Three glutamate transporters, EAAC1, GLT-1, and GLAST, have been found in the spinal cord.6,8,11–13
They are expressed most densely in the superficial dorsal horn, which is the primary center of nociceptive processing. However, their localization and distribution at synaptic and non-synaptic sites has not been reported. Our study provides evidence that EAAC1 is present in dorsal horn neurons and axonal terminals at both synaptic and non-synaptic sites and in nociceptive primary afferents. In line with the present observation, we previously showed that EAAC1 co-localized with calcitonin gene-related peptide and isolectin B4
in the dorsal root ganglion neurons and that EAAC1 immunoreactivity in the superficial dorsal horn was reduced on the side ipsilateral to the dorsal rhizotomy.11
In contrast to EAAC1, GLT-1 and GLAST proteins were not detected in the dorsal root ganglion, although GLAST messenger RNA was observed in this region. Under electron microscopy, GLT-1 and GLAST were abundantly distributed in glial cells at perisynaptic sites in the superficial dorsal horn. This finding is consistent with a recent report that showed co-localization of GLT-1 and GLAST with both glial fibrillary acidic protein (a marker of astrocytes) and OX-42 (a marker of microglia) in the dorsal horn.44
Thus, our findings provide a new morphologic basis for the functional role of spinal glutamate transporters in normal sensory transmission and pathologic pain (including inflammatory pain).
We and others have reported that intrathecal injection of glutamate transporter inhibitors produces hyperactivity of dorsal horn neurons, spontaneous nociceptive behaviors, and thermal and mechanical pain hypersensitivities in naïve animals.14,15
Based on these pronociceptive effects, it is reasonable to infer that enhancing spinal glutamate transporter uptake activity might produce antinociception in pathological conditions. Unexpectedly, however, glutamate transporter activator MS-15333–39
had no effect on formalin-induced pain behaviors, even at the highest dose (1,000 μg). In contrast, intrathecal administration of three different glutamate transporter inhibitors, TBOA, dihydrokainate, and DL-THA, significantly blocked formalin-induced nociceptive behaviors in the second phase. Intrathecal TBOA also attenuated CFA-induced thermal hyperalgesia. Consistent with these results, Niederberger et al.12,17
reported that pharmacologic inhibition of spinal glutamate transporter with L-trans
-pyrrolidine-2,4-dicarboxylate or transient knockdown of GLT-1 and GLAST decreased the number of formalin-induced flinches. Interestingly, intrathecal administration of riluzole, a glutamate transporter activator, resulted in antinociception in rat neuropathic pain.13
This controversial result might be attributed to the fact that riluzole has a number of other effects, such as blocking sodium channels, glutamate receptors, and γ-aminobutyric acid uptake.45
In a clinical study, riluzole was ineffective against peripheral neuropathic pain.46
Although dihydrokainate and DL-THA used in the present study might act as agonists or antagonists at glutamate receptors, TBOA is the most potent competitive blocker of glutamate transporters and did not show any significant effects on either the ionotropic or metabotropic glutamate receptors.16,35,47–49
Thus, the reduced pain-related behaviors that we observed (at least those caused by TBOA) cannot be explained by direct interaction between glutamate transporter blockers and glutamate receptors.
Here, we examined potential mechanisms by which glutamate transporter inhibitors might have antinociceptive effects on inflammatory pain. We first demonstrated that all rats treated with TBOA, DL-THA, and dihydrokainate displayed normal placing, grasping, and righting reflexes, indicating that the antinociception cannot be attributed to impaired motor functions. We further found that spinal glutamate transporter uptake activity was significantly reduced on the ipsilateral side 6 h after CFA injection and 1 h after formalin injection. A similar reduction in glutamate transport activity was reported in the dorsal horn ipsilateral to peripheral nerve injury.13,50
It is unclear how inflammatory and neuropathic inputs cause a reduction in spinal glutamate uptake. Although neither CFA nor formalin altered total expression of spinal glutamate transporter proteins, peripheral noxious insults might reduce plasma membrane expression of spinal glutamate transporters, a possibility that remains to be confirmed.
Reduction of spinal glutamate transporter uptake may elevate glutamate concentration in the synaptic cleft and non-synaptic extracellular space and contribute to the development and maintenance of inflammatory and neuropathic pain.6,13
It is possible that in our study, MS-153 was unable to effectively activate impaired spinal glutamate transporters to clear synaptic glutamate accumulation under inflammatory pain conditions. Interestingly, contralateral spinal glutamate transporter uptake activity increased to varying extents after CFA and formalin injection. The relevance of this increase is unknown but might be an attempt to compensate for the reduction in glutamate transporter uptake on the ipsilateral side to clear excess glutamate.
One might expect that under inflammatory pain conditions, intrathecal administration of glutamate transporter inhibitors would further block clearance of glutamate and enhance the increases in synaptic and non-synaptic extracellular glutamate in the dorsal horn. This enhancement might cause excitotoxicity, destroying susceptible dorsal horn neurons and interfering with transmission of dorsal horn nociceptive information.6
However, transient treatment with TBOA did not induce any detectable spinal neuronal damage in rats subjected to the formalin or CFA model. This finding suggests that the antinociceptive effect of spinal glutamate transporter inhibition is not a result of dorsal horn neuronal excitotoxicity. Importantly, we found that intrathecal co-administration of MSOP blocked the TBOA-produced antinociceptive effect in both pain models, suggesting the involvement of spinal inhibitory presynaptic group III mGluRs. Spinal group III mGluRs are activated under pathologic pain conditions, but not under normal conditions.4,5
If spinal glutamate transporter inhibition enhances the activation of inhibitory presynaptic group III mGluRs, it would limit further glutamate release from primary afferents and result in an antinociceptive effect in inflammatory pain.6
This possibility is supported by a previous experiment in which inhibition of spinal glutamate uptake decreased evoked postsynaptic excitatory potentials of dorsal horn neurons in inflammatory pain; the decrease was reversible by a group III mGluR antagonist.51
Inhibition of glutamate uptake has also been reported to decrease synaptic release of glutamate.48
It is noteworthy that intrathecal MSOP did not completely reverse the antinociceptive effect of TBOA, suggesting that other potential mechanisms might exist. Glutamate transporter uptake function is dependent on the membrane potential and the transmembrane ion gradients established by the Na+
pump. Under inflammatory pain conditions, hyperactive dorsal horn neurons and glial cells might consume large amounts of cellular energy, disturb energy metabolism, and result in energy insufficiency.6
Such insufficiency might impair the Na+
pump and reverse glutamate transporter operation. Indeed, during brain ischemia, glutamate transporter operation was reversed, causing glutamate release due to ATP depletion.49
TBOA reduced glutamate release and had neuroprotective actions in brain ischemia.49
We previously reported that ischemia-stimulating medium reversed the operation of glutamate transporters of dorsal horn neurons in vitro
, causing glutamate to be released.52
Thus, it is possible that the antinociceptive effect of glutamate transporter inhibitors might also be the result of the blockade of reversed transport and consequent release of glutamate in dorsal horn.6
However, direct evidence regarding reversed operation of spinal glutamate transporters in inflammatory pain in vivo
is lacking and remains to be further confirmed. In addition, other potential mechanisms, such as postsynaptic desensitization of dorsal horn glutamate receptors and disturbance of the glutamate/glutamine cycle in spinal glial cells might also contribute to the role of spinal glutamate transporter inhibitors in inflammatory pain.6
In summary, here we have ascertained the synaptic and non-synaptic localization and distribution of EAAC1, GLT-1, and GLAST in the superficial dorsal horn. In addition, behavioral studies showed that spinal glutamate transporter inhibition produces an antinociceptive effect in formalin- and CFA-induced inflammatory pain. This effect was attenuated by a group III mGluR antagonist. Our findings suggest that spinal glutamate transporter inhibition can relieve inflammatory pain through a mechanism that involves the activation of inhibitory pre-synaptic group III mGluRs.