Korean J Physiol Pharmacol.  2021 Jan;25(1):79-85. 10.4196/kjpp.2021.25.1.79.

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

• Cai WT
• Han J
• Kim WY
• Kim JH

• KMID: 2509657
• DOI: http://doi.org/10.4196/kjpp.2021.25.1.79

Abstract

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.

Keyword

Cocaine; Glutamate receptor; Nucleus accumbens; Tissue fixation

Figure

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  Fig. 1 Experiment outline. An experimental timeline indicating the cocaine sensitization scheme and the point at which immersion-fixation was performed. A schematic diagram of a matched coronal brain section (left) is adapted from the brain atlas of Paxinos and Watson [24], and a representative fluorescent image of the NAcc shows some of the sampled regions (white square boxes) selected for GluA1 analysis (right). Only one or two sample regions from both core and shell, respectively, were obtained from each slice of brain sections. The scale bar is 500 µm.

• 

  Fig. 2 Locomotor activity counts during exposure. All rats were habituated to the locomotor activity chamber for 1 h and their locomotor activity measured for an additional 1 h following their respective injections. Locomotor activity was measured only at day 1 and 7 as remaining injections were administered in the home cage (once daily, a total of 7 injections). Data are expressed as mean + standard error of the mean. **p < 0.01, significant differences compared to saline exposed animals. †††p < 0.001, significant difference at day 7 compared to day 1 in cocaine exposed group as revealed by post-hoc Bonferroni comparisons following two-way repeated ANOVA.

• 

  Fig. 3 Differential GluA1 expression in the NAcc after cocaine exposure is detectable using immersion-fixation immunohistochemistry. (A) Representative images showing co-localization of GluA1 and PSD95 in the NAcc core and shell, obtained with 1-h immersion-fixation on withdrawal day 21 after repeated exposure to saline or cocaine. Green and red indicates PSD95 and GluA1 expression, respectively, while yellow indicates co-localization of the two proteins. The scale bar is 20 µm. (B) Analysis of GluA1 intensity in the NAcc core and shell obtained with 1-h immersion-fixation on withdrawal day 21 after repeated exposure to saline or cocaine. Data are expressed as mean + standard error of the mean. relative to GluA1 intensity in the PSD area of the saline-exposed group. Symbols indicate significant differences as revealed by post-hoc Bonferroni comparisons following two-way ANOVA analysis. *p < 0.05, **p < 0.01; significantly different compared to saline pre-exposed group within each area (PSD or extra-PSD). †††p < 0.001; significantly different compared to PSD area. The total numbers of slices used in the analysis for the NAcc core and shell from 4 animals are 22 and 24 for saline- and cocaine-exposed rats, respectively. The sample numbers for PSD and extra-PSD are the same in all cases.

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