A role for Kalirin-7 in nociceptive sensitization via activity-dependent modulation of spinal synapses

Abstract

Synaptic plasticity is the cornerstone of processes underlying persistent nociceptive activity-induced changes in normal nociceptive sensitivity. Kalirin-7 is a multifunctional guanine-nucleotide-exchange factor (GEF) for Rho GTPases that is characterized by its localization at excitatory synapses, interactions with glutamate receptors and its ability to dynamically modulate the neuronal cytoskeleton. Here we show that spinally expressed Kalirin-7 is required for persistent nociceptive activity-dependent synaptic long-term potentiation as well as activity-dependent remodelling of synaptic spines in the spinal dorsal horn, thereby orchestrating functional and structural plasticity during the course of inflammatory pain.

Figure 1. Expression of diverse Kalirins in the spinal dorsal horn and specific knockout of Kal-7 in spinal neurons.

(a) Structure of the mouse Kalrn gene and the major protein products resulting via alternative splicing. Sites targeted by the reagents used in this study are shown. (b) Western blotting with antibody against the spectrin-repeat region detects expression of Kalirin in the spinal dorsal horn (SDH), with lysates from cortex and hippocampus as positive controls. (c) Transcripts for truncated or full-length Kalirins together with full-length Kalirin-7 (Kal-7) detected in the spinal dorsal horn via RT–PCR. (d) Unilateral microinjection of recombinant adeno-associated virus (rAAV) virus expressing Cre recombinase and EGFP into the mouse lumbar spinal dorsal horn (Scale bar, 300 μm). (e) Validation of Cre-mediated Kal-7 conditional knockout in mouse spinal dorsal horn (SDH-Kal-7^−/− mice) by quantitative RT–PCR, showing 50% reduction of Kal-7, but not Kal-9 or Kal-12 isoforms, in total lysate from spinal L4-L5 segments, as compared with AAV-EGFP-injected Kal-7^fl/fl mice (Kal-7^fl/fl-EGFP control mice) (n=3 independent experiments). *P<0.05 as compared with control group; two-way ANOVA followed by Bonferroni post-hoc test. Error bars represent s.e.m.

Figure 2. Functional impact of loss of all Kalirins or specifically Kal-7 in spinal dorsal horn neurons on pain-related behavior.

(a) Suppression of Phase II, but not phase I, nocifensive behaviour induced by intraplantar formalin in mice lacking all Kalirins in spinal dorsal horn neurons (SDH-pan Kal^−/− mice) (n=7–8 per group). (b) CFA-induced mechanical hypersensitivity represented as area under the curve (AUC) of the entire von Frey stimulus-response curve over von Frey forces 0.04 to 1.4 g tested at various time points post-CFA injection in SDH-pan Kal^−/− mice as compared with control littermates (n=8–10 animals per group). (c) Suppression of Phase II nocifensive behaviour induced by intraplantar formalin in mice lacking Kal-7 specifically in spinal dorsal horn neurons (SDH-Kal-7^−/− mice) as compared with control littermates (n=8–9 per group). (d) Change in response threshold to mechanical force via von Frey filaments following intraplantar injection of complete Freund's adjuvant (CFA) (n=10–11 animals per group). (e) CFA-induced mechanical hypersensitivity represented as area under the curve (AUC) of the entire von Frey stimulus-response curve over von Frey forces 0.04 to 1.4 g tested at various time points post-CFA injection (n=10–11 animals per group). In all panels, *P<0.05 as compared with control group; ^†P<0.05 as compared with basal condition; two-way ANOVA followed by Bonferroni post hoc test. Error bars represent s.e.m.

Figure 3. Role of Kal-7 signalling in dendritic spine remodelling in spinal dorsal horn neurons.

(a) Typical examples of activity-dependent increase in dendritic spines following peripheral paw inflammation in control mice, but not in mice with spinal deletion of Kal-7 (overview scale bar, 50 μm, inset scale bar, 10 μm). (b) Quantification of spine density in Kal-7 knockout and control group under naive or CFA-treated condition in vivo (16–20 neurons were analysed from three animals each per treatment group). (c,d) Typical examples (c) and quantitative summary (d) of dendritic spines on specific deletion of Kal-7 in excitatory spinal cord neurons in culture via treatment with AAV-CKII-EGFP and AAV-Synapsin-Cre; double-positive neurons (merge) showed a lesser density of spines than Cre-negative neurons (scale bar, 50 μm in upper and 10 μm in lower panels); lower right, typical examples of different types of dendritic spines (scale bar, 1 μm). *P<0.05 as compared with control group; two-way ANOVA, Bonferroni post hoc test. Error bars represent s.e.m.

Figure 4. Bidirectional modulation of expression of Rac1 in spinal dorsal horn neurons in vivo and its structural and functional impact.

(a) Western blotting of spinal lysates for validation of spinal knockdown and overexpression of Rac1 via AAV-mediated shRNA delivery or Rac1 cDNA delivery in comparison with corresponding controls. Lower panel represents quantitative summary of Rac1 expression normalized to corresponding Tubulin control. (b,c) Typical examples of traces derived from Golgi-impregnated spinal neurons (b) and their quantitative summary (c) in mice with spinal AAV-mediated knockdown or overexpression of Rac1, using mice spinally expressing a non-targeting shRNA or EGFP as controls (Scale bar, 50 μm; 15–18 neurons from three animals per treatment group). (d) Attenuation of phase II nocifensive behaviour, but not of phase I, in the formalin test by spinal knockdown of Rac1 (n=7–8 per group). (e) Spinal neuron-specific knockdown of Rac1 led to a reduction of mechanical hypersensitivity induced by intraplantar CFA injection as compared with mice expressing control RNA (n=6 per group). (f) Overexpression of Rac1 in neurons of the spinal dorsal horn led to enhanced basal sensitivity to mechanical nociceptive stimuli (n=6–8 mice). (g) Functional impact of overexpression of Rac1 or EGFP (control) in the spinal dorsal horn of SDH-Kal-7^−/− mice on sensitivity to mechanical nociceptive stimuli and CFA-induced hypersensitivity (n=6 mice per group). (h) Quantification of changes in spine density on overexpression of Rac1 or EGFP in spinal neurons lacking Kal-7 at 24 h post-CFA (25–30 neurons were analysed from from animals each per treatment group). *P<0.05 as compared with control group; ^†P<0.05 as compared with basal condition; two-way ANOVA, Bonferroni post hoc test. Error bars represent s.e.m.

Figure 5. Functional impact of disrupting synaptic interactions of Kal-7 in spinal neurons or loss of Kal-7 expression on synaptic transmission.

(a–c) At synapses between C nociceptors and spinal projection neurons, intracellular application of a Kal-7 C-terminal (CT) interfering peptide, but not a control peptide, blocked spinal long-term potentiation (n=8–10 slices). (d–f) Analysis of synaptic long-term potentiation in spinal neurons of mice lacking Kal-7 (Kal-7^−/− mice) and wild-type littermates (n=8–10 slices). Typical traces of evoked excitatory postsynaptic currents (eEPSCs) (a,d), time-course (b,e) and quantitative summary of % change in EPSC magnitude (c,f) are shown. *P<0.05 as compared with control group, Student's t-test. Error bars represent s.e.m.