Effect of 7,8-dihydroxyflavone, a small-molecule TrkB agonist, on emotional learning

Abstract

Objective: Despite increasing awareness of the many important roles played by brain-derived neurotrophic factor (BDNF) activation of TrkB, a fuller understanding of this system and the use of potential TrkB-acting therapeutic agents has been limited by the lack of any identified small-molecule TrkB agonists that fully mimic the actions of BDNF at brain TrkB receptors in vivo. However, 7,8-dihydroxyflavone (7,8-DHF) has recently been identified as a specific TrkB agonist that crosses the blood-brain barrier after oral or intraperitoneal administration. The authors combined pharmacological, biochemical, and behavioral approaches in a preclinical study examining the role of 7,8-DHF in modulating emotional memory in mice.

Method: The authors first examined the ability of systemic 7,8-DHF to activate TrkB receptors in the amygdala. They then examined the effects of systemic 7,8-DHF on acquisition and extinction of conditioned fear, using specific and well-characterized BDNF-dependent learning paradigms in several models using naive mice and mice with prior traumatic stress exposure.

Results: Amygdala TrkB receptors, which have previously been shown to be required for emotional learning, were activated by systemic 7,8-DHF (at 5 mg/kg i.p.). 7,8-DHF enhanced both the acquisition of fear and its extinction. It also appeared to rescue an extinction deficit in mice with a history of immobilization stress.

Conclusions: These data suggest that 7,8-DHF may be an excellent agent for use in understanding the effects of TrkB activation in learning and memory paradigms and may be attractive for use in reversing learning and extinction deficits associated with psychopathology.

FIGURE 1. Activation of p-TrkB and MAPK in Mouse Amygdala by Systemic 7,8-Dihydroxyflavone (7,8-DHF)^a

^a MAPK=mitogen-activated protein kinase. Panel A shows immunoblots of mouse amygdala punches examining total TrkB protein (top), activated p-TrkB (second row), total MAPK (third row), and activated p-MAPK (bottom row). Each condition is represented in duplicate, with amygdala punches from mice injected intraperitoneally with vehicle (control) or 7,8-DHF (5 mg/kg) 1 hour or 2 hours prior to sacrifice. Full-length TrkB is detected at both ~95 kDa (nonglycosylated) and ~140–145 kDa (glycosylated) forms. Although total levels of TrkB and MAPK do not change, systemic 7,8-DHF led to robust activation/phosphorylation of amygdala TrkB (p-TrkB Y706) and MAPK (p-MAPK). Panel B shows the quantification (mean values with standard deviations) of the p-TrkB706:TrkB ratio represented in the top two immunoblots of panel A, demonstrating increased p-TrkB706 with 7,8-DHF treatment. In panel C, receptor autoradiography demonstrates that ³H-7,8-DHF binds to brain regions known to express TrkB protein, including the amygdala (arrow). Panel D shows in situ hybridization of TrkB mRNA expression from the Allen Brain Atlas (www.allenbrainatlas.com). In panel E, ³H-7,8-DHF shows no significant binding when tissue is pretreated with excess cold 7,8-DHF, suggesting relative specificity. Panel F shows dense expression of TrkB protein on pyramidal neurons in basolateral rat amygdala identified with immunocytochemistry. Scale bar represents ~50 μm.

FIGURE 2. Effects of 7,8-Dihydroxyflavone (7,8-DHF) on Fear Conditioning in Wild-Type Mice^a

^a Panel A shows results of a dose-response study performed with 7,8-DHF (N=10 in each dose group). The graph shows the average proportion of animals freezing during the first three conditioned stimulus trials (^#p≤0.05 relative to vehicle [control]). Panel B shows the mean total distance and the distance traveled in the center of the open field for 10 minutes with 7,8-DHF or vehicle injected 1 hour before testing; there were no differences between groups. Panel C shows the mean shock reactivity in the startle apparatus during the cue-dependent fear conditioning; there were no differences between groups. Panel D shows the mean percentage of time spent freezing, in three conditioned stimulus trials per group (across the entire 15 conditioned stimuli), in testing for cue-dependent fear memory 24 hours after fear conditioning. Mice that had received 7,8-DHF prior to fear conditioning showed increased freezing. Panel E shows the mean percentage of time spent freezing within-session for each test trial for the first 10 trials of session. Within-session fear differences were particularly pronounced in the first conditioned stimulus trial of the testing session. Panel F shows that the mean total time spent freezing during testing (conditioned stimulus and no-stimulus intertrial periods) was significantly greater in the group of mice that had received 7,8-DHF 1 hour prior to fear conditioning. Error bars in panels B–F indicate standard deviations. In panels D–F, *p<0.05 between 7,8-DHF and vehicle groups.

FIGURE 3. Effect of 7,8-Dihydroxyflavone (7,8-DHF) on Fear Extinction in Wild-Type Mice^a

^a Panel A shows the mean percentage of total time spent freezing during the testing. Pre-extinction was performed 24 hours after cue-dependent fear conditioning, and within-extinction (in the presence of drug) was performed 24 hours after that. Mice that received 7,8-DHF showed enhanced extinction of fear relative to those that received vehicle. Panel B shows the mean percentage of time spent freezing in response to conditioned stimulus presentation during the within-extinction session, represented in blocks of three trials. Mice that received 7,8-DHF 1 hour beforehand had similar initial freezing but showed rapid extinction of fear compared to those that received vehicle. In panel C, after all animals had been extinguished to a minimal level of fear (post-extinction test in the absence of drug, 24 hours after within-session), they were then subject to reinstatement (24 hours after post-extinction). Total freezing in post-extinction and post-reinstatement was compared between animals that had received 7,8-DHF or vehicle 1 hour before the extinction training. Error bars indicate standard deviations. *p<0.05 between the 7,8-DHF and vehicle groups.

FIGURE 4. Effect of 7,8-Dihydroxyflavone (7,8-DHF) on Extinction in a Traumatic Stress Model^a

^a Mice underwent 2-hour immobilization 6 days before the first session of fear conditioning, followed by repeated sessions of extinction. Panel A shows that shortly after immobilization, there is a robust activation of the hypothalamic-pituitary-adrenal axis, as demonstrated by a greater mean acute level of plasma corticosterone relative to control mice. In panel B, the immobilized group showed delayed extinction in analyses of conditioned freezing in extinction sessions 2 and 3 (panels C and D), whereas both groups had equivalent low levels of freezing by session 4, suggesting a delay in extinction following immobilization stress. (In panels A–D, ***p<0.001, **p<0.01, *p<0.05 between control and immobilization groups.) In panel E, animals also showed impaired extinction when fear-conditioned 12 days after immobilization stress. Because of the delayed nature of extinction in immobilization-treated animals, we examined the last session to compare differential freezing during pre-extinction, within-session extinction, and post-extinction sessions across groups (with and without immobilization, and with and without 7,8-DHF). Mice that were given 7,8-DHF prior to extinction showed a significant drug-by-session effect of enhancement of extinction across sessions (**p<0.01 session-by-group interaction between pre-extinction, within-extinction, and post-reinstatement). Panel F shows that 7,8-DHF was associated with decreased freezing in the within-session extinction trials in animals with a prior history of immobilization. Freezing (average of six trials within each bin) is shown during within-extinction session, 1 hour after administration of 7,8-DHF or vehicle (***p<0.001, **p<0.01 for the immobilization-vehicle compared with immobilization-7,8-DHF groups). Error bars indicate standard deviations.