an Intensity Gradient of Anxious Behavior

in the

Summers' Laboratory of Molecular Neuropsychoecoendochemistry

Figure 1. - (A) Timelines of the experimental protocols all begin with seven days of acclimation to cages, followed thereafter by anxiogenic or anxiolytic treatments. Interaction with SAM apparatus occurred over four days, and may have taken place in the presence (middle and bottom timelines) or absence (top) of a larger conspecific aggressor. (Top) For experiments in which the anxiolytic neuropeptide S (NPS) is delivered icv, intraventricular cannula placement was executed on day eight followed by recover until icv injection of NPS and interaction with the empty SAM apparatus of each of days eleven through fourteen to test for open field (OF) anxious behavior and escape. (Middle) Social anxiety and escape was tested by ip injection of the know anxiogenic a2-adrenoreceptor antagonist yohimbine in escaping animals (determined on days 1 and 2 of SAM interaction; days 8 and 9 overall) or the anxiolytic CRF1 antagonist antalarmin in submitting animals, just prior to day 3 and measuring latency to escape or inhibition of escape on days 3 and 4 of SAM social interactions (days 10 and 11 overall). (Bottom) For the voluntary exercise experiments, mice were tested for predisposition for anxious behavior on the EPM on day 8 and then provided a running wheel for 6 days. Social interactions in the SAM begin after 2 days of running, and continue for another 4 days (days 10–13 overall). The icv NPS experiment ended on day 14, the yohimbine/antalarmin experiments on day 11, and the running wheel experiments ended on day 13. (B) The SAM apparatus includes an open field (OF) arena that can be adjusted for size. The OF includes two escape routes, which lead to safe zones only accessible to small test mice. Test mice are added within the opaque cylindrical divider. Large aggressors are added outside the cylindrical divider. The divider is removed to allow social interaction. (C) The continuum or gradient of intensity of anxious behavior as revealed by the Stress-Alternatives Model.

Figure 2. - Anxiogenic (A) and Anxiolytic (B) drug treatments on day 3 of SAM exposure changed escaping (A) or submissive (B) behavioral phenotypes in a substantial proportion of socially interactive mice. (A) The anxiogenic a2-adrenoreceptor antagonist yohimbine inhibited escape behavior. Prior to ip delivery of yohimbine, all of the mice that would receive the anxiogenic drug escaped (light gray wide hatched bars; escaping animals were chosen for this treatment), but on the day of yohimbine administration only approximately one fourth of the animals escaped (dark gray bars with narrow hatching), and over 70% remained submissively. On the following day (4), with no additional a2-adrenoreceptor inhibition, most (60%) returned to escape behavior, with 40% continuing to remain submissively. (B) The anxiolytic CRF1 receptor antagonist antalarmin promoted escape behavior in previously non-escaping submissive mice. Prior to ip delivery of antalarmin, none of the mice that would receive the anxiolytic drug escaped (no bars are evident; submissive animals were chosen for this treatment), but on the day of antalarmin administration 40% of the animals escaped (cross hatched bars). On day 4 (no drug treatment) 30% continued to escape.

Figure 3. - Mice escape more rapidly (mean latency ± SEM) from the SAM apparatus / SAM social interactions with familiarity, which is enhanced by anxiolytic treatments, and inhibited by anxiogenic treatments. A) Mice injected icv with neuropeptide S (NPS, dashed line with circles) escape significantly faster from the SAM open field (OF) alone (in the absence of social interaction) than aCSF treated controls (solid line/triangles) on the initial trial (#) and during trials on days 3 and 4 (*). B) Mice given access to exercise (running wheel, dashed line/circles) exhibit reduced latency to escape from social aggression on the initial (*) and 3rd trials (#) of SAM social interaction compared to animals that received aggression in the absence of the opportunity for voluntary exercise (solid line/triangles). C) Escaping mice injected ip with the anxiogenic a2-adrenoreceptor antagonist yohimbine on day 3 of SAM social interaction, but continued to escape (See Fig. 3A), did so significantly more slowly (*) on day 3 than mice injected with vehicle. D) Non-escaping submissive mice injected ip with the anxiolytic CRF1 receptor antagonist antalarmin that began escaping (See Fig. 3B) on day 3 did so significantly more rapidly in initial escape (*) than the initial escape (day 1) of escaping animals injected with vehicle, but more slowly than vehicle controls escaped on day 3.

Figure 4. - Plasma corticosterone (mean ± SEM) reflects anxiogenic conditions. (A) In the absence of social interaction in the SAM, icv injection (vehicle, hatched bars) significantly elevated corticosterone concentrations, which is reduced somewhat by NPS icv injection (cross hatched bars). (B) During social aggressive interactions in the SAM, corticosterone concentrations are elevated; highest in submissive animals (cross hatched bars), and significantly reduced in those that escape (left hatching) compared with controls (open bar). Social stress-induced elevation of corticosterone secretion is ameliorated by access to voluntary exercise on a running wheel for submissive animals (*, gray cross hatched bar) and also for escaping mice (#, gray left hatched bar); while presence of the running wheel in the absence of social interaction did not affect plasma corticosterone concentrations. Bars marked with differing letters above the mean/error bar (e.g., A–C; or X and Y) are significantly different, whereas bars marked with the same letter (such as X) are not significantly different. Bars marked # reflect significant differences between escaping animals without access to running wheels and escaping animals with running wheels (Otherwise bars marked A, B, or C, are not compared with bars labeled X, Y, or Z); and * designates significant reduction in corticosterone in submissive animals with running wheels compared to submissive animals without access.

Figure 5. - Intra-amygdalar (central amygdala, CeA) neuropeptide S (NPS) gene expression (mean ± SEM) is elevated by anxiogenic/stressful conditions, and alleviated by anxiolytic conditions. (A) Injection (icv) of NPS (cross hatched bars) stimulated increased NPS mRNA in CeA compared to cage controls (clear bars) and icv aCSF (vehicle) injection (hatched bar), but (B) had no effect on NPS receptor (NPSR). (C) During social aggressive interactions in the SAM, NPS mRNA is elevated; highest in submissive animals (cross hatched bars; see C, E, G), and significantly reduced in those that escape (left hatching) compared with controls (open bar). Social stress-induced elevation of NPS expression is somewhat ameliorated by access to voluntary exercise on a running wheel for submissive animals; while presence of the running wheel in the absence of social interaction did not affect NPS expression. (D) Social aggressive interactions in also stimulated enhanced NPSR mRNA compared with controls (open bar). Presence of the running wheel in the absence of social interaction did not affect NPSR expression. (E, F) Social interaction with escape and the anxiogenic drug yohimbine stimulate elevated expression of both NPS and NPSR mRNA. (G, H) Socially submissive interaction significantly stimulates elevated NPS and NPSR gene expression (higher for both in submissive mice than escaping mice, see E, F), both of which are ameliorated by the anxiolytic drug antalarmin. Bars marked with differing letters above the mean/error bar (e.g., A–C; or X and Y) are significantly different, whereas bars marked with the same letter (such as X) are not significantly different.

Figure 6. - Intra-amygdalar (central amygdala, CeA) brain-derived neurotrophic factor (BDNF) gene expression (mean ± SEM) is elevated by anxiogenic/stressful conditions, and alleviated by anxiolytic conditions. There were no effects of treatments on BDNF TrKB receptor mRNA. (A) Injection (icv) of NPS (cross hatched bars) returned BDNF mRNA in CeA to cage control expression levels (clear bars), compared to reduced expression in icv aCSF (vehicle) injection (hatched bar). (B) Running wheels (*) and submission increased BDNF fold expression in the CeA, compared with cage controls (clear bars) and escaping mice without exercise (hatched bars). (C) Increased CeA BDNF mRNA in submissive animals (similar to B; cross hatched bars) is ameliorated by anxiolytic CRF1 antagonist antalarmin treatment (gray bars). (D) The anxiogenic a2-adrenoreceptor antagonist yohimbine stimulated increased BDNF gene expression in the CeA, compared to cage controls (clear bar) and escaping mice (hatched bar). Bars marked with differing letters above the mean/error bar (e.g., A–C; or X and Y) are significantly different, whereas bars marked with the same letter (such as A and AB) are not significantly different.

Figure 1. - Schematic depictions of developmental iterations of the Stress-Alternatives Model (SAM) designed for mice, trout, rats, and hamsters. A) The current SAM apparatus designed for rodents (dimensions are for the mouse version), including mice, rats, and hamsters. This version has no corners, an oval open field with flexible dimensions and two escape routes only large enough for the smaller test animal. A test rodent is placed inside the center circular opaque divider (dashed line); the divider is removed after presentation of a tone (conditioned stimulus, CS) to initiate trials. Trials may be social interactions with the addition of a larger conspecific placed outside the circular divider prior to the start. Arena and escape routes are novel for the initial trial, and L-shaped tunnels obscure the destination, creating some anxious behavior (delayed latency to escape) on their initial use. B) The SAM was first tested with rainbow trout in a 284 L aquarium where a test animal is fed. A removable opaque divider (dashed line) was used to separate the larger aggressor and intruder prior to interaction; its removal followed cessation of water flow (CS). A second removable opaque divider (dashed line) allows access to an escape hole large enough for only the smaller test fish, located near the top of the water column, which leads to the safety chamber. C) Initial experiments with rats utilized a square arena, and a single escape hole and safety chamber. A second escape hole and safety chamber were added to prevent the larger aggressive animal from blocking access to the safety chamber. A diagonal opaque divider (dashed line) separated the large rat from test rats, and was removed following a short tone (CS), prior to social interaction. D) Golden hamsters reliably escape social aggression, and this version of the SAM was designed to study the effect of forced interaction and submission versus escape. A removable divider (dashed line) could be used to block access to the escape hole and safety chamber for smaller individuals, forcing some hamsters to submit to the larger aggressor.

Figure 2. - Across species tested in the SAM, approximately 50% escape, and 50% remain submissively. Animals escape or remain submissively when presented with a larger aggressive conspecific in SAM social interactions, where an escape route is available that is too small for the larger animal to pass. Means are averaged over nine experiments (3 for trout, one for rats, and 5 for mice). The ranges were greatest for mice, and extend from 43.3 to 60% for escape and for submission extend from 40 to 56.7%

Figure 3. - Intensity of aggression doesn't drive escape behavior. The original raw data have been normalized to the initial/baseline levels of aggression, such that the means represent change in intensity of aggression received (derived by: maximum intensity/average total intensity of aggression/time) as related to escape or submission in SAM social interactions. The data have also been normalized for time spent in the arena. Data from all trials have been collapsed, as there were no significant differences between trials. Top) No significant differences in the increase in intensity of aggression between escaping (gray bar, upward hatching) and submissive (white bar, downward hatching) rainbow trout. Bottom) Mice that escaped aggression received significantly (P < 0.05) reduced aggression intensity over time, while those that submitted received more.

Smith JP, MA Prince, JK Achua, JM Robertson, RT Anderson, PJ Ronan, and CH Summers 2016 Intensity of anxiety is modified via complex
 	integrative stress circuitries. Psychoneuroendocrinology 63: 351-361

Robertson JM, MA Prince, JK Achua, RE Carpenter, DH Arendt, JP Smith, TL Summers, TR Summers, and CH Summers 2015 Nuance and behavioral
	cogency: How the Visible Burrow System inspired the Stress-Alternatives Model and conceptualization of the continuum of anxiety. 
	Physiology and Behavior 146: 86-97 

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