MicroRNA134 of Ventral Hippocampus Is Involved in Cocaine Extinction-Induced Anxiety-like and Depression-like Behaviors in Mice

Abstract

We previously found that cocaine abuse could increase microRNA134 (miR134) levels in the hippocampus; yet the roles of miR134 in cocaine-related abnormal psychiatric outcomes remain unknown. In this study, using the cocaine-induced conditioned place preference (CPP) mice model, we found that mice exhibit enhanced anxiety-like and depression-like behaviors during the cocaine extinction (CE) period of CPP, accompanied by obviously increased miR134 levels and decreased levels of 19 genes that are associated with synaptic plasticity, glia activity, and neurochemical microenvironments, in the ventral hippocampus (vHP). Knockdown of miR134 in vHP in vivo reversed the changes in 15 of 19 potential gene targets of miR134 and rescued the abnormal anxiety-like and depression-like behavioral outcomes in CE mice. In parallel, knockdown of miR134 reversed CE-induced changes in dendritic spines and synaptic proteins and increased the field excitatory postsynaptic potential (fEPSP) of CA1 pyramidal neurons in the vHP of CE mice. In addition, knockdown of miR134 suppressed the CE-enhanced microglia activity, inflammatory, apoptotic, and oxidative stress statuses in the vHP. With the data taken together, miR134 may be involved in cocaine-associated psychiatric problems, potentially via regulating the expressions of its gene targets that are related to synaptic plasticity and neurochemical microenvironments.

Figure 1

miR134-5P Level Is Increased by CE in the vHP of Mice (A) Proposed model of the present study. (B) Experimental design and timeline. (C) CPP scores (left, n = 16) and miR134-5P levels (right, n = 6) of vHP. CPP, conditioned place preference; CE, cocaine extinction mice; EPM, elevated plus maze; OFT, open field test; TST, tail suspension test; FST, forced swim test; SE, saline extinction mice; vHP, ventral hippocampus. **p < 0.01, versus SE mice; N.S., p > 0.05, versus SE mice.

Figure 2

miR134-Expressed and miR134-RNAi-virus-Transfected Cell Types in the vHP (A) Illustration of miR134-expressed cell types in the vHP of native mice (left). White arrows indicate positive staining of biomarkers for cell types (green indicates CAMKII, GFAP, and Iba-1); white triangles indicate colocalization with miR134 (red) and biomarkers (green). Scale bars, 100 μm. Ratio of colocalization with miR134 and biomarkers to miR-134-positive cells (right). (B) Illustration of miR134-RNAi-virus-transfected cell types in the vHP. Top panels: the injection site of virus (green, left; scale bar, 1 μm), expression of virus (green, middle; scale bar, 100 μm), and miR-134-5P levels (right) in the vHP of CE mice. Middle panels: injection site of virus (green; scale bars, 1 mm). Bottom panels: miR134-RNAi-virus-transfected cell types in the vHP (left). White arrows indicate positive staining of biomarkers for cell types (red indicates NeuN, GFAP, and Iba-1); white triangles indicate colocalization with GFP (green) and biomarkers (red). Scale bars, 50 μm. Ratio of colocalization with GFP and biomarkers to GFP-positive cells (right).

Figure 3

Knockdown of miR134 in the vHP Rescues CE-Enhanced Anxiety-like and Depression-like Behaviors in Mice (A) EPM. Percentage of time spent in the open arms of EPM in SE and CE mice (left, n = 24), as well as in miR134 RNAi and controlled RNAi of CE mice (right, n = 24). (B) OFT. Distance traveled in the OFT apparatus (left); percentage of time spent in the central zone of the OFT (middle) in SE and CE mice (n = 24), as well as that in miR134 RNAi and controlled RNAi of CE mice (right, n = 24). (C) TST. Immobility time during the TST in SE and CE mice (left, n = 24), as well as in miR134 RNAi and controlled RNAi of CE mice (right, n = 24). (D) FST. Immobility time during the FST in SE and CE mice (left, n = 24), as well as in miR134 RNAi and controlled RNAi of CE mice (right, n = 24). **p < 0.01 versus SE mice; ^##p < 0.01 versus CE-control mice. CE, cocaine extinction mice; SE, saline extinction mice; CE-control, controlled RNAi-injected CE mice; CE-miR134-RNAi, miR134-RNAi-injected CE mice.

Figure 4

Target Gene Candidates of miR134 Are Scanned in Mice (A) mRNA levels of potential miR134 target genes in SE and CE mice (left, n = 12), as well as in miR134 RNAi and controlled RNAi of CE mice (right, n = 12). (B) Protein levels of miR134 target gene candidates in SE and CE mice (top, n = 6), as well as in miR134 RNAi and controlled RNAi of CE mice (bottom, n = 6). *p < 0.05, and **p < 0.01, versus SE mice; ^#p < 0.05, and ^##p < 0.01, versus CE-control mice. CE, cocaine extinction mice; SE, saline extinction mice; CE-control, controlled RNAi-injected CE mice; CE-miR134-RNAi, miR134-RNAi-injected CE mice.

Figure 5

Knockdown of miR134 in the vHP Ameliorates CE-Induced Changes in Synaptic Plasticity of the vHP (A) Dendritic spines. Representative images of dendritic spines in the vHP of SE and CE mice (left, n = 12; scale bars, 5 μm), as well as in miR134 RNAi and controlled RNAi of CE mice (right, n = 12; scale bars, 1 μm). Red arrows indicate the spines. (B) Synapses. Representative images of synapses (left) and graph of synaptic density (right, n = 12) in the vHP of SE and CE mice. Red arrows indicate synapses. Scale bars, 1 μm. (C) Protein levels of synapsin I (SYP I) and PSD-95 in the vHP of SE and CE mice (left, n = 6), as well as in miR134 RNAi and controlled RNAi of CE mice (right, n = 6). (D) fEPSP of the vHP in miR134 RNAi and controlled RNAi of CE mice. Representative traces of EPSP (top) and slopes of EPSP (bottom; n = 12) of vHP in controlled RNAi and miR134 RNAi mice. *p < 0.05 and **p < 0.01, versus SE mice; ^#p < 0.05 and ^##p < 0.01, versus CE-control mice. CE, cocaine extinction mice; SE, saline extinction mice; CE-control, controlled RNAi-injected CE mice; CE-miR134-RNAi, miR134-RNAi-injected CE mice.

Figure 6

Knockdown of miR134 in the vHP Relieves the Enhancements in Microglia Activity and Inflammatory, Apoptotic, and Oxidative Stress Statuses of CE Mice (A) Protein levels of GFAP and Iba-1 in the vHP of SE and CE mice (left, n = 6), as well as in miR134 RNAi and controlled RNAi of CE mice (right, n = 6). (B) Protein levels of pro-inflammatory factors in the vHP of SE and CE mice (left, n = 6), as well as in miR134 RNAi and controlled RNAi of CE mice (right, n = 6). (C) Protein levels of apoptotic molecules in the vHP of SE and CE mice (left, n = 6), as well as in miR134 RNAi and controlled RNAi of CE mice (right, n = 6). (D) Activities of SOD (top) and level of MDA (bottom) in the vHP of SE and CE mice (left, n = 12), as well as in miR134 RNAi and controlled RNAi of CE mice (right, n = 12). *p < 0.05 and **p < 0.01, versus SE mice; ^#p < 0.05 and ^##p < 0.01, versus CE-control mice. CE, cocaine extinction mice; SE, saline extinction mice; CE-control, controlled RNAi-injected CE mice; CE-miR134-RNAi, miR134-RNAi-injected CE mice.