Global Gene Knockout of Kcnip3 Enhances Pain Sensitivity and Exacerbates Negative Emotions in Rats

Abstract

The Ca²⁺-binding protein Kv channel interacting protein 3 (KChIP3) or downstream regulatory element antagonist modulator (DREAM), a member of the neuronal calcium sensor (NCS) family, shows remarkable multifunctional properties. It acts as a transcriptional repressor in the nucleus and a modulator of ion channels or receptors, such as Kv4, NMDA receptors and TRPV1 channels on the cytomembrane. Previous studies of Kcnip3 ^-/- mice have indicated that KChIP3 facilitates pain hypersensitivity by repressing Pdyn expression in the spinal cord. Conversely, studies from transgenic daDREAM (dominant active DREAM) mice indicated that KChIP3 contributes to analgesia by repressing Bdnf expression and attenuating the development of central sensitization. To further determine the role of KChIP3 in pain transmission and its possible involvement in emotional processing, we assessed the pain sensitivity and negative emotional behaviors of Kcnip3 ^-/- rats. The knockout rats showed higher pain sensitivity compared to the wild-type rats both in the acute nociceptive pain model and in the late phase (i.e., 2, 4 and 6 days post complete Freund's adjuvant injection) of the chronic inflammatory pain model. Importantly, Kcnip3 ^-/- rats displayed stronger aversion to the pain-associated compartment, higher anxiety level and aggravated depression-like behavior. Furthermore, RNA-Seq transcriptional profiling of the forebrain cortex were compared between wild-type and Kcnip3 ^-/- rats. Among the 68 upregulated genes, 19 genes (including Nr4a2, Ret, Cplx3, Rgs9, and Itgad) are associated with neural development or synaptic transmission, particularly dopamine neurotransmission. Among the 79 downregulated genes, 16 genes (including Col3a1, Itm2a, Pcdhb3, Pcdhb22, Pcdhb20, Ddc, and Sncaip) are associated with neural development or dopaminergic transmission. Transcriptional upregulation of Nr4a2, Ret, Cplx3 and Rgs9, and downregulation of Col3a1, Itm2a, Pcdhb3 and Ddc, were validated by qPCR analysis. In summary, our studies showed that Kcnip3 ^-/- rats displayed higher pain sensitivity and stronger negative emotions, suggesting an involvement of KChIP3 in negative emotions and possible role in central nociceptive processing.

Keywords: KChIP3; RNA-Seq analysis; anxiety; conditioned place aversion; depression; inflammatory pain; negative emotions; nociceptive pain.

FIGURE 1

Validation of Kcnip3 gene knockout in Kcnip3^-/- rats by quantitative real-time PCR (qPCR) and Western blot. (A) qPCR analysis of Kcnip3 gene expression in the cerebral cortex of wild-type (WT) and Kcnip3^-/- rats. ^∗∗p < 0.01, paired t-test. (B) Western blot analysis of KChIP3 protein expression in the central nervous system of wild-type and Kcnip3^-/- rats. KO, knockout; Hippo, hippocampus. (C) Western blot analysis of KChIP1, 2 and 4 expression in the peripheral and central nervous system of wild-type and Kcnip3^-/- rats under normal condition. DRG, dorsal root ganglion. (D) Relative quantification of KChIP1, 2 and 4 protein levels in the peripheral and central nervous system of wild-type and Kcnip3^-/- rats. n = 3 for both groups. ^∗p < 0.05, paired t-test.

FIGURE 2

Increased pain sensitivity after intraplantar injection of formalin or complete Freund’s adjuvant (CFA) in Kcnip3^-/- rats. (A) Schematic diagram of the establishment of formalin or CFA-induced pain models. (B,C) Flinching counts (B) and licking duration (C) of hindpaw at 5-min intervals for 1 h in the formalin pain model of wild-type and Kcnip3^-/- rats. n = 8 for both groups. ^∗p < 0.05, ^∗∗∗p < 0.001, two-way repeated-measures ANOVA followed by Sidak’s multiple comparisons test. (D) Analysis of the cumulative number of flinching behaviors (left) and licking time (right) of hindpaw in the second phase (20–50 min post injection) of formalin pain model of wild-type and Kcnip3^-/- rats. ^∗∗p < 0.01, ^∗∗∗p < 0.001, unpaired t-test. (E) Time course of hindpaw licking latency in the 52°C hot plate test in the CFA pain model of wild-type and Kcnip3^-/- rats. n = 8 for both groups. ^∗∗p < 0.01, comparison between the genotypes at the indicated time points; ^###p < 0.001, comparison between the two curves, two-way repeated-measures ANOVA followed by Sidak’s multiple comparisons test. (F) Time course of lifting counts of hindpaw in the 4°C cold plate test within 1 min post CFA injection in wild-type and Kcnip3^-/- rats. n = 8 for both groups. ^∗p < 0.05, comparison between the genotypes at the indicated time points; ^###p < 0.001, comparison between the two curves, two-way repeated-measures ANOVA followed by Sidak’s multiple comparisons test.

FIGURE 3

Kcnip3 gene deletion exacerbates the aversive emotional response of pain. (A) Schematic diagram of a three-chamber conditioned place avoidance (CPA) apparatus and training procedure. (B) Percentage of the time spent in the formalin-paired compartment before and after the conditioning procedure in wild-type and Kcnip3^-/- rats. n = 8 for both groups. ^∗∗∗p < 0.001, comparison between preconditioning and postconditioning; ^#p < 0.05, comparison between the two curves, two-way repeated-measures ANOVA followed by Sidak’s multiple comparisons test. (C) Change in the percentage of the time spent in the formalin-paired compartment after the conditioning procedure in comparison to that prior to the conditioning procedure in wild-type and Kcnip3^-/- rats. n = 8 for both groups. ^∗∗p < 0.01, unpaired t-test. (D) Total distance traveled before (left, preconditioning) and after (right, postconditioning) the conditioning procedure in wild-type and Kcnip3^-/- rats. n = 8 for both groups. p > 0.05, unpaired t-test.

FIGURE 4

Enhanced anxiety-like behavior in Kcnip3^-/- rats during inflammatory pain. (A,B) The elevated plus-maze test. Kcnip3^-/- rats made fewer visits to the open arms (A) and spent less time in the open arms (B) in a 5-min test 1 day after CFA injection compared to the wild-type rats. n = 8 for both groups. ^∗p < 0.05, unpaired t-test. (C,D) The open field test. (C) Kcnip3^-/- rats displayed less time spent in the center of the open field in a 5-min test 1 day after CFA injection compared to the wild-type rats. n = 8 for both groups. ^∗∗p < 0.01, unpaired t-test. (D) Wild-type and Kcnip3^-/- rats exhibit similar locomotor activity in the open field test 1 day after CFA injection. n = 8 for both groups, unpaired t-test.

FIGURE 5

Enhanced depression-like behavior in Kcnip3^-/- rats under both normal and inflammatory pain conditions. (A) Schematic diagram of the forced swimming test. (B) Immobility time of wild-type and Kcnip3^-/- rats before (left) and 1 day after CFA injection (right). n = 7 for both groups. ^∗p < 0.05, ^∗∗p < 0.01, unpaired t-test. (C) Schematic diagram of the sucrose preference test. (D) Preference for sucrose, expressed as a percentage of the volume of sucrose intake over the total volume of fluid intake of wild-type and Kcnip3^-/- rats before (left) and 1 day after CFA injection (right). n = 7 for both groups. ^∗p < 0.05, ^∗∗p < 0.01, unpaired t-test.

FIGURE 6

(A) Volcano plots illustrating log₁₀(adjusted p-value) in relation to the log₂(fold change) for the differentially expressed genes in Kcnip3^-/- rats compared to wild-type rats. Genes that passed the significance threshold (FDR < 0.05) and the expression cut-off log₂(fold change) > 0.58 are colored red (upregulated, fold change > 1.5) or green (downregulated, fold change < 0.67), while genes outside this range are colored blue. (B) qPCR analysis of upregulated genes in the forebrain cortex of wild-type (WT) and Kcnip3^-/- rats. n = 4–5, ^∗p < 0.05, paired t-test. (C) qPCR analysis of downregulated genes in the forebrain cortex of WT and Kcnip3^-/- rats. n = 3, ^∗p < 0.05, ^∗∗p < 0.01, paired t-test.