Extinction memory improvement by the metabolic enhancer methylene blue

Abstract

We investigated whether postextinction administration of methylene blue (MB) could enhance retention of an extinguished conditioned response. MB is a redox compound that at low doses elevates cytochrome oxidase activity, thereby improving brain energy production. Saline or MB (4 mg/kg intraperitoneally) were administered to rats for 5 d following extinction training of tone-footshock conditioning. Postextinction freezing was lower in rats receiving MB compared with saline, suggesting that MB improved retention of the extinction memory. The MB effect was specific to tone-evoked freezing because there were no differences in pretone freezing. Control subjects similarly injected with MB showed no evidence of nonspecific effects on measures of motor activity and fearfulness. MB-treated rats exhibited both greater retention of extinction and greater overall brain metabolic activity. Rats with higher retention of extinction also showed a relative increase in cytochrome oxidase activity in prefrontal cortical regions, especially anterior infralimbic cortex, dorsal and medial frontal cortex, and lateral orbital cortex. These regional metabolic increases were also correlated to the behavioral freezing index used to assess retention of extinction. It was concluded that MB administered postextinction could enhance retention of extinction memory through an increase in brain cytochrome oxidase activity.

Figures

Figure 1

Mean ± SE freezing counts during the 2 d of extinction before the beginning of MB and saline injections.

Figure 2

Behavioral effects of methylene blue on the extinction of conditioned freezing. (A) Mean ± SE freezing scores (CS - preCS freezing counts) after 2 d of acquisition training before the onset of MB/saline treatments. (B) Mean ± SE total postextinction freezing scores in the MB-treated and saline-treated groups. (C) Mean ± SE freezing scores in the MB-treated and saline-treated groups in postextinction probes I to V. Asterisks indicate significant differences at P < 0.05.

Figure 3

Behavioral effects of methylene blue on fear-related behavior. (A) Mean ± SE time spent in center and periphery prior to any treatment. (B) Mean ± SE time spent in center and periphery 24 h following one MB injection. (C) Mean ± SE time spent in center and periphery 24 h after five daily MB injections.

Figure 4

Overall absolute brain cytochrome oxidase (CO) activity units (μM/min/g) in the brains of MB-treated and saline-treated groups. Asterisk indicates significant group difference at P ≤ 0.001.

Figure 5

Coronal brain diagrams (right) and cytochrome oxidase stained sections (left) illustrating the location of the regions of interest by Bregma level according to the Paxinos and Watson (1997) atlas. Abbreviations are listed in Table 3.