Polysynaptic excitatory postsynaptic potentials that trigger spasms after spinal cord injury in rats are inhibited by 5-HT1B and 5-HT1F receptors

Abstract

Sensory afferent transmission and associated spinal reflexes are normally inhibited by serotonin (5-HT) derived from the brain stem. Spinal cord injury (SCI) that eliminates this 5-HT innervation leads to a disinhibition of sensory transmission and a consequent emergence of unusually long polysynaptic excitatory postsynaptic potentials (EPSPs) in motoneurons. These EPSPs play a critical role in triggering long polysynaptic reflexes (LPRs) that initiate muscles spasms. In the present study we examined which 5-HT receptors modulate the EPSPs and whether these receptors adapt to a loss of 5-HT after chronic spinal transection in rats. The EPSPs and associated LPRs recorded in vitro in spinal cords from chronic spinal rats were consistently inhibited by 5-HT(1B) or 5-HT(1F) receptor agonists, including zolmitriptan (5-HT(1B/1D/1F)) and LY344864 (5-HT(1F)), with a sigmoidal dose-response relation, from which we computed the 50% inhibition (EC(50)) and potency (-log EC(50)). The potencies of 5-HT receptor agonists were highly correlated with their binding affinity to 5-HT(1B) and 5-HT(1F) receptors, and not to other 5-HT receptors. Zolmitriptan also inhibited the LPRs and general muscle spasms recorded in vivo in the awake chronic spinal rat. The 5-HT(1B) receptor antagonists SB216641 and GR127935 and the inverse agonist SB224289 reduced the inhibition of LPRs by 5-HT(1B) agonists (zolmitriptan). However, when applied alone, SB224289, SB216641, and GR127935 had no effect on the LPRs, indicating that 5-HT(1B) receptors do not adapt to chronic injury, remaining silent, without constitutive activity. The reduction in EPSPs with zolmitriptan unmasked a large glycine-mediated inhibitory postsynaptic current (IPSC) after SCI. This IPSC and associated chloride current reversed at -73 mV, slightly below the resting membrane potential. Zolmitriptan did not change motoneuron properties. Our results demonstrate that 5-HT(1B/1F) agonists, such as zolmitriptan, can restore inhibition of sensory transmission after SCI without affecting general motoneuron function and thus may serve as a novel class of antispastic drugs.

Figures

Fig. 1.

Polysynaptic reflexes and their underlying excitatory postsynaptic potential (EPSP) in chronic spinal rats. A: a long-lasting reflex triggered by dorsal root stimulation [0.1-ms pulse, 3 times threshold (3×T)] and recorded from the ventral roots, with the reflex components long polysynaptic reflex (LPR) and long-lasting reflex (LLR) quantified during periods indicated by horizontal arrows (top trace). Inset: short polysynaptic reflex (SPR) on expanded time scale. Bottom trace shows elimination of LLR, but not LPR, after blocking the L-type Ca2+ channel with isradipine (15 μM). Bkg, background root activity. B: persistent inward current (PIC)-mediated plateau potential and sustained firing (LLR) evoked by dorsal root stimulation (3×T) in motoneuron at rest (without injected current; top trace). With a hyperpolarizing bias current to prevent PIC activation, the same stimulation only evoked polysynaptic EPSPs, with short and long EPSP components, corresponding to the SPR and the LPR (bottom trace).

Fig. 2.

5-HT1B receptor activity inhibits the polysynaptic reflexes in chronic spinal rats. A: long-lasting polysynaptic reflex triggered by dorsal root stimulation (0.1-ms pulse, 3×T) and recorded from the ventral roots, with LPR and LLR components indicated by horizontal bars. B: reduction of LPR and LLR with application of the 5-HT1B/1D/1F agonist zolmitriptan (300 nM; >50% reduction). C and D: reduction of LPR and LLR, respectively, with increasing zolmitriptan dose (decrease over ∼100-fold change in dose; left). Best-fit sigmoidal curves are shown with subsequent estimation of EC50. Prior application of a single blocking dose of the selective 5-HT1B antagonist SB224289 (5 μM) or the 5-HT1B/1D antagonist SB216641 (5 μM) antagonized the inhibitory action of zolmitriptan (shifting EC50 to the right). Each plot shows the typical response from a single rat, with a different rat for each condition, because agonists are not feasible to washout and repeat after antagonist application (taking many hours to wash).

Fig. 3.

Mixed 5-HT1 and 5-HT2 receptor agonists have a biphasic response, only inhibiting reflexes at high doses. A–C: dose-response relations for the 5-HT1 and 5-HT2 receptor agonist α-methyl-5-HT and 5-HT itself, with increased reflexes (LPR and LLR) at low doses (5-HT2 mediated) and decreased reflexes at high doses (5-HT1 mediated). In A and C, the heavy line is a sigmoidal curve fit to the inhibitory phase of the dose-response relation and used to estimate the EC50 for the 5-HT1 receptor-mediated inhibitory action. In B, the heavy line is a sigmoidal curve fit to the excitatory phase of the dose-response relation, mediated by 5-HT2 receptors. D: dose-response relation for the 5-HT effect on the LPR after 5-HT2 receptor block with methysergide (10 μM) and 5-HT3 receptor block with granisetron (GR; 0.3 μM), with a similar EC50 to that obtained in C.

Fig. 4.

Potency of 5-HT receptor agonists at inhibiting the LPR is only related to binding to 5-HT1B and 5-HT1F receptors. A: 5-HT1B receptor agonist potency (pEC50 = −log EC50) for inhibiting the LPR plotted against the agonist binding affinity to that receptor (pKi). Each agonist is indicated next to its data point: BW, BW723C86; Zolm, zolmitriptan; EMD, EMD386088. Thin line indicates significant linear correlation between potency and affinity (r = 0.96, P < 0.05, n = 6). Dashed line represents the best fit line with unit slope (potency = binding affinity + C, where C ≈ −1). B–D: similar potency-affinity scatter plots for the remaining 5-HT receptors. Thin line indicates significant linear correlation between agonist potency and affinity for 5-HT1F receptors (solid circles; r = 0.91, P < 0.05, n = 5). Dashed lines represent the unit slope line. Other receptors had no significant correlation between potency and affinity (open symbols; P > 0.05). ND and shaded zone indicate no detected effect of agonist on the LPR. Agonists used and affinities are listed in Table 1, with agonists assumed to act at a receptor only if Ki < 400 nM. Potencies are from Table 2. Potencies for 5-HT and zolmitriptan action in the presence of 5-HT1B antagonists were used (plotted) for comparison to 5-HT1D, 5-HT1E, and 5-HT1F receptor binding affinity, because these antagonists removed confounding effects of 5-HT1B receptors. Table 3 also summarizes agonists/antagonists used for each receptor.

Fig. 5.

5-HT1B/1D/1F agonist zolmitriptan inhibits the SPR. A: SPR evoked in the ventral root of a chronic spinal rat after dorsal root stimulation (0.1 ms, 3×T), quantified during the period indicated by the horizontal bar. B: inhibition of the SPR by zolmitriptan (300 nM).

Fig. 6.

Potency of 5-HT receptor agonists at inhibiting the SPR is only related to binding to 5-HT1B and 5-HT1F receptors. A: 5-HT1B receptor agonist potency (pEC50) for inhibiting the SPR plotted against the agonist binding affinity to that receptor (pKi). Format is identical to that described in Fig. 4. Thin line indicates significant linear correlation between potency and affinity (r = 0.95, P < 0.05, n = 6). Dashed line represents the best fit line with unit slope. B and C: similar potency-affinity scatter plots for the remaining 5-HT receptors. Thin line indicates significant linear correlation between agonist potency and affinity for 5-HT1F receptors (filled circles; r = 0.94, P < 0.05, n = 5). Dashed lines represent the unit slope line. The remaining receptors had no significant correlation between potency and affinity (open symbols; P > 0.05). ND and shaded zone indicate no detected effect of agonist on the LPR. Agonists used, potencies, and affinities are detailed in Fig. 4. Table 3 also summarizes agonists/antagonists used for each receptor.

Fig. 7.

5-HT1B receptor is not endogenously active in chronic spinal rats. A: a block of possible endogenous 5-HT1B receptor activity with SB224289 (3 μM, horizontal bar) produced no increase (or change) in the LPR or SPR. Reflexes were measured at about 15-min intervals (●). B: in contrast, SB224289 (3 μM) increased the LPR and SPR after 5-HT1B receptors were exogenously activated by zolmitriptan (1 μM), which initially deceased these reflexes.

Fig. 8.

Zolmitriptan inhibits polysynaptic EPSPs in motoneurons of chronic spinal rats. A: PIC-mediated plateau potential and sustained firing (LLR) evoked by dorsal root stimulation (0.1-ms pulse, 3×T) in a motoneuron at rest (top trace; −72 mV, without injected current; spikes clipped). With a hyperpolarizing bias current to prevent PIC activation, the same stimulation only evoked a polysynaptic EPSP, with short- and long-duration components indicated (bottom trace; motoneuron at −80 mV). B: in the same motoneuron, zolmitriptan (1 μM) eliminated the plateau and LLR evoked by dorsal root stimulation (top trace) and inhibited the short and long EPSPs (hyperpolarized, bottom trace).

Fig. 9.

Zolmitriptan inhibits excitatory postsynaptic currents but not PICs in motoneurons of chronic spinal rats. A and C: PIC in a motoneuron, activated by slowly increasing the membrane potential under voltage clamp and quantified at its initial peak, where it produced a downward deflection in the recorded current (at arrow) relative to the leak current (thin line). The PIC was unaffected by zolmitriptan application (1 μM). Dashed marks indicate rest (−71 mV) and −50 mV. B: in the same motoneuron, short and long excitatory postsynaptic currents (EPSC; downward current deflections) and inhibitory postsynaptic current (IPSC; upward current deflections) evoked by dorsal root stimulation (0.1-ms pulse, 3×T) in voltage-clamp mode at rest (bottom trace) and above rest (−60 mV). Expanded time scale is shown at right. Note the large IPSC that arises just after the short EPSC at depolarized potentials (−60 mV), which essentially interrupts the EPSCs. D: zolmitriptan (1 μM) reduced the long and short EPSCs (at rest) and revealed a longer and larger IPSC.

Fig. 10.

Zolmitriptan reduces long EPSC, further revealing IPSCs, with reversal potential at rest. A, top plot: long EPSC (negative currents), measured at 300 ms poststimulation, plotted against the holding potential, for the same motoneuron and stimulation as in Fig. 9 (reversing above −60 mV). Linear regression line, fit to the data, crosses the voltage axis at about −57 mV, the reversal potential for this mixed current. Middle plot: early peak of IPSC, measured at 20–30 ms poststimulation, plotted against holding potential, again during voltage clamp as in Fig. 9. Linear regression line crosses the voltage axis near rest (shaded bar, −71 mV), the reversal potential for this pure IPSC. Bottom plot: short-latency transient EPSP peak, measured at about 5 ms poststimulus, with reversal potential at about −40 mV. B, top plot: zolmitriptan (1 μM) inhibited the long EPSC (negative currents reduced), revealing a pure long-duration IPSC (positive currents; measured again 300 ms poststimulation), with a reversal potential near rest (regression line axis crossing). Middle plot: zolmitriptan did not affect the early peak of the IPSC measured 20–30 ms poststimulation. Bottom plot: zolmitriptan inhibited the short EPSC.

Fig. 11.

Zolmitriptan reduces spasms in the awake chronic spinal rat. A: spasm in chronic spinal rat evoked by electrical-cutaneous stimulation of the tail (3×T) and recorded with electromyogram. B: intrathecal application of zolmitriptan (0.1 mM in 30 μl of saline) reduced the LPR and LLR, quantified at horizontal bars.