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Anxiolytic-like Effects of Translocator Protein (TSPO) Ligand ZBD-2 in an Animal Model of Chronic Pain

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Anxiolytic-like Effects of Translocator Protein (TSPO) Ligand ZBD-2 in an Animal Model of Chronic Pain

Dong-Sheng Wang et al. Mol Pain.

Abstract

The activation of Translocator protein (18 kDa) (TSPO) has been demonstrated to mediate rapid anxiolytic efficacy in stress response and stress-related disorders. This protein is involved in the synthesis of endogenous neurosteroids that promote γ-aminobutyric acid (GABA)-mediated neurotransmission in the central neural system. However, little is known about the functions and the underlying mechanisms of TSPO in chronic pain-induced anxiety-like behaviors. The novel TSPO ligand N-benzyl-N-ethyl-2-(7,8-dihydro-7-benzyl-8-oxo-2-phenyl-9H-purin-9-yl) acetamide (ZBD-2) was used in the present study. We found that ZBD-2 (0.15 or 1.5 mg/kg) significantly attenuated anxiety-like behaviors in mice with chronic inflammatory pain induced by hindpaw injection of complete Freund's adjuvant (CFA). However, the treatment did not alter the nociceptive threshold or inflammation in the hindpaw. Hindpaw injection of CFA induced the upregulation of TSPO, GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and NR2B-containing N-methyl-D-aspartate (NMDA) receptors in the basolateral amygdala (BLA). ZBD-2 administration reversed the alterations of the abovementioned proteins in the BLA of the CFA-injected mice. Electrophysiological recording revealed that ZBD-2 could prevent an imbalance between excitatory and inhibitory transmissions in the BLA synapses of CFA-injected mice. Therefore, as the novel ligand of TSPO, ZBD-2 induced anxiolytic effects, but did not affect the nociceptive threshold of mice under chronic pain. The anxiolytic effects of ZBD-2 were related to the regulation of the balance between excitatory and inhibitory transmissions in the BLA.

Figures

Figure 1
Figure 1
ZBD-2 reduces anxiety-like behaviors. (A) Chemical structures of ZBD-2. (B) EPM test were performed on Day 21 after CFA injection. ZBD-2 (0.15 and 1.5 mg/kg) reversed the time in the open arms in CFA-injected mice. (C) ZBD-2 (0.15 and 1.5 mg/kg) reversed the open arm entries in CFA-injected mice. (D) No difference of the total number of arm entries in each group. (E) In OF test, ZBD-2 (0.15 and 1.5 mg/kg) reversed the time in center areas in CFA-injected mice. (F) No difference of the total distance traveled in each group. n = 6 in each group, *p < 0.05, **p < 0.01 compared to the control group; # p < 0.05, ## p < 0.01 compared to the CFA+ saline group.
Figure 2
Figure 2
Effects of ZBD-2 on pain perception. Mechanical allodynia and thermal hyperalgesia were detected on Day 0, 1, 7, 14 and 21 after CFA injection. ZBD-2 (0.15 and 1.5 mg/kg) did not change the mechanical allodynia in the ipsilateral (A) and contralateral hindpaw (B). ZBD-2 (0.15 and 1.5 mg/kg) did not change the thermal hyperalgesia in the ipsilateral (C) and contralateral hindpaw (D). (E) ZBD-2 did not reduce edema in CFA-injected hindpaws. n = 6 in each group, **p < 0.01 compared to the control group.
Figure 3
Figure 3
Effects of ZBD-2 on TSPO levels. (A) Representative results of Western blot analysis for TSPO in the BLA on the Day 21 after hindpaw CFA-injection. (B) ZBD-2 (0.15 and 1.5 mg/kg) for one week significantly decreased the upregulation of TSPO in the BLA. n = 6 in each group, **p < 0.01 compared to the control group; # p < 0.05, ## p < 0.01 compared to the CFA+ saline group.
Figure 4
Figure 4
Effects of ZBD-2 on protein expression in the BLA. (A) Representative results of Western blot analysis in the BLA on the Day 21 after hindpaw CFA-injection. (B) ZBD-2 (0.15 and 1.5 mg/kg) for one week slightly decreased the levels of NR2A-containing NMDARs. (C) ZBD-2 (1.5 mg/kg) reversed the upregulation of NR2B-containing NMDARs. (D-E) ZBD-2 (0.15 and 1.5 mg/kg) reversed the upregulation of GluR1 and CaMKII-α. (F) Representative results of Western blot analysis for GABAA-α2 and GAD67. (G and H) Levels of GABAA-α2 and GAD67 were not altered in groups. n = 6 in each group, *p < 0.05, **p < 0.01 compared to the control group; # p < 0.05, ## p < 0.01 compared to the CFA+ saline group.
Figure 5
Figure 5
Effects of ZBD-2 on mEPSC in the BLA. (A) Representative mEPSCs recorded in pyramidal neurons of BLA at a holding potential of-70 mV. (B) Cumulative frequency and amplitude histogram of the mEPSCs from the slices in each group. (C) Summery of mEPSCs frequency (left) and amplitude (right) in control (n = 11 slices/4 mice), CFA-vehicle (n = 10 slices/4 mice), CFA-ZBD-2 (n = 12 slices/4 mice) treated mice. ZBD-2 (1.5 mg/kg) for one week reversed the increases of mEPSCs frequency and amplitude in CFA-treated mice. *p < 0.05 compared to the control group; # p < 0.05 compared to the CFA+ saline group.
Figure 6
Figure 6
Effects of ZBD-2 on mIPSC in the BLA. (A) Representative mIPSCs recorded in pyramidal neurons of BLA at a holding potential of 0 mV. (B) Cumulative frequency and amplitude histogram of the mIPSCs from the slices in each group. (C) Summery of mIPSCs frequency (left) and amplitude (right) in control (n = 12 slices/4 mice), CFA-vehicle (n = 12 slices/4 mice), CFA-ZBD-2 (n = 11 slices/4 mice) treated mice. ZBD-2 (1.5 mg/kg) for one week reversed the decreases of mIPSCs amplitude in CFA-treated mice. *p < 0.05 compared to the control group; # p < 0.05 compared to the CFA+ saline group.

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