The cAMP Response Element- Binding Protein/Brain-Derived Neurotrophic Factor Pathway in Anterior Cingulate Cortex Regulates Neuropathic Pain and Anxiodepression Like Behaviors in Rats

Abstract

Neuropathic pain is often accompanied by anxiety and depression-like manifestations. Many studies have shown that alterations in synaptic plasticity in the anterior cingulate cortex (ACC) play a critical role, but the specific underlying mechanisms remain unclear. Previously, we showed that cAMP response element-binding protein (CREB) in the dorsal root ganglion (DRG) acts as a transcription factor contributing to neuropathic pain development. At the same time, brain-derived neurotrophic factor (BDNF), as important targets of CREB, is intricate in neuronal growth, differentiation, as well as the establishment of synaptic plasticity. Here, we found that peripheral nerve injury activated the spinal cord and ACC, and silencing the ACC resulted in significant relief of pain sensitivity, anxiety, and depression in SNI rats. In parallel, the CREB/BDNF pathway was activated in the spinal cord and ACC. Central specific knockdown and peripheral non-specific inhibition of CREB reversed pain sensitivity and anxiodepression induced by peripheral nerve injury. Consequently, we identified cingulate CREB/BDNF as an assuring therapeutic method for treating neuropathic pain as well as related anxiodepression.

Keywords: BDNF; CREB; anterior cingulate cortex; anxiety; depression; neuropathic pain.

FIGURE 1

Selectively nerve injured rats exhibit pain hypersensitivity and anxiodepression like behaviors. (A,B) SNI significantly reduced the PWT (A) and the PWL (B) in the ipsilateral paws (n = 8 rats/group). (C) The weight growth rate of SNI rats was lower than that of sham rats (n = 8 rats/group). (D) The rats showed a significant reduction in the number of entries into the center area and the time spent in the center area after SNI in the OFT (n = 12 rats/group). (E) The proportion of entries into the open arms and the proportion of time in the open arms were significantly decreased in the SNI rats in the EPM (n = 12 rats/group). (F,G) The eating latency in the NFST (F) and the immobility time in the FST (G) of SNI rats were significant increase of SNI rats (n = 12 rats/group). (H) There was no significant difference in the total distance traveled by SNI rats in the OFT (n = 12 rats/group). (I) Schematic representation of the locomotor trajectories of sham and SNI rats at 21 days after surgery in the OFT and EPM. (J) Probability of SNI rats exhibiting anxiety and depression like behaviors at 21 days after surgery. *P < 0.05, ^**P < 0.001, ^***P < 0.0001 for sham vs. SNI.

FIGURE 2

There is a reciprocal projection between the ACC and the spinal cord, and peripheral nerve injury activates the spinal cord and the ACC. (A) CTB-555 (green, 250 nl) and CTB-488 (red, 250 nl), respectively, were stereotaxically injected into the ipsilateral spinal dorsal horn and contralateral ACC (Scar bar = 50 μm). (B) Immunofluorescence showed that coexpression signals (yellow) of CTB-555 and CTB-488 were observed in the injected area of the spinal dorsal horn and ACC. (C) The expression of c-fos and PSD-95 was increased in the spinal cord of SNI rats (n = 8 rats/group). (D) The expression of c-fos, PSD-95 and NLGN2 was increased in the ACC of SNI rats (n = 8 rats/group). (E) Peripheral nerve injury activated ipsilateral spinal cord and contralateral ACC (Scar bar = 50 μm). (F) Peripheral nerve injury elevated PSD-95 in the ipsilateral spinal cord and contralateral ACC (Scar bar = 50 μm). ^**P < 0.001, ^***P < 0.0001 for sham vs. SNI.

FIGURE 3

Silencing the ACC by chemicalgenetic techniques reduced neuropathic pain and anxiodepression like behaviors. (A,B) Injection of rAAV-hSyn-hM4D (Gi)-EGFP into the contralateral ACC of SNI rats (A) and experimental timing (B). (C) Immunofluorescence showed that rAAV-hSyn-hM4D (Gi)-EGFP was injected into the ACC and was successfully transfected (Scar bar = 50 μm). (D,E) Intraperitoneal injection of CNO (3.3 mg/kg) to silence ACC attenuated PWT (D) and PWL (E) in SNI rats (n = 10 rats/group). (F,G) Intraperitoneal injection of CNO (3.3 mg/kg) to silence ACC did not influence the number of entries into the center area, the time in the center area (F), or the total distance (G) in the OFT (n = 10 rats/group). (H) Silencing ACC by intraperitoneal injection of CNO (3.3 mg/kg) increased the proportion of entries into the open arms and the proportion of time in the open arms in the EPM (n = 10 rats/group). (I) Intraperitoneal injection of CNO (3.3 mg/kg) to silence ACC decreased the immobility time in the FST (n = 10 rats/group). *P < 0.05, ^**P < 0.001, ^***P < 0.0001 for sham + Control vs. SNI + Control; ^#P < 0.05, ^##P < 0.001 for SNI + Control vs. SNI + CNO.

FIGURE 4

Peripheral nerve injury activates the CREB/BDNF pathway in the spinal cord and ACC. (A) The expression of CREB, p-CREB, BDNF and CaMKII α was significantly increased in the ipsilateral spinal cord at all-time points (7, 14, 21, and 28 days) after the nerve injury in rats (n = 8 rats/group). (B) The expression of CREB, p-CREB, BDNF and CaMKII α was significantly increased in the contralateral ACC at all-time points (7, 14, 21, and 28 days) after the nerve injury in rats (n = 8 rats/group). (C) The expression of CREB mRNA and BDNF mRNA was significantly increased in the spinal cords of SNI rats (n = 5 rats/group). (D) The expression of CREB mRNA and BDNF mRNA was significantly increased in the ACC of SNI rats (n = 7 rats/group). (E,F) Immunofluorescence showed that peripheral nerve injury induced an increase in the expression of CREB, p-CREB, and BDNF in the ipsilateral spinal cord (E) and contralateral ACC (F) (Scar bar = 50 μm). *P < 0.05, ^**P < 0.001, ^***P < 0.0001 for sham vs. SNI.

FIGURE 5

Characterization of expression profile of CREB and BDNF in the ACC. (A,B) Representative examples (A) and quantitative summary (B) showing that CREB was highly coexpressed with neuronal nuclear antigen (NeuN), sparsely with either glial fibrillary acidic protein (GFAP) or Iba1 (n = 3) (Scar bar = 50 μm). (C,D) Representative examples (C) and quantitative summary (D) showing that BDNF was highly coexpressed with glial fibrillary acidic protein (GFAP), sparsely with either neuronal nuclear antigen (NeuN) or Iba1 (n = 3) (Scar bar = 50 μm). (E,F) Peripheral nerve injury increases the coexpression of BDNF and GFAP in the spinal cord (E) and ACC (F) (Scar bar = 50 μm).

FIGURE 6

Knockdown of CREB in the ACC alleviates pain hypersensitivity and anxiodepression like behaviors in SNI rats. (A,B) Injection of rAAV-u6-shRNA Creb1-CMV-EGFP into the contralateral ACC of SNI rats (A) and experimental timing (B). (C) The virus was successfully injected into the ACC and stably transfected (Scar bar = 50 μm). (D) CREB was significantly knocked down within the ACC (Scar bar = 50 μm). (E) Knockdown of CREB in the ACC decreased CREB/BDNF mRNA in the ACC (n = 4 rats/group). (F,G) Knockdown of CREB in the ACC alleviated PWT (F) and PWL (G) in SNI rats (n = 12 rats/group). (H) Knockdown of CREB in the ACC increased the number of entries into the center area and time in the center area in SNI rats at 14 days after surgery (n = 12 rats/group). (I) Knockdown of CREB in the ACC increased the proportion of entries into the open arms and the proportion of time in the open arms in SNI rats at 14 days after surgery (n = 12 rats/group). (J) Knockdown of CREB in the ACC increased the number of entries into the center area, but not the time in the center area in SNI rats at 21 days after surgery (n = 12 rats/group). (K) Knockdown of CREB in the ACC was no significant difference in the total distance traveled by SNI rats at 21 days after surgery (n = 12 rats/group). (L) Knockdown of CREB in the ACC increased the proportion of entries into the open arms and the proportion of time in the open arms in SNI rats at 21 days after surgery (n = 12 rats/group). (M,N) Knockdown of CREB in the ACC decreased the eating latency (M) and the immobility time (N) in SNI rats at 21 days after surgery (n = 12 rats/group). *P < 0.05,^**P < 0.001,^***P < 0.0001 for SNI + Scramble vs. SNI + shCreb.

FIGURE 7

Knockdown of CREB in the ACC increased CREB/BDNF expression in the spinal cord in SNI rats. (A) Knockdown of CREB in the ACC activated the CREB/BDNF pathway in the spinal cord (n = 8 rats/group). (B) Knockdown of CREB in the ACC inhibited the CREB/BDNF pathway in the ACC (n = 8 rats/group). (C) Representative examples showing that knockdown of CREB in the ACC increased the expression of CREB and BDNF in the spinal cord (Scar bar = 50 μm). (D) Representative examples showing that knockdown of CREB in the ACC decreased the expression of CREB and BDNF in the ACC (Scar bar = 50 μm). *P < 0.05, ^**P < 0.001, ^***P < 0.0001 for SNI + Scramble vs. SNI + shCreb.

FIGURE 8

Intraperitoneal administration of the CREB inhibitor (666–15) reduced neuropathic pain and anxiodepression like behaviors in SNI rats. (A) 666–15 (10 mg/kg) was administered intraperitoneally 1 day before surgery and at 7 days intervals thereafter. (B,C) Intraperitoneal injection of 666–15 attenuated PWT (B) and PWL (C) in SNI rats (n = 12 rats/group). (D) Intraperitoneal injection of 666–15 increased the number of entries into the center area and the time in the center area in SNI rats (n = 12 rats/group). (E) Intraperitoneal injection of 666–15 increased the proportion of entries into the open arms and the proportion of time in the open arms in SNI rats (n = 12 rats/group). (F,G) Intraperitoneal injection of 666–15 decreased the eating latency (F) and the immobility time (G) in SNI rats (n = 12 rats/group). (H) Intraperitoneal injection of 666–15 suppressed the CREB/BDNF pathway in the spinal cord (n = 8 rats/group). (I) Intraperitoneal injection of 666–15 decreased the expression of p-CREB, BDNF, and PSD, but not CREB, in the ACC (n = 8 rats/group). *P < 0.05, ^**P < 0.001, ^***P < 0.0001 for SNI + Control vs. SNI + 666–15.

FIGURE 9

Intraperitoneal administration of LPS induced anxiodepression like behaviors in rats and activated the CREB/BDNF signaling pathway in the ACC. (A) Lipopolysaccharide (LPS) was injected intraperitoneally (i.p.) every 2 days for 14 days. (B) Intraperitoneal administration of LPS did not affect PWT and PWL in rats (n = 9 rats/group). (C) Intraperitoneal administration of LPS reduced the number of entries into the center area, but not the time in the center area in rats (n = 9 rats/group). (D) Intraperitoneal administration of LPS reduced the proportion of entries into the open arms and the proportion of time in the open arms in rats (n = 9 rats/group). (E) Intraperitoneal administration of LPS did not alter the locomotor activity of the rats (n = 9 rats/group). (F,G) Intraperitoneal administration of LPS increased the eating latency (F) and the immobility time (G) in rats (n = 9 rats/group). (H) Intraperitoneal administration of LPS activated the CREB/BDNF pathway in the ACC (n = 6 rats/group). (I) Intraperitoneal administration of LPS Suppressed the CREB/BDNF pathway in the hippocampus (n = 6 rats/group). *P < 0.05, ^**P < 0.001,^***P < 0.0001 for Control vs. LPS.