoneuron of chronic spinal rat, activated by slowly increasing the membrane potential under voltage-clamp in presence of 2 μM tetrodotoxin (TTX) and quantified at its initial peak, where it produced a downward deflection in the recorded current (thick black plot, at arrow, Ca²⁺ PIC) relative to the leak current (thin line). Bottom plot, small Ca²⁺ PIC after SB206553 application (5 μM). (c) Ca²⁺ PIC in another motoneuron (arrow), which is unaffected by SB242084 application (5 μM). (d) Top, PIC-mediated plateau and sustained firing (LLR) evoked by dorsal root stimulation (3×T; without TTX) in a motoneuron at rest (without injected current; top). Bottom, with a hyperpolarizing bias current to prevent PIC activation, the same stimulation only evoked a polysynaptic EPSP (lower plot). (e) Response of same motoneuron as in d to dorsal root stimulation after application of SB206553 (5 μM), at rest (top) and with a hyperpolarizing bias current (bottom). (f) Group means of Ca²⁺ PIC (normalized to predrug Ca²⁺ PIC in chronic spinal rats, control), with SB206553 (SB206; n = 7), cyproheptadine (cypro, 20 μM; n = 16) and SB242084 (SB242; n = 5) in chronic spinal rats and in acute spinal rats (white bar, no drugs, n = 7). (g) Normalized group means of EPSP amplitude (middle bar; control mean 4.4 mV) and duration (right bar, control 480 ms) with inverse agonists cyproheptadine or SB206553 (chronic). **P < 0.01 relative to control, 100%. Error bars represent s.e.m.

Figure 4

A highly constitutively active 5-HT_2C receptor isoform is upregulated with injury. (a) Schematic showing 5-HT_2C receptor with various isoforms produced by changing three amino acids on its intracellular loop (green; isoforms named by amino acid triplet). These three amino acids (underlined) are changed by post-transcriptional editing of RNA at five sites (A–E; adenosine editing), leading to various native receptor isoforms, of which the unedited isoform (INI) is most highly constitutively active. (b) Total 5-HT_2C receptor mRNA (normalized to an internal control, 18S rRNA) in chronic spinal rats (n = 6) and normal uninjured rats (n = 6). (c) Proportion of 5-HT_2C receptor mRNA with editing at sites A, B and D (editing efficiency) in chronic spinal and normal rats (C and E site editing efficiency < 30% and not changed, data not shown). (d) Distribution of 5-HT_2C receptor isoform mRNA in the spinal cord of normal and chronic spinal rats (15 isoforms detected; the five most prevalent are shown). (e) Comparison of change in INI isoform expression (top) and Ca²⁺ PIC (bottom, recorded in vitro) after chronic spinal injury. *P < 0.05, **P < 0.01, significant change with injury. Error bars indicate s.e.m.

Figure 5

5-HT₂ receptor inverse agonist blocks spasms in spinal cord injured humans. (a) Leg spasm triggered by brief electrical stimulation of the medial arch of the foot (3–5×T). TA, tibialis anterior. (b) Spasm recorded with tibialis anterior muscle surface EMG and quantified over the time windows indicated (LLR and SLR), before and 2 h after blocking constitutively active 5-HT₂ receptors with cyproheptadine (8 mg administered orally). The inset on a different scale shows SLR. (c) Gradual reduction in the spasms (LLRs), but not SLRs, over time after inverse agonist application. (d,e) Normalized group means for LLRs (d) and SLRs (e) with cyproheptadine (n = 7 subjects). **P < 0.01 relative to control, 100%. Error bars represent s.e.m.

Figure 6

Spontaneous recovery of locomotion in staggered-hemisected rats depends on constitutively active 5-HT₂ receptors. (a) Schematic of staggered-hemisection SCI, which transects all descending axons from the brain, including 5-HT neurons (white circles), but leaves local propriospinal neurons (black) that transverse the injury and help relay descending signals for initiation of locomotion (gray). (b) Rat walking with good weight support and toe clearance three weeks after the staggered-hemisection (after second hemisection). (c) Same rat with little hindlimb weight support (just foot paddling motions), while the forelimbs dragged the hindquarters during walking after blocking constitutively active 5-HT₂ receptors with SB206553 (3 mM in 30 μl saline, i.t.; same dose as in Fig. 1). Scale bar, 2 cm. (d) Group means of BBB locomotor scores before and after SB206553 injection (n = 8) and control SB242084 injection (3 mM in 30 μl saline, i.t.; n = 8 rats). **P < 0.01 relative to preinjection. Error bars represent s.e.m.