Modulation of spinal GABAergic analgesia by inhibition of chloride extrusion capacity in mice

Abstract

Spinal gamma-aminobutyric acid receptor type A (GABA(A)) receptor modulation with agonists and allosteric modulators evokes analgesia and antinociception. Changes in K(+)-Cl(-) cotransporter isoform 2 (KCC2) expression or function that occur after peripheral nerve injury can result in an impairment in the Cl(-) extrusion capacity of spinal dorsal horn neurons. This, in turn, alters Cl(-)-mediated hyperpolarization via GABA(A) receptor activation, contributing to allodynia or hypersensitivity associated with nerve injury or inflammation. A gap in knowledge exists concerning how this loss of spinal KCC2 activity differentially impacts the analgesic efficacy or potency of GABA(A) agonists and allosteric modulators. We utilized intrathecal drug administration in the tail flick assay to measure the analgesic effects of general GABA(A) agonists muscimol and Z-3-[(aminoiminomethyl)thio]prop-2-enoic acid (ZAPA), the ∂-subunit-preferring agonist 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol (THIP), and allosteric modulators of the benzodiazepine (midazolam) and neurosteroid (ganaxolone) class, alone or in the presence of K(+)-Cl(-) cotransporter isoform (KCC) blockade. Intrathecal muscimol, ZAPA, THIP midazolam, and ganaxolone all evoked significant analgesia in the tail flick test. Coadministration of either agonists or allosteric modulators with [(dihydroindenyl)oxy] alkanoic acid (DIOA) (a drug that blocks KCC2) had no effect on agonist or allosteric modulator potency. On the other hand, the analgesic efficacy of muscimol and ZAPA and the allosteric modulator ganaxolone were markedly reduced whereas THIP and midazolam were unaffected. Finally, in the spared nerve injury model, midazolam significantly reversed tactile hypersensitivity while ganaxolone had no effect. These results indicate that the KCC2-dependent Cl(-) extrusion capacity differentially regulates the analgesic efficacy of agonists and allosteric modulators at the GABA(A) receptor complex.

Perspective: Our work suggests that drug discovery efforts for the treatment of chronic pain disorders should target benzodiazepine or ∂-subunit-containing sites at the GABA(A) complex.

Figure 1

Dose response curves for intrathecal GABA_A agonists and allosteric modulators with and without DIOA treatment in the tail flick test. Log doses of drug in grams are shown on x-axis while sum of %MPE for 10 and 20 min time points are shown on y-axis. Dose response curves are shown with agonist or allosteric modulators ± DOIA. ED₅₀s calculated from these curve fits (log(agonist) vs. response – Variable slope (four parameters)) are shown in Table 1.

Figure 2

Peak-effect responses of intrathecal GABA_A agonists and allosteric modulators with and without DIOA treatment in the tail flick test. Maximal effective doses for each agonist or allosteric modulator are shown ± DIOA expressed as %MPE over a 60 min time course. DIOA reduced analgesic efficacy for agonists muscimol (A) and ZAPA (B) but did not influence THIP (C). DIOA did not influence midazolam (D) efficacy but did reduce ganaxolone efficacy (E). Clonidine (F) was unaffected by DIOA. Two-way ANOVA with Bonferroni post-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Peak-effect responses for intrathecal THIP and ganaxolone with and without furosemide treatment. Furosemide had no effect on THIP (A) analgesic efficacy but significantly reduced ganaxolone analgesia (B). Two way ANOVA with Bonferroni post-test. ** p < 0.01, *** p < 0.001.

Figure 4

Effect of intrathecal midazolam and ganaxolone on spared nerve injury-induced neuropathic allodynia. SNI surgery was performed and mice were tested for mechanical allodynia on the ipsilateral hindpaw 10–14 days post surgery. (A) intrathecal injection of midazolam significantly reversed mechanical allodynia in the mouse SNI model whereas ganaxolone (B) was without effect. One way ANOVA with Dunnet post-test comparing to baseline. * p < 0.05, *** p < 0.001.