Immunohistochemical detection of GluA1 subunit of AMPA receptor in the rat nucleus accumbens following cocaine exposure

α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are differentially regulated in the nucleus accumbens (NAcc) of the brain after cocaine exposure. However, these results are supported only by biochemical and electrophysiological methods, but have not been validated with immunohistochemistry. To overcome the restriction of antigen loss on the postsynaptic target molecules that occurs during perfusion-fixation, we adopted an immersion-fixation method that enabled us to immunohistochemically quantify the expression levels of the AMPA receptor GluA1 subunit in the NAcc. Interestingly, compared to saline exposure, cocaine significantly increased the immunofluorescence intensity of GluA1 in two sub-regions, the core and the shell, of the NAcc on withdrawal day 21 following cocaine exposure, which led to locomotor sensitization. Increases in GluA1 intensity were observed in both the extra-post synaptic density (PSD) and PSD areas in the two sub-regions of the NAcc. These results clearly indicate that AMPA receptor plasticity, as exemplified by GluA1, in the NAcc can be visually detected by immunohistochemistry and confocal imaging. These results expand our understanding of the molecular changes occurring in neuronal synapses by adding a new form of analysis to conventional biochemical and electrophysiological methods.

Immunohistochemical detection of GluA1 subunit of AMPA receptor in the rat nucleus accumbens following cocaine exposure

α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are differentially regulated in the nucleus accumbens (NAcc) of the brain after cocaine exposure. However, these results are supported only by biochemical and electrophysiological methods, but have not been validated with immunohistochemistry. To overcome the restriction of antigen loss on the postsynaptic target molecules that occurs during perfusion-fixation, we adopted an immersion-fixation method that enabled us to immunohistochemically quantify the expression levels of the AMPA receptor GluA1 subunit in the NAcc. Interestingly, compared to saline exposure, cocaine significantly increased the immunofluorescence intensity of GluA1 in two sub-regions, the core and the shell, of the NAcc on withdrawal day 21 following cocaine exposure, which led to locomotor sensitization. Increases in GluA1 intensity were observed in both the extra-post synaptic density (PSD) and PSD areas in the two sub-regions of the NAcc. These results clearly indicate that AMPA receptor plasticity, as exemplified by GluA1, in the NAcc can be visually detected by immunohistochemistry and confocal imaging. These results expand our understanding of the molecular changes occurring in neuronal synapses by adding a new form of analysis to conventional biochemical and electrophysiological methods.

Ezrin-radixin-moesin proteins are regulated by Akt-GSK3β signaling in the rat nucleus accumbens core

The ezrin-radixin-moesin (ERM) proteins are a family of membrane-associated proteins known to play roles in cell-shape determination as well as in signaling pathways. We have previously shown that amphetamine decreases phosphorylation levels of these proteins in the nucleus accumbens (NAcc), an important neuronal substrate mediating rewarding effects of drugs of abuse. In the present study, we further examined what molecular pathways may be involved in this process. By direct microinjection of LY294002, a PI3 kinase inhibitor, or of S9 peptide, a proposed GSK3β activator, into the NAcc core, we found that phosphorylation levels of ERM as well as of GSK3β in this site are simultaneously decreased. These results indicate that ERM proteins are under the regulation of Akt-GSK3β signaling pathway in the NAcc core. The present findings have a significant implication to a novel signal pathway possibly leading to structural plasticity in relation with drug addiction.

Mechanism of bradykinin on inflammations of retinal pigment epithelium cells / 国际眼科杂志(Guoji Yanke Zazhi)

Abstract?AIM: To investigate mechanism of bradykinin ( BK) on inflammations of retinal pigment epithelium ( RPE) cells.?METHODS: ARPE -19 cells were cultured in vitro, stimulated by 100nM BK for 24h. Cell morphology changes were observed by microscope, and BK receptor localization was detected through cell immunofluorescence. Changes of Ca2+in BK and BR antagonist stimuli were detected by laser scanning confocal microscopy.The expressions of COX-1, COX-2, eNOS and iNOS protein in control group and BK group were detected by Western Blot.?RESULTS: After the stimulation of BK, there was no significant changes of ARPE-19 cells in morphology.Kinin B1 receptors ( B1R ) and B2 receptors ( B2R ) could be detected in ARPE-19 cells.Compared with control group, Ca2+concentrations significantly increased in BK group; in B1R antagonist group and B2R antagonist group Ca2+concentrations increased less than BK group; B1R and B2R antagonist group showed no obvious changes in Ca2+concentrations.Compared with control group, COX-2 and iNOS protein concentrations were significantly increased in BK group (P<0.001).?CONCLUSION:BK induces the increasing expression of COX-2 and iNOS in the cultured ARPE cells through binding with either B1R or B2R.