Anxiolytic reversal of classically conditioned / chronic stress-induced gene expression and learning in the Stress Alternatives Model

Abstract

Social decision-making is critically influenced by neurocircuitries that regulate stress responsiveness. Adaptive choices, therefore, are altered by stress-related neuromodulatory peptide systems, such as corticotropin releasing factor (CRF). Experimental designs that take advantage of ecologically salient fear-inducing stimuli allow for revelation of neural mechanisms that regulate the balance between pro- and anti-stress responsiveness. To accomplish this, we developed a social stress and conditioning protocol, the Stress Alternatives Model (SAM), that utilizes a simple dichotomous choice, and produces distinctive behavioral phenotypes (Escape or Stay). The experiments involve repeated social aggression, a potent unconditioned stimulus (US), from a novel larger conspecific (a 3X larger Rainbow trout). Prior to the social interaction, the smaller test fish is presented with an auditory conditioning stimulus (water off = CS). During the social aggression, an escape route is available, but is only large enough for the smaller test animal. Surprisingly, although the new aggressor provides vigorous attacks each day, only 50% of the test fish choose Escape. Stay fish, treated with the CRF₁ antagonist antalarmin, a potent anxiolytic drug, on day 4, promotes Escape behavior for the last 4 days of the SAM protocol. The results suggest that the decision to Escape, required a reduction in stress reactivity. The Stay fish that chose Escape following anxiolytic treatment, learned how to use the escape route prior to stress reduction, as the Escape latency in these fish was significantly faster than first time escapers. In Escape fish, the use of the escape route is learned over several days, reducing the Escape latency over time in the SAM. Fear conditioning (water off + aggression) resulted in elevated hippocampal (DL) Bdnf mRNA levels, with coincident reduction in the AMPA receptor subunit Glua1 expression, a result that is reversed following a one-time treatment (during SAM aggression on day 4) with the anxiolytic CRF₁ receptor antagonist antalarmin.

Keywords: Anxiety; Corticotropin-releasing factor; Depression; Fear conditioning; PTSD; SAM; Social aggression.

Fig. 1.

Experimental design and SAM aquatic social fear conditioning and behavioral arena. (A) The investigation design timeline included a week of acclimation to experimental tanks, followed by pretest blood sample 3 days of recovery, and then SAM conditioning. (B) Pulling the opaque dividers obscuring combatants and escape route, following CS presentation (water off), allowed for (C) social interactions with a novel large aggressor (US) occurred daily in the SAM over the next week, with CR testing on the 8^th day. Fifteen minutes after the initiation of testing terminal blood and brain samples were collected for analysis. Individual trout exhibiting (D) Stay or (E) Escape phenotypes were reliably stable across the training period.

Fig. 2.

The CRF₁ antagonist Antalarmin shifts pro-stress behavior and neuroendocrine responsiveness toward anti-stress phenotypes and outcomes. (A) Individual trout originally exhibiting Stay or Escape phenotypes were roughly equivalent: 45% Escape, 55% Stay. While antalarmin treatment did not affect the behavioral phenotype of Escape fish (data not shown), (B) most Stay animals (67%) began, and continued, using the escape route following CRF₁ antagonist treatment on day 4. A minority (33%) of Stay fish continued to Stay following antalarmin treatment.

Fig. 3.

Plasma cortisol concentration [F] was similar in all fish before treatments or group placement (blood drawn 3 days prior to SAM exposure and 7 days before drug treatment; left bars). In the same animals, following all 7 days of SAM exposure, saline-treated control Stay fish without antalarmin treatment (hatched white bar) produced a significant physiological conditioned response (CR), elevated plasma [F], in response to the CS only (water off; no aggression) on day 8 (from a second blood draw), compared to saline-treated control Escape fish (white bar). Day 4 CRF₁ receptor antagonist treatment (gray bars) eliminated increased [F] in Stay fish that Escape from day 4 onward (St/Esc; gray right-down hatched bar), but not in Stay fish that retained the Stay phenotype (St/St), which had elevated plasma [F] compared to fish treated with Antalarmin that Escape (*), and fish that began as Stay trout then Escape after Antalarmin pretreatment (*), Pre-SAM Stay controls [F] (+), and compared to all controls (#).

Fig. 4.

Latency to escape (mean ± SEM) over 7 days SAM social interaction with CS:US pairings. Black line with circles indicates escape latency in seconds for saline-treated control fish that escaped during social interactions. Black hashed line with squares indicates fish that escaped during the first 4 training sessions and were treated with CRF₁ receptor antagonist on day 4. Diamonds with dotted line indicates fish that did not escape during the first 4 training sessions, were treated with CRF₁ receptor antagonist on day 4 and began escaping.

Fig. 5.

(A) Hippocampal Bdnf gene expression (mean fold ± SEM) is significantly but differentially enhanced in saline-treated control Escape (Esc; white tilt-hatched bar) vs saline-treated control Stay (white flat-hatched bar) fish following CS only presentation on test day (day 8). Escape and Stay fish expressed significantly elevated (*) Bdnf gene expression compared to individuals that received the conditioned stimulus only (CS; water off; white bar), to individuals receiving daily exposure to the unconditioned stimulus only (US; aggression from larger, novel conspecific; white dotted bar), and to Escape and Stay fish treated (day 4) with the CRF₁ antagonist antalarmin (gray bars). Importantly, Escape Bdnf mRNA levels were also significantly (+) elevated compared to Stay fish; and stimulated hippocampal Bdnf gene expression is reversed by treatment with the CRF₁ antagonist antalarmin. (B) Hippocampal Glua1 gene expression is significantly (*) reduced in saline-treated control Stay fish following presentation of the CS alone on test day compared to all other groups. Importantly Reduced hippocampal Glua1 mRNA expression is reversed following treatment with the CRF₁ antagonist antalarmin. (C) Hippocampal Glua2 gene expression is not influenced by presentation of the CS only on test day. Following presentation of the CS only on test day (day 8), no differences in Glua2 mRNA were detected between groups. Additionally, treatment with the CRF₁ antagonist did not influence Hippocampal GluA2 mRNA expression following presentation of the CS only on test day (day 8).