γ1 GABAA Receptors in Spinal Nociceptive Circuits

Abstract

GABAergic neurons and GABA_A receptors (GABA_ARs) are critical elements of almost all neuronal circuits. Most GABA_ARs of the CNS are heteropentameric ion channels composed of two α, two β, and one γ subunits. These receptors serve as important drug targets for benzodiazepine (BDZ) site agonists, which potentiate the action of GABA at GABA_ARs. Most GABA_AR classifications rely on the heterogeneity of the α subunit (α1-α6) included in the receptor complex. Heterogeneity of the γ subunits (γ1-γ3), which mediate synaptic clustering of GABA_ARs and contribute, together with α subunits, to the benzodiazepine (BDZ) binding site, has gained less attention, mainly because γ2 subunits greatly outnumber the other γ subunits in most brain regions. Here, we have investigated a potential role of non-γ2 GABA_ARs in neural circuits of the spinal dorsal horn, a key site of nociceptive processing. Female and male mice were studied. We demonstrate that besides γ2 subunits, γ1 subunits are significantly expressed in the spinal dorsal horn, especially in its superficial layers. Unlike global γ2 subunit deletion, which is lethal, spinal cord-specific loss of γ2 subunits was well tolerated. GABA_AR clustering in the superficial dorsal horn remained largely unaffected and antihyperalgesic actions of HZ-166, a nonsedative BDZ site agonist, were partially retained. Our results thus suggest that the superficial dorsal horn harbors functionally relevant amounts of γ1 subunits that support the synaptic clustering of GABA_ARs in this site. They further suggest that γ1 containing GABA_ARs contribute to the spinal control of nociceptive information flow.

Keywords: GABAA receptor subtype; dorsal horn; gephyrin; nociception; pain; receptor clustering.

Figure 1.

GABA_AR γ subunit expression in mouse spinal cord and DRG. A, qRT-PCR measurements of the three GABA_AR γ subunits relative to β-actin in lumbar DRGs of naive mice. Developmental changes from E15 to adulthood (P50) (n = 5–7 mice). B, Same as A but lumbar spinal cord.

Figure 2.

Cellular expression pattern of the GABA_AR γ1 and γ2 subunits. A, mFISH experiments showing expression of Gabrg1 (left) and Gabrg2 (right) in transverse lumbar spinal cord sections costained with DAPI to visualize cell nuclei. Scale bar, 200 µm. B, Expression of Gabrg1 and Gabrg2 in glutamatergic (vGluT2) and GABAergic (vGAT) neurons in the superficial dorsal horn. Arrows indicate cells with coexpression. C, Statistical analysis: g1, Gabrg1; g2, Gabrg2; sdh, superficial dorsal horn; ddh, deep dorsal horn. Mean ± SD. D, Coexpression of Gabrg1 and Gabrg2 with Gabra2 and Gabra3. Arrows indicate cells with coexpression. Scale bar, 20 µm. E, Statistical analysis. g1, Gabrg1; g2, Gabrg2; a2, Gabra2; a3, Gabra3. Eight sections were analyzed per condition. Two sections were taken per mouse. Each dot represents a single section. F, G, Expression of Gabrg1 in different types of non-neuronal cells. F, mFISH using probers for Gabrg1, GFAP (astrocytes), Olig2 (oligodendrocytes), and Aif1 (microglia). Arrows indicate cells coexpressing the respective marker with Gabrg1. Scale bar, 20 µm. G, Statistical analysis. Mean ± SD.

Figure 3.

Nociceptive sensitivity of hoxB8γ2^−/− mice. A, B, Verification of the loss of γ2 subunit expression in hoxB8γ2^−/− mice and unchanged Gabrg1 expression in hoxB8γ2^−/− mice. A, Left, Transverse lumbar spinal cord sections of a γ2^fl/fl (left) and a hoxB8γ2^−/− mouse (right) stained for gephyrin and GABA_AR γ2 subunit protein. Right, Comparison of Gabrg1 expression (FISH) in γ2^fl/fl and hoxB8γ2^−/− mice. Scale bar, 100 µm. B, Left, Quantification of Gabrg2 mRNA relative to β-actin in lumbar DRGs of γ2^fl/fl (n = 4–6) and hoxB8γ2^−/− (n = 8–9) mice. Gabrg2 mRNA was detected with a probe binding to the sequence flanked by the two loxP sites. Unpaired test. Right, Quantification of the GABA_AR γ subunit expression in DRGs of γ2^fl/fl (n = 4–6) and hoxB8γ2^−/− mice (n = 6). Here, Gabrg2 mRNA was detected with a probe binding a sequence outside the region flanked by the two loxP sites. t tests followed by Bonferroni’s correction for multiple testing. C, Same as B, but lumbar spinal cord tissue (n = 8, for both γ2^fl/fl and hoxB8γ2^−/− mice). D, Nociceptive and somatic sensitivity. Mechanical sensitivity was tested in the pin prick, the von Frey test, and with a soft paint brush. Thermal sensitivity was assessed in the Hargreaves test, in the cold plantar test (dry ice) and the acetone test. Unpaired t tests, n = 10–14 and 10–16 for hoxB8γ2^−/− mice and γ2^fl/fl mice, respectively. Closed and open symbols indicate male and female mice, respectively. E, Muscle strength, motor coordination, and effects of TPA023B (1 mg/kg, p.o.) on locomotor activity assessed in the horizontal wire test, the rotarod test, and the actimeter test, respectively. Muscle relaxation, unpaired t test, n = 10 and 10 for hoxB8γ2^−/− mice and γ2^fl/fl mice. Motor coordination, unpaired t test, n = 8 and 8 for hoxB8γ2^−/− mice and γ2^fl/fl mice. Locomotor activity. Two-way ANOVA followed by Bonferroni’s post hoc test. Treatment * genotype interaction F_(1,18) = 0.014, n = 6–7 and 4–6 for hoxB8γ2^−/− mice and γ2^fl/fl mice. Circles and squares represent individual mice. Closed and open symbols indicate male and female mice, respectively. Mean ± SEM.

Figure 4.

GABA_AR clustering in the dorsal horn of hoxB8γ2^−/− mice. A, Deep dorsal horn. Immunofluorescent staining of gephyrin (green) and GABA_AR α2 subunits (top) or GABA_AR α3 subunits (bottom) in γ2^fl/fl and hoxB8γ2^−/− mice. Statistics: Cluster density of gephyrin and GABA_AR α2 and α3 subunits. Unpaired t tests. Mean ± SEM. B, Same as A but superficial dorsal horn. C, High-resolution images illustrating colocalization of gephyrin with GABA_AR α2 and α3 subunits in the deep dorsal horn. Scale bars, 10 µm (left) and 3 µm (right). D, Same as C but superficial dorsal horn. Individual dots represent one section. In total 9–10 sections from three mice were analyzed per condition.

Figure 5.

Activity of HZ-166 at γ1 and γ2GABA_ARs. A, Schematic representation of the experiment. Potentiation of recombinant γ2 (α2β3γ2) and γ1 (α2β3γ1) GABA_AR currents by HZ-166 was assessed in HEK 293 cells. GABA concentration was EC₅ (1 μM for α2β3 γ2 and 10 μM for α2β3γ1). B, Example trances of GABA evoked membrane currents in the presence and absence of a saturating concentration of HZ-166 (10 and 100 µM for γ2 and γ1 containing GABA_ARs. C, Concentration response curve fitted to the Hill equation with a baseline fixed to 0. Number of cells, n = 6–11. Data are mean ± SEM.

Figure 6.

Antihyperalgesic actions of HZ-166 in mice with neuropathic hyperalgesia. A, Changes in GABA_AR γ subunit expression after peripheral nerve damage. qRT-PCR measurements of mRNA encoding for the three GABA_AR γ subunits in lumbar DRG (left) and lumbar spinal cords (right), before and 7 d after CCI surgery (n = 8–10 mice per group). mRNA expression is expressed relative to β-actin expression. Statistics, DRG: ANOVA followed by Bonferroni’s post hoc test. F_(2,39) = 6.25. Spinal cord: F_(2,33) = 1.44. Error bars indicate mean ± SEM. B, Dose-dependent reversal of mechanical hyperalgesia by HZ-166 7 d after CCI surgery. n = 9, 6, 7, 6, and 6 mice for vehicle, 0.01, 0.03, 0.1, and 0.3 mg/kg HZ-166 intrathecally. C, Left panel (paw withdrawal threshold vs time), Partially retained antihyperalgesia by HZ-166 (0.3 mg/kg, i.t.) in hoxB8γ2^−/− mice. Repeated-measures ANOVA followed by Dunnett's post hoc test with predrug baseline as reference F_(2,12) = 73.5. Right panel (statistical analysis). Percent maximum possible effect averaged for time points 0.5 and 1 h. Unpaired t test. n = 7, for both hoxB8γ2^−/− mice and γ2^fl/fl mice, respectively. Closed and open symbols indicate male and female mice. D, Expression of Gabrg1 and Gabrg3 in DRGs and spinal cords of γ2^fl/fl and hoxB8γ2^−/− mice 7 d after CCI surgery (mean ± SEM). t tests followed by Bonferroni’s correction for multiple testing. Data are mean ± SEM.