Gabapentin increases extracellular glutamatergic level in the locus coeruleus via astroglial glutamate transporter-dependent mechanisms

Abstract

Gabapentin has shown to be effective in animals and humans with acute postoperative and chronic pain. Yet the mechanisms by which gabapentin reduces pain have not been fully addressed. The current study performed in vivo microdialysis in the locus coeruleus (LC) in normal and spinal nerve ligated (SNL) rats to examine the effect of gabapentin on extracellular glutamate concentration and its mechanisms of action with focus on presynaptic GABA-B receptors, astroglial glutamate transporter-1 (GLT-1), and interactions with α2δ subunits of voltage-gated Ca(2+) channels and endogenous noradrenaline. Basal extracellular concentration and tissue content of glutamate in the LC were greater in SNL rats than normal ones. Intravenously administered and LC-perfused gabapentin increased extracellular glutamate concentration in the LC. The net amount of glutamate increased by gabapentin is larger in SNL rats compared with normal ones, although the percentage increases from the baseline did not differ. The gabapentin-related α2δ ligand pregabalin increased extracellular glutamate concentration in the LC, whereas another α2δ ligand, 3-exo-aminobicyclo [2.2.1] heptane-2-exo-carboxylic acid (ABHCA), did not. Selective blockade by the dihydrokainic acid or knock-down of GLT-1 by the small interfering RNA abolished the gabapentin-induced glutamate increase in the LC, whereas blockade of GABA-B receptors by the CGP-35348 and depletion of noradrenalin by the dopamine-β-hydroxylase antibody conjugated to saporin did not. These results suggest that gabapentin induces glutamate release from astrocytes in the LC via GLT-1-dependent mechanisms to stimulate descending inhibition. The present study also demonstrates that this target of gabapentin in astrocytes does not require interaction with α2δ subunits in neurons.

Keywords: Astrocyte; Gabapentin; Glutamate; Glutamate transporters; Locus coeruleus; Peripheral nerve injury.

Fig. 1

Effects of systemically and locally administered gabapentin (GBP) on extracellular glutamate concentrations in the LC in normal and SNL rats. (A) Saline (Normal: n=10, SNL: n=8) or GBP (50 mg/kg, Normal: n=11, SNL: n=12) was intravenously injected. (B) Vehicle (Normal: n=11, SNL: n=7), GBP 1 mM (Normal: n=11), GBP 10 mM (Normal: n=8, SNL: n=12), or GBP 100 mM (Normal: n=12, SNL: n=8) was perfused into the LC for 90 min through the microdialysis probe. Changes in glutamate concentrations in microdialysates from the LC are presented over time as percentage of baseline. *P<0.05 vs. saline or vehicle.

Fig. 2

Effects of α₂δ subunit ligands on extracellular glutamate concentrations in the LC. Vehicle (n=11), gabapentin (10 mM, n=8), pregabalin (10 mM, n=11), or ABHCA (10 mM, n=8) was perfused into the LC for 90 min through the microdialysis probe in normal rats. Changes in glutamate concentrations in microdialysates from the LC are presented over time as percentage of baseline. *P<0.05 vs. vehicle. #P<0.05 vs. gabapentin.

Fig. 3

Effects of GLT-1 and GABA-B receptor blockers on gabapentin-induced increase in extracellular glutamate in the LC. (A) Vehicle or gabapentin (10 mM) was perfused into the LC for 90 min through the microdialysis probe in normal rats in the presence of buffer (vehicle: n=11, gabapentin: n=11), dihydrokainic acid (DHK, 1 mM, vehicle: n=7, gabapentin: n=7), or CGP 35345 (CGP, 1 mM, vehicle: n=9, gabapentin: n=9). Changes in glutamate concentrations in microdialysates from the LC are presented over time as percentage of baseline. *P<0.05 vs. vehicle. #P<0.05 vs. gabapentin.. (B) KCl (100 mM) was perfused into the LC for 90 min through the microdialysis probe in normal rats in the absence (n=6) or presence of CGP (n=6). *P<0.05 vs. KCl alone.

Fig. 4

Knockdown of GLT-1 abolished gabapentin-induced increase in extracellular glutamate in the LC. Normal rats were treated with intra-LC injections of non-targeting (8.3 pmol/rat, n=11) or GLT-1 selective siRNA (8.3 pmol/rat, n=11) for 5 consecutive days prior to the experiment. (A) Representative western blotting images of GLT-1 in the LC from normal rats treated with non-targeting (left images) or GLT-1 selective siRNA (right images). (B) Changes in glutamate concentrations in microdialysates from the LC during perfusion of gabapentin (GBP, 10 mM) are presented over time as percentage of baseline. *P<0.05 vs. non-targeting siRNA. #P<0.05 vs. baseline.

Fig. 5

Depletion of noradrenergic neurons did not alter gabapentin-induced increase in extracellular glutamate in the LC. Normal rats were treated with an intra-LC injection of IgG-saporin (0.25 μg/rat, n=9) or DBH-saporin (0.25 μg/rat, n=8) at 2 weeks prior to the experiment. Changes in glutamate concentrations in microdialysates from the LC during perfusion of gabapentin (GBP, 10 mM) are presented over time as percentage of baseline. *P<0.05 vs. baseline.

Fig. 6

Effect of locally administered gabapentin on extracellular glutamate concentrations in the spinal cord. Vehicle or gabapentin (10 mM) was perfused into the spinal dorsal horn for 90 min through the microdialysis probe in normal rats (n=9 in each group). Changes in glutamate concentrations in microdialysates from the spinal cord are presented over time as percentage of baseline. *P<0.05 vs. vehicle.