Pharmacogenetic Activation of Spinal Glycinergic Neurons Ameliorates CCI-Induced Neuropathic Pain and Chloroquine- or Histamine-Induced Itch

Pharmacogenetic Activation of Spinal Glycinergic Neurons Ameliorates CCI-Induced Neuropathic Pain and Chloroquine- or Histamine-Induced Itch

Pharmacogenetic Activation of Spinal Glycinergic Neurons Ameliorates CCI-Induced Neuropathic Pain and Chloroquine- or Histamine-Induced Itch

(A) Diagram illustrating the AAV genome containing the flex.hM3Dq-mCherry cassette.

(B) Expression of hM3Dq in the spinal cord of GlyT2::Cre;GlyT2::eGFP double-transgenic mice is indicated by mCherry fluorescence. Expression is limited to the ipsilateral dorsal horn and is present in somata (see detail, bottom) and neurites of eGFP+ neurons. Scale bars, 200 μm (top) and 50 μm (bottom).

(C) Antihyperalgesic effects. All experiments were made in GlyT2::Cre+ mice. AAV.flex.hM3Dq-mCherry was injected at day 0, CCI surgery was performed on day 7 on virus-injected mice, and vehicle or CNO (1 mg/kg, i.p.) was injected on day 14. Mechanical PWTs (g) were assessed using electronic von Frey filaments before CCI surgery (pre-CCI), after CCI surgery immediately before CNO/vehicle injection (post-CCI), for 5 hr after CNO/vehicle injection, and 1 day later (post-drug). Repeated measures ANOVA, F(6,66) = 4.47; p = 0.001 for treatment x time interaction. Post hoc comparisons revealed significant differences between CNO and vehicle-treated groups for time points 2 and 3 hr (n = 6 and 7 for vehicle and CNO, respectively).

(D) Acute antinociceptive effects (repeated-measures ANOVA). Significant treatment effects were observed in the Hargreaves test (F(1,9) = 43.1, p < 0.001 [n = 5 and 6 for CNO and vehicle, respectively]), for cold hyperalgesia (F(1,9) = 56.2, p < 0.001 [n = 6 and 5]), and for pinprick stimulation (F(1,8) = 21.0, p = 0.002 [n = 5 each]). (E) Horizontal wire test. Repeated measures ANOVA (F(1,7) = 0.001, p = 0.98). (F) Blockade of chloroquine- and histamine-induced itch in AAV.flex.hM3Dq-mCherry-injected GlyT2::Cre+ mice by CNO. ∗∗∗p < 0.001; ∗∗p < 0.01, unpaired t test, n = 7 for all four groups. All error bars indicate SEM.