Korzan WJ and CH Summers 2021 Evolution of stress responses refine mechanisms of social rank. 
	Neurobiology of Stress 100328: 1-19

Pang T, JDW Yaeger, CH Summers, and R Mitra 2021 Cardinal role of the environment in stress induced changes
	across life stages and generations. Neuroscience and Biobehavioral Reviews 124: 137-150 

Yaeger JDW, KT Krupp, JJ Gale, CH Summers 2020 Counter-balanced microcircuits for Orx1 and Orx2 regulation
	of stress reactivity. Medicine in Drug Discovery 100059:  1-20

Summers CH, JDW Yaeger, CD Staton, DH Arendt, TR Summers 2020 Orexin/hypocretin receptor modulation of anxiolytic and antidepressive 
	responses during social stress and decision-making: potential for therapy. Brain Research 1731: 146085: 1-15

Data from: Staton CD, JDW Yaeger, DD Khalid, F Haroun, BS Fernandez, JS Fernandez, BK Summers, TR Summers, M Sathyanesan, 
	SS Newton, CH Summers 2018 Orexin 2 receptor stimulation enhances resilience, while orexin 2 inhibition promotes 
	susceptibility, to social stress, anxiety and depression. Neuropharmacology 143: 79-94.



Fig. 1. Experimental design, Stress-Alternatives Model (SAM), and Social Interaction/Preference test (SIP). A) Experimental timeline: 5-day acclimation, icv surgeries , recovery for 7-10 days, followed by aggressive social interaction (SAM). Days 1-4 included the pairing of the CS+ (tone) with US+ (aggressive social interaction) prior to social interaction.  Drug treatments were given prior to social interaction on day 3. On day 5, test mice underwent an SIP test followed by a final SAM exposure that included the CS+ but not the US+, and sampled thereafter.  B) Design of the SAM apparatus: a clear rectangular (91 x 22 x 26 cm) apparatus, containing an oval escapable open field (OF) arena, divided into three sections by curved opaque barriers with L-shaped tunnel escape routes at the apex (only large enough for the test mouse to pass through) that lead to safe areas. The test mouse is placed in an opaque cylinder (16.75 x 20 cm) in the middle of the OF and the aggressor is placed outside this area. The cylinder is removed just prior to social interaction. C) Design of SIP apparatus; an opaque square arena (40 x 40 x 40 cm) with a clear perforated container set at the middle of one wall. Test mice are first placed in the apparatus at the opposite end from an empty container (left) for 2.5 min before being removed. The empty container is then replaced with a container that has a novel CD1 mouse strain/size matched to mice used as aggressors in SAM (right). The test mouse is placed back into the apparatus in the same manner as previously described for another 2.5 min before being removed.


Fig. 2. Escape behavior is promoted by Orx2 agonism, and limited by Orx2 antagonism, while latency to escape is unchanged.  A) Escape behavior was significantly reduced, following icv injection of Orx2 receptor antagonist (MK-1064) on day 3 (light gray hatched bar) in mice that were previously Escape phenotype (light gray hatched bar; # indicates significance in antagonist treated animals from Days 1&2), but remained unchanged in vehicle treated animals (light gray bar).  B) Escape behavior was promoted following day 3 icv injection of Orx2 receptor agonist ([Ala11,D-Leu15]-Orexin B), on day 4 (dark gray hatched bar; not significant) in mice that were previously Stay phenotype.  (C) Escape latency was significantly reduced (*) on day 4 of SAM interaction compared to day 1. There was no change seen between Escape animals treated with vehicle and animals treated with Orx2 antagonist. 
Fig. 3. Fear-conditioning is represented by freezing produced by pairing a conditioned stimulus (CS = tone) with an unconditioned stimulus (US = aggression). It is only increased in Stay mice, and is reversed by Orx2 agonism.    A) Mean (± SEM) fear freezing ratio (FFR = [ (freezing time after tone/15 seconds) / (freezing time before tone/30 seconds)] x 100) of Stay phenotype mice (dark gray bar) on test day (two days after vehicle injection) reflects a conditioned response (CR) which is significantly (‡) greater than the FFR for Escape phenotype animals. B) Treatment with an Orx2 antagonist (hatched bars) did not significantly alter the fear freezing ratio 2 days after injection, compared with vehicle treated mice.  C)  In Stay mice, which exhibit a CR (significantly elevated FFR) in vehicle treated animals, icv injection of an Orx2 agonist (dark gray hatched bars) blocks the fear freezing conditioned response, producing a FFR which is significantly (§) reduced compared with vehicle controls (dark gray bars).  D)  Home cage activity is not altered by icv injection of Orx2 agonist or antagonist.
Fig. 4.    Freezing time is elevated in Stay mice during SAM interaction and can be increased by Orx2 receptor antagonism and decreased by Orx2 receptor agonism. A) Mean (± SEM) contextual freezing time in vehicle treated Stay animals (solid dots) is significantly increased (*) compared to day one on days 2-4 of SAM interaction; and compared to vehicle treated Escape mice (square ‡) on injection day (3; arrow ‡).  B) Mean (± SEM) % freezing time increases in all groups on day 2, compared to day 1, prior to treatment (+ significance in agonist treated animals, # significance in antagonist treated animals).  Injection of Orx2 antagonist (icv; triangles) increased freezing on day 3 compared to day 1, day 2, and day 4, and icv Orx2 agonist (diamonds, arrow) significantly decreased freezing relative to vehicle (§) and antagonist treatments (†) on day 3 (arrow).  
Fig. 5. Startle responses in Stay mice are increased, but can be diminished by stimulating Orx2 receptors. A) Mean (± SEM) number of startle responses/time in SAM for vehicle treated Stay animals (solid dots) is significantly elevated on day 4 compared to days 1, 2 and 3 (*) and to Escape vehicle treated animals (‡, solid squares) on the day of vehicle injection (arrow, ‡). B) Compared across days, Stay mice treated with vehicle (solid dots) have an increased number of startle responses on day 4 compared to all other days (* indicates significance in vehicle treated animals). Stay mice treated with Orx2 agonist (diamonds) are significantly (§) different from vehicle on day 3 (arrow) and day 4; but not compared to Orx2 antagonist treatment (triangles). 
Fig. 6. Attentiveness to the escape route is low in Stay mice but can be improved with icv administration of Orx2 agonist. A) Vehicle treated mice exhibiting the Stay phenotype (solid dots) spend significantly (‡) less time attentive to the escape hole compared to Escape phenotype animals (squares), across days, and on the day of vehicle injection (arrow).  B) Vehicle and Orx2 antagonist (triangles) treated Stay mice are equally disinterested in the escape route. However, mice treated with Orx2 agonist (diamonds) display a significant increase in % time attentive to the escape hole on days 3 and 4 when compared to day 1 (+).  Specifically on day 3, Orx2 agonist treated mice (arrow) spend a significant more percentage of time attentive to escape hole compared to both vehicle (§) and Orx2 antagonist (†). 
Fig. 7. Time near container plus social target / time near a novel container lacking a social target  (stress resilience) is significantly greater in Escape compared to Stay mice, which is diminished by Orx2 antagonism (Escape mice) and reversed by Orx2 agonist treatment. A, B) Vehicle treated Stay mice (dark gray bar and dots) are significantly (‡) less stress-resilient compared to Escape mice (light gray bar and dots), but when Orx2 antagonist is administered to a cohort of Escape mice (hatched light gray bar and triangles) there is a significant (§) decrease compared to vehicle treated Escape mice. C, D) When Orx2 agonist is administered icv to Stay mice (hatched dark gray bar and diamonds) there is a significant (§) increase in stress-resilience compared to vehicle-treated Stay mice (dark gray bar and dots).  
Fig. 8. Social stress and Orx2 antagonist treatment increase plasma corticosterone concentrations. A) Mean (± SEM) plasma corticosterone concentrations are significantly (bars with differing letters above, such as A vs B) elevated by aggressive social interactions, in both Escape (light gray bar) and Stay (dark gray bar) phenotype mice compared to home-cage controls (white bar). B) Among Escape mice, icv injection with an Orx2 antagonist 2 days prior (on day 3, light gray left hatched bar) does not affect plasma corticosterone on test (5) day. In contrast, among Stay phenotypes, icv injection on day 3 with an Orx2 antagonist (dark gray, left hatched bar) stimulates increased plasma corticosterone measured on day 5 compared to vehicle treated Stay mice (B vs A), antagonist treated Escape mice (‡), and C) Orx2 agonist (dark gray, right hatched bar; C vs B) Stay mice.
Fig. 9. Anxiolysis in Stay mice is accompanied by activation of BLA or ItC inhibitory interneurons. Photomicrographs (magnification 40x) of the amygdala in the AP -1.06, centered on ML + or - 2.65 and DV 4.50 relative to bregma were triple labeled with A) the immediate–early  gene product early growth response protein 1 (EGR1, Zif268, NGF1A, Krox24, or TIS8), B) the GABA neuron calcium-binding protein Parvalbumin (Pv), and C) the nuclear marker 4',6-diamidino-2-phenylindole (DAPI).  D) The EGR1/Pv/DAPI triple labeling is visible in the merged image; scale bar equals 50 µm.  This confluence of labeling, particularly the presence of EGR1, is only evident in Stay mice that have been injected icv with the Orx2 agonist [Ala15, D-Leu11]-OrxB. In Pv-positive neurons E) only in Stay mice treated with Orx2 agonist [Ala15, D-Leu11]-OrxB was the EGR1 labeling significantly increased (dark gray right-hatched bar) compared to cage control (clear bar), vehicle (dark gray bar) and Orx2 antagonist (MK-1064)-treated mice (dark gray left-hatched bar).  F) In Pv-negative neurons, a much smaller, but similar increase in EGR1 labeling occurred. G) Labeling of Pv was similar in all treatment groups.


Data from: Robertson JM, JK Achua, JP Smith, MA Prince, CD Staton, PJ Ronan, TR Summers, CH Summers 2017 Anxious behavior induces 
				elevated hippocampal Cb2 receptor gene expression.  Neuroscience 352: 273-284


Figure 3. - Social stress modifies Cb2 receptor gene expression in the ventral hippocampus. A) Socially aggressive interactions plus fear conditioning in the SAM significantly (* compared with cage controls [white bar], # compared with escape [gray bar]) increased ventral dentate gyrus (vDG) Cb2 receptor mRNA (mean ± SEM) in submissive (dark gray bar) mice. B) In the vCA1 the Cb2 receptor mRNA is significantly (# compared with escape) increased in submissive animals (no change from control animals).



Data from: Achua... CH Summers 2017	Behavioral Neuroscience : submitted 


Figure 3. - Social stress + fear conditioning (FC) diminishes Orx1, but enhances Orx2, receptor gene expression in the ventral hippocampus. Regardless of subregion, submissive mice always show reduced Orx receptor mRNA compared with escaping mice. A) Interactions in the SAM + FC significantly (*compared with cage controls [white bar]) decreased ventral dentate gyrus (vDG) Orx1 receptor mRNA (mean ± SEM) equally in escaping (gray bar) and submissive (dark gray bar) mice, submissive mice Orx1 receptor mRNA significantly (# compared with escape) decreased from escaping mice. B) Social stress + FC diminishes Orx1 receptor gene expression in the dorsal hippocampus. Interactions in the SAM significantly (*compared with cage controls, # compared with escape) reduced dorsal dentate gyrus (dDG) Orx1 receptor mRNA in submissive mice. C) The mRNA for Orx2 receptor in vDG is significantly (*compared with cage controls) increased in escaping and submissive animals, but with submissive Orx2 mRNA significantly (#) reduced compared with escaping mice. D) Social stress diminishes Orx2 receptor gene expression in the dorsal hippocampus. Interactions in the SAM +FC significantly (# compared with escape) decreased dorsal CA1 Orx2 receptor mRNA in submissive mice.



Data from: Arendt DH... CH Summers 2014	Psychoneuroendocrinology 40C: 17-26 


Figure 1. - Social Defeat Protocol (A) Sequence and timing of social defeats (by a novel CD1 mouse once/day for 10 days) and following social preference test (day 11). (B) Social preference scores based on the amount of time test mice spent near and away from a social target delineate animals that were resilient and susceptible to social defeat. “Resilient” animals were defeated, but still showed a social preference (>100% time spent with social target/time spent away from the social target × 100) similar to “Controls”. Defeated animals that displayed a social aversion (<100%) relative to controls and were defined as “Susceptible”.





Figure 2. Susceptible mice have increased BLA Orx1 but decreased Orx2 receptors In the basolateral amygdala (BLA), (A) susceptible animals had significantly elevated amounts of Orx1 mRNA relative to control (*) and resilient (#) animals. (B) This effect in susceptible animals occurred with a concomitant decrease in BLA Orx2 mRNA. In the prelimbic cortex (PrL), there were no differences between the groups for the (C) Orx1 or (D) Orx2 receptor transcripts. *,#p < 0.05.





Figure 3. Orx1 negatively correlated with resilience Orx2 positively correlated  Social preference scores were significantly correlated with BLA expression of (A) Orx1 and (B) Orx2 mRNA. In the BLA there was a significant (p < 0.001, r2 = 0.635) negative regression between the Orx1 mRNA and social preference, and a significant (p < 0.001, r2 = 0.661) positive regression between the Orx2 mRNA and social preference. These correlations were absent in the PrL for (C) Orx1 and (D) Orx2 receptor transcripts.





Figure 4.BLA IC viral injections of shRNA Viral shRNA constructs significantly (*) reduced the expression of the endogenous mRNA sequence for the respective orexin receptor. (A) Gene expression comparisons were made between the control group that received a nonspecific “scramble” virus and the experimental group which received the “knockdown” virus targeting the Orx1 or Orx2 receptor. (B) Behavioral data were analyzed for animals with expression of GFP (?) in the basolateral complex. (C) Sequence and timing of a series of tests commonly used to detect depression and anxiety related behaviors to which transfected animals were exposed. *p < 0.05.




  
Figures 5 & 6. Orexin 2 receptors in the BLA are anxiolytic Knocking down the Orx2 receptor in the BLA increased anxiety-like behaviors. (A) While none of the treatment groups exhibited altered EPM performance, (B) the viral shRNA Orx2 knockdown significantly decreased (*) social preference relative to controls. Locomotion was unaffected in the social preference test with Orx2 knockdown. There was no significant change in social preference for animals that received the Orx1 knockdown despite a significant decrease in the total amount of locomotor activity. (C) Orx2 knockdown also decreased time spent in the center of an open field apparatus. There were no significant changes in locomotion due to Orx1 or Orx2 knockdown in the open field. *p < 0.05. Knockdown of either orexin receptor in the BLA produced no significant differences in behavioral measures of depression for either the (A) sucrose preference test or (B) forced swim test relative to the control treatment.



Funding
NIH R15 MH104485 Amygdalar Orexin Modulation of Affective Disorders $426,477 2015-2017
Published papers resulting from NIH R15 MH125306
Carpenter RE, B Sabirzhanov, TR Summers, TG Clark, J Keifer and CH Summers 2023 Anxiolytic reversal of classically conditioned / 
	chronic stress-induced gene expression and learning in the Stress Alternatives Model. Behavioural Brain Research 
	114258: 440: 1-10 

Yaeger JDW, KT Krupp, BM Jacobs, BO Onserio, BL Meyerink,  JT Cain, PJ Ronan, KJ Renner, RJ DiLeone, CH Summers  2022 
	Orexin 1 receptor antagonism in basolateral amygdala shifts the balance from pro- to anti-stress signaling and behavior. 
	Biological Psychiatry 91: 841-852
    
Yaeger JDW, KT Krupp, TR Summers, CH Summers 2022 Contextual generalization of social stress learning is modulated by
	orexin receptors in basolateral amygdala. Neuropharmacology 215: 109168:1-12



Published papers resulting from NIH R15 MH104485

Arendt DH, PJ Ronan, KD Oliver, LB Callahan,TR Summers and CH Summers 2013 Depressive
 	behavior and activation of the orexin/hypocretin system Behav Neurosci 127:86-94

Arendt DH, J Hassell, Hao Li, JK Achua, DJ Guarnieri, RJ DiLeone, PJ Ronan, and CH Summers 2014
  	Anxiolytic function of the orexin 2 /hypocretin A receptor in the basolateral amygdala.
 	Psychoneuroendocrinology 40C: 17-26

Keifer J and CH Summers 2016 Putting the "biology" back into "neurobiology: The strength of
	diversity in animal model systems for neuroscience research Front Syst Neurosci 10: 69: 1-9

Korzan WJ and CH Summers 2021 Evolution of stress responses refine mechanisms of social rank. 
	Neurobiology of Stress 100328: 1-19

Pang T, JDW Yaeger, CH Summers, and R Mitra 2021 Cardinal role of the environment in stress induced changes
	across life stages and generations. Neuroscience and Biobehavioral Reviews 124: 137-150 

Pearson BL, JN Crawley, D Eilam, NS Pentkowski, CH Summers 2017 Curiosity as an approach to 
	ethoexperimental analysis: Behavioral neuroscience as seen by students and colleagues of Bob Blanchard. 
	Neuroscience & Biobehavioral Reviews 76: 415-422

Smith JP, MA Prince, JK Achua, JM Robertson, RT Anderson, PJ Ronan, CH Summers 2016 Intensity of anxiety is modified via complex
 	integrative stress circuitries. Psychoneuroendocrinology 63: 351-361
    
Robertson JM, JK Achua, JP Smith, MA Prince, CD Staton, PJ Ronan, TR Summers, CH Summers 2017 Anxious behavior induces 
	elevated hippocampal Cb2 receptor gene expression.  Neuroscience 352: 273-284

Robertson JM, MA Prince, JK Achua, RE Carpenter, DH Arendt, JP Smith, TL Summers, TR Summers, 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 

Staton CD, JDW Yaeger, DD Khalid, F Haroun, BS Fernandez, JS Fernandez, BK Summers, TR Summers, M Sathyanesan, 
	SS Newton, CH Summers 2018 Orexin 2 receptor stimulation enhances resilience, while orexin 2 inhibition promotes 
	susceptibility, to social stress, anxiety and depression. Neuropharmacology 143: 79-94.

Summers CH, JDW Yaeger, CD Staton, DH Arendt, TR Summers 2020 Orexin/hypocretin receptor modulation of anxiolytic and antidepressive 
	responses during social stress and decision-making: potential for therapy. Brain Research 1731: 146085: 1-15
    
Summers TR, TL Summers,  RE Carpenter,  JP Smith,  SL Young,  B Meyerink,  TZ Orr,  DH Arendt, and  CH Summers 2017 Learning and CRF-induced 
	indecision during escape and submission in rainbow trout during socially aggressive interactions in the Stress-Alternatives Model
	Frontiers in Neuroscience 11: 515: 1-13
    
Waters RP, M Rivalan, D Bangasser, J Deussing, M Ising, SK Wood, F Holsboer, CH Summers 2015 Evidence for the role of 
	corticotropin-releasing factor in major depression disorder Neuroscience & Biobehavioral Reviews 58: 63-78
    
Yaeger JDW, KT Krupp, JJ Gale, CH Summers 2020 Counter-balanced microcircuits for Orx1 and Orx2 regulation
	of stress reactivity. Medicine in Drug Discovery 100059:  1-20




Research Web Pages:         
Orexin, Anxiety, and Depression            
Stress Alternatives Model            
Anxiety Intensity Gradient            



3D-SAM Rainbow Trout            
Evolution of Anxiety and Depression            
Gene Sequences





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