Ondansetron reverses antihypersensitivity from clonidine in rats after peripheral nerve injury: role of γ-aminobutyric acid in α2-adrenoceptor and 5-HT3 serotonin receptor analgesia

Abstract

Introduction: Monoaminergic pathways, impinging an α2-adrenoceptors and 5-HT3 serotonin receptors, modulate nociceptive transmission, but their mechanisms and interactions after neuropathic injury are unknown. Here we examine these interactions in rodents after nerve injury.

Methods: Male Sprague-Dawley rats following L5-L6 spinal nerve ligation (SNL) were used for either behavioral testing, in vivo microdialysis for γ-aminobutyric acid (GABA) and acetylcholine release, or synaptosome preparation for GABA release.

Results: Intrathecal administration of the α2-adrenoceptor agonist (clonidine) and 5-HT3 receptor agonist (chlorophenylbiguanide) reduced hypersensitivity in SNL rats via GABA receptor-mediated mechanisms. Clonidine increased GABA and acetylcholine release in vivo in the spinal cord of SNL rats but not in normal rats. Clonidine-induced spinal GABA release in SNL rats was blocked by α2-adrenergic and nicotinic cholinergic antagonists. The 5-HT3 receptor antagonist ondansetron decreased and chlorophenylbiguanide increased spinal GABA release in both normal and SNL rats. In synaptosomes from the spinal dorsal horn of SNL rats, presynaptic GABA release was increased by nicotinic agonists and decreased by muscarinic and α2-adrenergic agonists. Spinally administered ondansetron significantly reduced clonidine-induced antihypersensitivity and spinal GABA release in SNL rats.

Conclusion: These results suggest that spinal GABA contributes to antihypersensitivity from intrathecal α2-adrenergic and 5-HT3 receptor agonists in the neuropathic pain state, that cholinergic neuroplasticity after nerve injury is critical for α2-adrenoceptor-mediated GABA release, and that blockade of spinal 5-HT3 receptors reduces α2-adrenoceptor-mediated antihypersensitivity via reducing total GABA release.

Figure 1

(A) Effects of γ-amino butyric acid (GABA) antagonists on clonidine-induced analgesia in spinal nerve ligated (SNL) rats. Animals received intrathecal injection of saline, clonidine (15 μg/rat), bicuculline (Bic, 0.25 μg/rat), CGP35348 (CGP, 15 μg/rat), or their combination. Withdrawal threshold in the paw ipsilateral to SNL is presented over the time. N = 6-8. * p < 0.05 versus saline. # p < 0.05 versus clonidine alone. (B) Microdialysis for spinal GABA release. Buffer or clonidine (30-300 μM) was perfused into the spinal dorsal horn through the microdialysis probe in normal and SNL rats. N = 7-9. Data are presented over time as percentage of baseline. * p < 0.05 versus buffer.

Figure 2

(A) Effects of α2-adrenergic and cholinergic antagonists on clonidine-induced γ-amino butyric acid (GABA) release in the spinal dorsal horn ipsilateral to spinal nerve ligation (SNL). Buffer (control), idazoxan (300 μM), atropine (100 μM), or mecamylamine (100 μM) in the absence or presence of clonidine (100 μM) was perfused into the spinal dorsal horn through the microdialysis probe. Data are presented over time as percentage of baseline. N = 6-9. * p < 0.05 versus control. (B) Effects of α2-adrenergic and cholinergic agonists on GABA release in the synaptosomes from the spinal dorsal horn ipsilateral to SNL surgery. Synaptosomes were treated with epibatidine (Epi, 1-10 μM), nicotine (Nic, 30-300 μM), muscarine (Mus, 5-50 μM), or dexmedetomidine (Dex, 10 nM) in the presence or absence of 12 mM KCl. Data are presented as percentage of baseline. N = 6-10. * p < 0.05 versus buffer or KCl alone.

Figure 3

Microdialysis for spinal acetylcholine release. Buffer or clonidine (100 μM) was perfused into the spinal dorsal horn through the microdialysis probe in normal and spinal nerve ligated (SNL) rats. Data are presented over time as percentage of baseline. N = 6-7. * p < 0.05 versus buffer.

Figure 4

(A) Effects of γ-amino butyric acid (GABA) antagonists on chlorophenylbiguanide (mCPBG)-induced analgesia in spinal nerve ligated (SNL) rats. Animals received intrathecal injection of saline, mCPBG (15 μg/rat), mCPBG + bicuculline (Bic, 0.25 μg/rat), or mCPBG + CGP35348 (CGP, 15 μg/rat). Withdrawal threshold in the paw ipsilateral to SNL is presented over the time. N = 7-8. * p < 0.05 versus saline. # p < 0.05 versus mCPBG alone. (B) Microdialysis for spinal GABA release. Buffer, ondansetron (100 μM), or mCPBG (100 μM) was perfused into the spinal dorsal horn through the microdialysis probe in normal and SNL rats. N = 6-8. Data are presented over time as percentage of baseline. * p < 0.05 versus buffer.

Figure 5

(A) Effects of 5-HT3 antagonists on clonidine-induced analgesia in spinal nerve ligated (SNL) rats. Animals received intrathecal injection of saline (Sal.), clonidine (Cl, 15 μg/rat), ondansetron (0.3-30 μg/rat), dolasetron (100 μg/rat), or their combination 1 h prior to the von Frey test. Withdrawal threshold in the paw ipsilateral to SNL is presented. N = 6-8. * p < 0.05 versus saline. # p < 0.05 versus clonidine alone. (B) Effects of ondansetron on clonidine-induced antinociception in normal rats. Animals received intrathecal injection of saline (Sal.), clonidine (Cl, 3-30 μg/rat), ondansetron (3-30 μg/rat), or their combination 1 h prior to the Randall-Sellitto test. Average of withdrawal threshold in the both paws is presented. N = 8-14. * p < 0.05 versus saline.

Figure 6

(A) Microdialysis for γ-amino butyric acid (GABA) release following perfusion of clonidine (100 μM), ondansetron (100 μM), or their combination in the spinal dorsal horn ipsilateral to spinal nerve ligation (SNL). Data are presented over time as percentage of baseline. N = 7. * p < 0.05 versus ondansetron alone. # p < 0.05 versus clonidine alone. (B) Effects of ondansetron on nicotine-induced GABA release in the synaptosomes from the spinal dorsal horn ipsilateral to SNL surgery. Synaptosomes were treated with vehicle, nicotine (Nic, 300 μM), ondansetron (1-10 μM), or their combination in the presence of 12 mM KCl. Data are presented as percentage of baseline. N = 9. * p < 0.05 versus vehicle alone.

Figure 7

Net effect of α2-adrenoceptor and 5-hydroxytryptamine-3 (5-HT3) receptors on nociceptive sensory transmission in the spinal cord of normal (left) and nerve injured (right) animals. The colored bar indicates the effect of each step in the proposed circuit of these neurotransmitters, with green depicting increased nociceptive neurotransmission and red depicting reduced nociceptive neurotransmission. Both norepinephrine and serotonin are released from pathways which descend to the spinal cord, and act on many receptor subtypes. As regards α2-adrenoceptors, norepinephrine release is minimal in the normal condition, and it’s net effect is slight inhibition, due to and actions on primary afferent endings and projection neurons (upper left panel). This effect is only mildly counterbalanced by inhibition of acetylcholine (ACh) release which would drive γ-amino benzoic acid (GABA) release and by direct inhibition of GABA release. After nerve injury, norepinephrine release increases and the effect of α2-adrenoceptors on ACh shifts from inhibition to excitation and the expression of inhibitory α2-adrenoceptors on afferents and projection neurons increases (highlighted in yellow), resulting in strong inhibition (upper right panel). As regards 5-HT3 receptors, serotonin release is minimal in the normal condition, and it’s net effect is slight inhibition, due to direct stimulation of GABA release (lower left panel). After nerve injury, serotonin release increases and the effect of 5-HT3 receptors results in enhanced nociception neurotransmission, due to increased excitatory signaling on afferents and projection neurons (highlighted in yellow, lower right panel).