Dissociable consequences of moderate and high volume stress are mediated by the differential energetic demands of stress.

Exposure to traumatic stress leads to persistent, deleterious behavioral and biological changes in both human and non-human species. The effects of stress are not always consistent, however, as exposure to different stressors often leads to heterogeneous effects. The intensity of the stressor may be a key factor in determining the consequences of stress. While it is difficult to quantify intensity for many stress types, electric shock exposure provides us with a stressor that has quantifiable parameters (presentation length x intensity x number = shock volume). Therefore, to test the procedural differences in shock volume that may account for some reported heterogeneity, we used two common shock procedures. Learned helplessness is a commonly reported behavioral outcome, highlighted by a deficit in subsequent shuttle-box escape, which requires a relatively high-volume stress (HVS) of about 100 uncontrollable shocks. Conversely, stress-enhanced fear learning (SEFL) is another common behavioral outcome that requires a relatively moderate-volume stress (MVS) of only 15 shocks. We exposed rats to HVS, MVS, or no stress (NS) and examined the effects on subsequent fear learning and normal weight gain. We found doubly dissociable effects of the two levels of stress. MVS enhanced contextual fear learning but did not impact weight, while HVS produced the opposite pattern. In other words, more stress does not simply lead to greater impairment. We then tested the hypothesis that the different stress-induced sequalae arouse from an energetic challenge imposed on the hippocampus by HVS but not MVS. HVS rats that consumed a glucose solution did exhibit SEFL. Furthermore, rats exposed to MVS and glucoprivated during single-trial context conditioning did not exhibit SEFL. Consistent with the hypothesis that the inability of HVS to enhance fear learning is because of an impact on the hippocampus, HVS did enhance hippocampus-independent auditory fear learning. Finally, we provide evidence that stressors of different volumes produce dissociable changes in glutamate receptor proteins in the basolateral amygdala (BLA) and dorsal hippocampus (DH). The data indicate that while the intensity of stress is a critical determinant of stress-induced phenotypes that effect is nonlinear.

1. Introduction

Acute, intense stressors can lead to various physiological and psychological conditions in both human and non-human species [1, 2]. Post-Traumatic Stress Disorder (PTSD) develops in about 20% of those that experience a traumatic stressor [3]. PTSD is a debilitating and heterogeneous disease marked by a wide variety of potential symptoms such as amnesia, anhedonia, avoidance behaviors, exaggerated fear-potentiated startle, hypervigilance, and insomnia [4]. PTSD patients also exhibit a diverse array of comorbidities, including major depressive disorder and substance use disorders [5-8]. Great strides have been made in understanding the neurobiological consequences of severe stress, yet little headway has been made in identifying effective treatment(s) of stress-induced psychiatric diseases such as PTSD.

One apparent roadblock to advancing basic research on stress and its translation to clinically effective treatment is the highly inconsistent findings among research groups. Inconsistent findings may be because stress models vary widely between research groups, leading to divergent behavioral and biological findings [9, 10]. Despite the disparity among groups, there has been little to no attempt to consolidate the stress literature. The many qualitatively different stressors used in the laboratory makes it nearly impossible to compare findings. However, electric shock exposure provides a stressor in which the parameters are quantifiable (duration, intensity, and number of presentations). There is prior evidence that the amount of shock acts as a determining factor for qualitative differences in stress-reactive behavior [11]. In a recent review, we looked at two comparable stress procedures due to their mutual use of inescapable and unpredictable electric shock as the stressor [9]. This review compared the behavioral and biological impacts of the stressors used to induce two often explored behavioral effects, learned helplessness and stress-enhanced fear learning.

The stressor that produces the learned helplessness (LH) effect typically consists of 100, 1 mA tail shocks of variable length (based on the yoked controls performance, this typically averages between 3–8 seconds over the course of the session) that occur during a 2-hour session [12, 13]. The hallmark behavior is the subsequent deficit in escape performance within the shuttle-box apparatus [14, 15]. However, rats exposed to the extensive 100 tailshock session also exhibit a wide array of behavioral characteristics that parallel several symptoms of PTSD and depression [16, 17]; for review, see [9]. For example, rats that are exposed to 100 tailshocks show anhedonia and lower weight than controls [18-21]. Several biological mediators of the shuttle-escape deficit have been identified, such as corticosterone, serotonin, and adenosine [12, 22, 23]. The impact of the energetically demanding fear state caused by this extensive stress session is also implicated in the deleterious behavioral consequences of stress. The 100-shock session transiently stresses energy homeostasis [24]. Access to a concentrated glucose solution reverses the shuttle-escape deficits produced by the stressor [25, 26], while artificial glucoprivation using 2-deoxy-D-glucose (2-DG) and adenosine agonists promote shuttle-escape deficits in unstressed rats [27, 28]. Furthermore, the activity of adenosine, which is closely linked to cellular energy homeostasis, is both necessary and sufficient for the stress-induced shuttle escape deficits [23, 27, 29, 30]. These findings led to our previous hypothesis that the energetic challenge induced by the stressor is a key mediator for the observed deleterious behavioral effects.

The stress-enhanced fear learning (SEFL) stressor consists of 15, 1 mA footshocks of fixed length (1 second) occurring during a 1.5-hour session. This stress procedure and subsequent behavioral phenomena became a focus of interest due to its ability to produce a long-lasting (e.g., at least 3 months) enhancement of subsequent contextual and auditory fear conditioning [31-33]. It was later discovered that this shock procedure also produced a robust array of anxiety-like behaviors [34, 35]. Rats exposed to 15 shocks also exhibit a wide array of behavioral characteristics similar to the symptoms of PTSD but do not exhibit depression-like behavior as reported following 100 shocks [9, 35]. Evidence of the neurobiological mediators for SEFL is relatively limited, but initial evidence points toward a rise in basolateral amygdala (BLA) GluA1, which forms a functionally unique tetramer of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, in the BLA, as a mediator for the sensitization effect [35].

The stressors that induce LH and SEFL produce several similar behavioral characteristics that model pathological fear and anxiety in the rat. The research also suggests that they may diverge in inducing depression-like behavior. However, research has not directly compared the LH and SEFL inducing stressors. Here we test the hypothesis that more extensive exposure to a stressor equates to greater behavioral and biological consequences. The LH and SEFL producing stressors are particularly useful in testing this because they are qualitatively similar yet vary on a major dimension: shock volume (shock number x current x length). Rats exposed to an LH-inducing stressor experience a total of about 300 to 800 seconds of 1 mA shock (100 shocks at an average of 3–8 seconds each, based on yoked performance), while SEFL-exposed rats receive a total of only 15 seconds of 1 mA shock (15 shocks at an average of 1 second each). To avoid confusion between the stressors and their behavioral consequences, and make it clear that we are not examining the effects of stressor controllability that have already been well characterized [12], we will subsequently refer to the 100-shock procedure as high-volume stress (HVS), the 15-shock procedure as moderate-volume stress (MVS), and the restraint controls as no shock (NS). While it is clear that both MVS and HVS produce similar levels of anxiety-like behavior in the animal, and only HVS produces consistent depressive-like behavior, their individual effects on fear have yet to be comprehensively examined. Here we test the hypothesis that enhanced fear learning is differentially expressed following MVS and HVS exposure.

Six experiments investigate the behavioral and neurobiological consequences of stress volume. Rats were restrained in tubes and exposed to either 0, 15, or 800 cumulative seconds of shock over a 90 or 113-minute interval. Rats were assessed for enhanced fear learning or sacrificed for tissue analysis one week after stress pretreatment. We included manipulations that increase or decrease glucose availability, with the hypothesis that glucose availability only mediates effects produced by the energetically demanding high-volume stressor. These manipulations either occurred immediately after the termination of the stress session or immediately before single-trial conditioning. Rats were weighed throughout the study, and weight gain was compared across groups.

2. Materials and methods

2.1. Subjects

Two hundred and eight Sprague-Dawley albino male rats (290–320 grams) from Envigo (Placentia, CA, USA) were housed in individual cages in a room maintained on a 12:12-hour light/dark cycle (6:00–17:59 lights on, 18:00–5:59 lights off). Animals were housed in the room for approximately two weeks prior to testing. Rats were housed in metal hanging cages. Each cage was equipped with a standard glass (250 mL) water bottle, with a rubber stopper and metal spout, and a metal food hopper that allowed to ad libitum consumption of water and standard rat chow. During this time, all animals had free access to food and water. All experimentation occurred during the early light cycle (7:00–10:00, approximately). The protocols in this paper received pre-approval by the UCLA Institutional Animal Care and Use Committee.

2.2. Apparatus

Rats were restrained in clear Plexiglass restraining tubes during stress pretreatment, as previously described [25]. Electric shock was administered via electrodes attached to a rat’s extended tail. During the session, each restraining tube was housed in an illuminated, sound-attenuating chamber. Testing occurred in Med Associates (St Alban, Vt) behavioral testing chambers. Each chamber was equipped with an infrared camera, speaker for tone delivery, shock scrambler, and fluorescent and infrared light sources. The behavioral testing chambers in each testing room were controlled by a PC using Med Associates Video Freeze software that automatically scored the animal’s shock-induced motion and freezing during the test session. Modification of the chamber’s contextual features used differential lighting, odors, ambient noise, and interchangeable grid floors and wall inserts to create distinct contexts when necessary.

2.3. Procedure

Rats were assigned randomly to groups of eight to ten rats each. Rats received restraint, fifteen (MVS) or one hundred (HVS) inescapable tailshocks. One day or one week later, rats underwent a fear conditioning procedure or were sacrificed for tissue analysis.

Rats that received HVS were exposed to 100, 1.0 mA variable-duration (mean = 8.0 s, range: 3 to 15 s) and inescapable tail shocks on a variable-time 60-s schedule (range: 20 to 150 s) in restraining tubes during a 113-min stress pretreatment session. Rats that received MVS shock were exposed to 15, 1.0 mA fixed-duration (1 second) and inescapable tail shocks on a variable-time 360-s schedule (range: 120 to 900 s) in restraining tubes during a 90-min stress pretreatment session. The specific HVS and MVS parameters were chosen to mimic previously published work that produced stress-induced shuttle-escape deficits [36] or stress-enhanced fear learning [31]. However, prior research with the MVS SEFL procedure always used footshock while prior work with HVS LH primarily used tailshock. To make the procedures more comparable we used tailshock for both our MVS and HVS stressors. The other groups were restrained in tubes for the same period (113 or 90 minutes) and received no shock. A home cage control (HCC) was added for all experiments involving tissue analysis. These animals were handled the same as other groups but were not exposed to the stress pretreatment.

The fear conditioning procedure was as follows. On the first day of testing, rats were placed in a novel environment and received a single, 1-second and 1 mA footshock after three minutes of free exploration. Rats were retrieved thirty seconds after shock exposure and returned to their home cage. The following day, rats were placed back into this context for eight minutes to test for contextual fear. Time spent freezing was assessed during both days. In the cued fear learning experiment, a 30 second, 65 dB, 2800 Hz tone preceded and co-terminated with shock. All rats were tested for contextual fear conditioning in the cued learning experiment as previously stated. All groups were then pre-exposed to a novel context one day after contextual fear conditioning testing. Preexposure consisted of three, 30-minute sessions across three days (1 session/day). Following preexposure, all rats received a tone test where, following a three-minute baseline period, three, 30-second tone presentations were spaced one minute apart.

In the experiments involving glucose manipulation, all stress and contextual fear conditioning procedures were identical to those previously described, but with the addition of glucose or 2-DG delivery. In the glucose intervention experiment, all rats were pre-exposed to a glucose cocktail over three consecutive days [26]. During glucose preexposure, each animal’s water bottle was replaced with a bottle containing the glucose cocktail. Glucose preexposure occurred 10 days prior to stress exposure. The cocktail consisted of 40% glucose and 5% sucrose dissolved in tap water (weight/volume). Rats received 18-hours of free access to glucose or water, based on experimental condition, immediately following the termination of stress pretreatment. In the experiment involving peripheral injection of 2-DG, rats were injected intraperitoneally with either vehicle or 100 mg/kg of 2-DG dissolved in distilled water twenty minutes before single-trial fear conditioning.

2.4. Western blot analyses

The dorsal hippocampus (DH), ventral hippocampus (VH), and BLA were dissected and flash frozen for western blot analysis. Tissue was homogenized and spun to separate crude and synapto-neurosome homogenate and diluted in a synaptic protein extraction reagent containing protease and phosphatase inhibitors (ThermoFisher, Cat #s 87793 & 78440). Protein concentrations of diluted homogenate were estimated using BCA assay (ThermoFisher, Cat # 23225). 15ug of protein was loaded into a 10% polyacrylamide gel for electrophoretic separation and then transferred to a PVDF membrane (Bio-Rad, Cat #s 5671035 & 1704157). Lanes were assessed for total protein using Ruby protein blot staining (ThermoFisher, Cat # S11791). Primary antibody was then applied overnight and secondary antibody (fluorescent or chemiluminescent) was applied for one to two hours the following day. Tissue was analyzed for GluA1 (Millipore cat # ABN241, 1:5000), GluA2 (Millipore cat # MABN1189, 1:1000), NR1 (Millipore cat # AB9864, 1:5000), NR2a (Millipore cat # AB1555P, 1:10000), NR2b (Abcam cat # AB28373, 1:5000), and GAPDH (Abcam cat # AB8245, 1:5000). Secondary antibodies were applied at a 1:10000 to 1:5000 dilution depending on primary antibody specifications (Abcam cat # AB205719, Bio-Rad cat #s 12005867 & 12004162). Blots were imaged using a ChemiDoc MP imager and analyzed using Image Lab software (Bio-Rad, cat #s 17001402 & 1709690).

2.5. Statistical analysis

Software package SPSS (SAS Institute, Inc., Version 16.0, Cary, NC, USA) was used for statistical analyses. One-way, two-way, three-way, and mixed-design ANOVAs were used when appropriate. Following significant interactions, Tukey post-hoc analyses were reported. Statistical significance was noted when p values were less than 0.05. Data are presented as individual data points overlaid by group means with error bars denoting group mean +/− SEM. No statistical outliers were removed from the data. Animals were excluded solely based on equipment malfunction.

3. Results

3.1 Moderate (MVS) and high-volume (HVS) stressors result in dissociable behavioral effects

We tested the hypothesis that rats exposed to HVS and MVS will exhibit distinct behavioral phenotypes. Due to the greater volume of shock exposure present in HVS, we initially hypothesized that rats exposed to HVS would exhibit heightened SEFL and greater persistent weight loss compared to rats exposed to MVS.

Fig 1 shows percent freezing to the conditioning context (A & C) and weight change (B & D) one day and one week following stress pretreatment. The MVS group showed higher levels of contextual fear-induced freezing compared to the NS and HVS groups during the test, regardless of the time that intervened between stress exposure and fear conditioning (A & C). One-way analyses of variance (ANOVA) on freezing during the context test yielded a significant main effect of group on freezing (%) both when the interval between stress exposure and fear conditioning was one day or one week, F(2, 20) = 8.010, p = .003 and F(2,20) = 7.095, p = .005. Tukey post-hoc comparisons on group means indicated a relationship among groups: MVS > HVS = NS. Both stress groups exhibited greater weight loss compared to NS 24-hours following stress exposure (B). A one-way ANOVA on Weight yielded a significant main effect of Group, F (2, 20) = 9.729, p = .001. Tukey post-hoc comparisons on groups means indicated a relationship among groups, such that: MVS = HVS < NS. Interestingly, the HVS group showed diminished normal weight gain one week following stress exposure compared to MVS and NS groups (D). A one-way ANOVA on Weight yielded a significant main effect of Group, F (2, 20) = 8.860, p = .002. Tukey post-hoc comparisons on group means indicated a relationship among groups, such that: MVS = NS > HVS.

Fig 1

Effects of stress volume on fear learning and weight maintenance.

Depicted: Percent freezing during context fear test one day (panel A) and one week (panel C) following stress exposure; weight change one day (panel B) and one week (panel D) following stress exposure. Rats were exposed to 0 (NS), 15 (MVS), or 100 (HVS) tailshocks one day or one week prior to single-trial fear conditioning. Testing consisted of exposure to a single, 1 mA shock in a novel context. Rats were then returned to this same context 24-hours later. Rats were weighed prior to stress exposure and prior to fear conditioning testing. The MVS group spent more time freezing during the context group when compared to NS and HVS groups. The HVS group showed significantly less weight gain when compared to MVS and NS groups one week after stress exposure. Both shock groups exhibited greater weight loss, compared to NS, one day following stress exposure. Error bars denote mean ± SEM. * p < .05, ** p < .01.

The rats exhibited no freezing prior to the single shock exposure. Therefore, there were no significant differences in baseline freezing (F < 1). This suggests that the differences observed in freezing during the context test are indeed due to differences in fear learning, not generalization between the stress and fear conditioning contexts. Furthermore, no significant differences in shock reactivity were observed as measured by the average motion index (F < 1).

3.2 Moderate and high-volume stress exposure results in enhanced cued fear conditioning

The previous experiment showed that differences in stress volume impact subsequent behavior and physiology of the animal. Specifically, when one week intervened between stress exposure and fear conditioning, we found a double-dissociation such that rats exposed to MVS exhibited SEFL but did not have suppressed weight gain; rats exposed to HVS showed the opposite pattern. HVS has a great physiological impact on the hippocampus [37, 38], but evidence of the stressor’s functional impact remains elusive [39]. While there is no evidence of HVS’s impact on hippocampal function during an unstressed state, a small body of evidence suggests that hippocampal processing may be impaired during any subsequent testing that also elicits the stress response [40, 41]. Contextual fear conditioning is reliant upon hippocampal processing [42, 43]. However, conditioning to discrete stimuli, such as a tone, typically does not rely on the hippocampus [44-47]. Here, we attempt to test the hypothesis that HVS impairs the enhancement of subsequent contextual fear conditioning by decreasing hippocampal function during a stressful event. Specifically, we hypothesize that while rats exposed to HVS do not express SEFL to a context, they will express SEFL to a tone- an association that does not require the hippocampus [48].

Fig 2 shows percent freezing to the 1-shock context (A), to the shock-associated tone (B), and during preexposure to a novel context (C). The MVS group showed higher freezing levels than the NS and HVS groups during the contextual fear test (A). A one-way ANOVA on freezing during the context test yielded a significant main effect of Group, F (2, 24) = 7.944, p = .0023. Tukey post-hoc comparisons on group means indicated a relationship among groups: MVS > HVS = NS. However, compared to the NS group, both MVS and HVS groups exhibited higher levels of freezing to the tone (B). A one-way ANOVA on freezing during tone presentation yielded a significant main effect of Group, F (2, 22) = 4.327, p = 0.0260. Tukey post-hoc comparisons on group means indicated a relationship among groups: NS < MVS = HVS. A mixed-design ANOVA on freezing during context preexposure (C) yielded a significant Group x Trial interaction, F (4, 26) = 3.185, p = .0296. However, Tukey’s post-hoc comparisons on group means did not indicate any statistically significant simple main effects. No differences in baseline freezing prior to the single shock exposure or tone presentation were observed (Fs<1).

Fig 2

Moderate and HVS exposure results in enhanced cued fear conditioning.

Depicted: Percent freezing during context conditioning test (panel A), presentation of the conditioned tone (panel B), and the first eight minutes of each context preexposure trial (panel C). Rats were exposed to 0 (NS), 15 (MVS), or 100 (HVS) tailshocks one week prior to single-trial fear conditioning. Testing consisted of exposure to a single, 1 mA shock in a novel context following presentation of a 30-second tone. Rats were then returned to this same context 24-hours later. Rats were then pre-exposed to another novel context. In this context, the previously-conditioned tone was presented and freezing was assessed. The MVS group spent more time freezing during the context test when compared to NS and HVS groups. Interestingly, both MVS and HVS groups showed higher levels of freezing to the tone compared to the NS group. Error bars denote mean ± SEM. * p < .05, ** p < .01.

3.3 Post-stress glucose rescues contextual SEFL behavior in rats exposed to high-volume stress

The previous experiment showed that while rats exposed to HVS did not exhibit enhancement of hippocampal-dependent contextual fear learning, they did express increased hippocampal-independent cued fear conditioning. Prior studies have indicated that glucose ingestion following HVS reverses several of the stressor’s behavioral impacts [24-Biol Psychiatry. 1997 ">26]. It has been hypothesized that hippocampal-encoding of the context may mediate glucose’s prophylactic effect [25, 40, 41]. We therefore hypothesized that post-stress glucose may induce the SEFL phenotype not previously observed in rats exposed to HVS. We also hypothesized that glucose may mitigate the suppression of weight gain observed following HVS.

Fig 3 shows percent freezing to the conditioned context (A) and weight change (B) one week following stress pretreatment. In rats given water following shock, the MVS group showed higher levels of freezing compared to the NS and HVS groups during the context test (as seen in the previous experiments). However, rats given glucose following HVS exhibited freezing levels higher than their water-drinking counterparts and similar to rats given MVS (A). A two-way ANOVA on freezing during the context test yielded a significant Stress x Fluid interaction, F(2, 42) = 3.499, p = .0393. Tukey post-hoc comparisons on group means indicated a relationship among groups: NS-W = NS-G = HVS-W < HVS-G = MVS-W = MVS-G. Weight gain was depressed in both HVS and MVS groups compared to NS. Furthermore, there appeared to be an overall depression of weight gain in groups that received access to post-stress glucose (B). A two-way ANOVA on weight change (%) yielded significant main effects of Stress, F(2, 40) = 12.34, p < .0001, and Fluid, F (1, 40) = 4.945, p = .0319. Tukey post-hoc comparisons on stress indicated a relationship among groups, such that NS > MVS = HVS. A one-way ANOVA showed no statistically significant effect of group on shock reactivity, F(2, 21) = 1.622, p = .221, or baseline freezing (F < 1).

Fig 3

Impacts of glucose ingestion on the fear learning and weight maintenance stress volume effects.

Depicted: Percent freezing during context conditioning test (panel A) and weight change one week following stress exposure (panel B). Rats were exposed to 0 (NS), 15 (MVS), or 100 (HVS) tail shocks one week prior to fear conditioning testing. Following stress exposure, all groups received 18-hour free access to a 40% glucose solution or tap water, based on experimental condition. All bottles were then switched back to tap water for the remainder of the experiment. Testing consisted of exposure to a single, 1 mA shock in a novel context. Rats were then returned to this same context 24-hours later. Rats were weighed prior to stress exposure and prior to fear conditioning testing. In groups that received water only, the MVS group spent more time freezing during the context test, when compared to NS and HVS groups. However, in groups that received post-stress glucose, both MVS and HVS groups exhibited freezing levels higher than the NS group. Regardless of fluid condition, both the HVS and MVS groups showed significantly less weight gain when compared to the NS group. Error bars denote mean ± SEM. * p < .05, ** p < .01, *** p < .001, **** p < .0001.

3.4 2-deoxy-D-glucose-induced glucoprivation inhibits the formation of contextual SEFL behavior in rats exposed to MVS

The previous experiment showed that consumption of a glucose solution is enough to produce contextual SEFL in rats exposed to HVS, which otherwise do not exhibit the phenotype. Here we test the opposite: is artificial glucose deprivation sufficient to inhibit the expression of SEFL in MVS animals? Previous research has shown that glucoprivation induced by 2-deoxy-D-glucose (2DG) in the absence of stress was sufficient to induce several of the behavioral phenotypes typically observed following HVS [28]. We test the hypothesis that injection of 2DG will suppress the expression of the SEFL phenotype in rats exposed to MVS stress.

Fig 4 shows percent freezing during the contextual fear test (A) and weight change (B) among groups one week following stress pretreatment. Rats that received vehicle and MVS showed higher freezing levels compared to the NS group during the context test 24 hours after 1-shock exposure (as previously seen). However, the group exposed to MVS and given an injection of 2-DG prior to fear conditioning exhibited decreased fear expression compared to their vehicle-injected counterparts (A). A one-way ANOVA on freezing during the context test yielded a significant main effect of Group, F(2, 37) = 10.740, p = .0002. Tukey post-hoc comparisons on group means indicated a relationship among groups, such that: NS-V = MVS-D < MVS-V. No significant differences in weight change were observed among groups (B), F (2, 29) = 0.6203, p = .545. A one-way ANOVA showed no statistically significant effect of group on shock reactivity or baseline freezing to the 1-shock context (F < 1).

Fig 4

Impacts of 2-deoxy-d-glucose injection on SEFL and weight maintenance in rats exposed to MVS.

Depicted: Percent freezing during context conditioning test (panel A) and weight change (panel B). Rats were exposed to 0 (NS), or 15 (MVS) tail shocks one week prior to fear conditioning testing. All groups received intraperitoneal injection of 2DG or vehicle prior to fear conditioning. Testing consisted of exposure to a single, 1 mA shock in a novel context. Rats were then returned to this same context 24-hours later. Rats were weighed prior to stress exposure and prior to fear conditioning testing. When 2DG was given prior to fear conditioning, the MVS-vehicle group spent more time freezing during the context group when compared to the NS and MVS-2DG groups. No weight maintenance effects of 2DG were observed at the time of testing. Error bars denote mean ± SEM. * p < .05, *** p < .001.

3.5 Moderate and high-volume stressors result in dissociable neurobiological effects

We have shown that stress volume impacts the subsequent behavioral phenotype in a dissociable manner. Here, we tested the hypothesis that stress also produces dissociable neurobiological effects. Our lab has previously shown that MVS stress increases GluA1 expression in the BLA [35]. Since we have shown that HVS inhibits hippocampal-dependent (context) SEFL, we hypothesized that HVS will produce a reduction in N-methyl-D-aspartate (NMDA) receptor concentrations in the hippocampus. We also hypothesized that HVS will induce a similar increase of GluA1 in the BLA, since HVS enhanced hippocampal-independent auditory SEFL.

Fig 5 shows AMPA and NMDA receptor subunit protein quantification in the BLA (E-H) and the DH (I-L) one week after stress treatment. As previously seen, rats exposed to HVS exhibited greater weight loss seven days after stress exposure (B). A one-way ANOVA on Weight Change (%) yielded a significant main effect of Group, F(3,20) = 4.413, p = .0155. Tukey post-hoc comparisons (α = .05) on Weight Change indicated the following ordered relationship among means: HCC = NS = MVS > HVS.

Fig 5

Neurobiological effects of stress volume.

Depicted: BLA (panels E-H) and DH (panels I-L) concentrations of GluA1 (panels E & I), GluA2 (panels F & J), NR1 (panels G & K), and NR2a/2b (panels H & L) as determined by western blot analysis. GluA1, GluA2, and NR1 are depicted as a ratio over GAPDH concentrations (panels C & D). Rats were exposed to 0 (NS), 15 (MVS), or 100 (HVS) tailshocks, or remained in their home cage (HCC) never exposed to context or a stressor, one week prior to sacrifice for tissue analysis. Rats were weighed prior to stress exposure and prior to sacrifice. MVS and HVS groups exhibited higher BLA concentrations of GluA1 compared to HCC and NS groups. The HVS exhibited higher concentrations of GluA2 compared to the HCC group. The HVS group had a lower concentration of DH NR1 when compared to NS. All groups exhibited a lower NR2a/2b ration in the DH when compared to the HCC group. Error bars denote mean ± SEM. * p < .05 (compared to HCC), # p < .05 (compared to NS).

Rats exposed to MVS or HVS exhibited greater levels of GluA1 in the BLA compared to HCC (E); rats exposed to HVS, but not MVS, also exhibited higher levels of GluA2 in the BLA compared to HCC (F). One-way ANOVAs on BLA protein analysis yielded significant main effects of Group on GluA1/GAPDH, F(3, 20) = 15.93, p < .0001, and GluA2/GAPDH, F(3, 20) = 3.214, p = .0449. Tukey post-hoc comparisons (α = .05) on GluA1/GAPDH indicated the following ordered relationship among group means: HCC = NS < MVS < HVS. Tukey post-hoc comparisons (α = .05) on GluA2/GAPDH indicated the following ordered relationship among group means: HCC < HVS. No group differences were observed in BLA NR1 or NR2a/2b ratios (G & H).

No group differences in DH GluA1 or GluA2 were observed (I & J). However, rats exposed to HVS exhibited decreased concentrations of NR1 in the DH compared to NS controls (K). Furthermore, all stressed groups exhibited a decreased NR2a:2b ratio in the DH compared to HCC (L). One-way ANOVAs on DH protein analysis yielded a significant main effect of Group on NR2a/2b, F(3, 20) = 3.980, p = .0234. Tukey post-hoc comparisons (α = .05) on NR2a/2b indicated the following ordered relationship among group means: HCC > NS = MVS = HVS. Due to high variability in the HCC, any group effect on DH NR1 was not statistically significant. However, if the HCC group is removed from the analysis, a one-way ANOVA on DH protein analysis yields a significant main effect of Group on NR1, F(2, 14) = 4.651, p = .0283. Tukey post-hoc comparisons on NR1 indicated the following ordered relationship among group means: NS = MVS > HVS. No significant main effects of Group were found during protein analysis of the VH.

4. Discussion

The experiments described above provide evidence that the volume of a stressor is a key factor in determining the behavioral and neurobiological consequences of stress and, this cannot simply be summarized as more stress leads to greater deleterious effects. We found evidence that supports the notion that HVS may model stress-induced conditions that have a depression component or comorbidity, while MVS may better model anxiety-only disorders. Furthermore, we found evidence that suggests that glucose exerts its behavioral effects exclusively in high volume-stressed rats. The effects of glucose appear to not only eliminate HVS-induced phenotypy [24-Biol Psychiatry. 1997 ">26], but in the case of SEFL, facilitate it in HVS-stressed animals. This is further exemplified by our finding that when rats exposed to MVS were glucoprivated during single-trial conditioning, the SEFL effect was eliminated. Finally, we provide evidence that stressors of different volumes produce dissociable changes in AMPA and NMDA receptor density in the BLA and dorsal hippocampus.

There are a number of potential mechanisms through which stress volume exerts its effects on subsequent fear learning. One possible explanation is that HVS is producing a general deficit in contextual fear learning that masks the sensitization effect of MVS. Contextual learning critically depends on hippocampal processing [42, 43, 49] and the hippocampus is profoundly affected by stress [50, 51]. An increase in circulating glucocorticoids during stress impairs glucose uptake transport into the hippocampus and severely impairs contextual processing [52-56]. The HVS procedure used in our experiments produces deficits in contextual discrimination [40] and long-term effects on hippocampal spine density [57], neurogenesis [38], synaptic plasticity, and long-term potentiation [37, 58]. Deficits in contextual learning are reversed by increasing hippocampal glucose concentrations by several means [59-61]. Therefore, while HVS may still induce the non-associative fear sensitization process within the BLA that occurs in MVS, the behavioral expression of this process may be nullified by an overall decrease in contextual fear learning (see Fig 6). This is, in part, supported by our finding that HVS did enhance fear conditioning to a tone. In addition, evidence suggests that cued fear conditioning is hippocampal-independent [44, 62]. While there are some conditions where the hippocampus plays a role in fear elicited by auditory cues, that effect is typically seen after CS termination [45] and our measure was confined to the 30 sec tone presentation. Our finding that cued, but not contextual, fear conditioning is enhanced by HVS therefore may suggest that hippocampal functioning may be impaired by exposure to an HVS. This hypothesis is further supported by our finding that HVS, but not MVS, decreases NR1 expression in the DH. NR1 is the obligatory NMDA receptor subunit and provides a reasonable estimate for NMDA receptor concentration [63]. Hippocampal NMDA receptor activity is essential for the acquisition of contextual fear [49, 62, 64]. Therefore, stress-enhanced contextual fear learning may be inhibited in HVS by decreasing the hippocampus’ ability to form new contextual memories. Follow-up studies investigating the differential impacts of MVS and HVS on peripheral and hippocampal energy homeostasis will be an important step in assessing this proposed mechanism. We have previously found that HVS uniquely challenges peripheral energy homeostasis, but these effects have not been compared to the effects following MVS exposure [24].

Fig 6

Schematic of hypothesized mechanism.

These results present but a few examples of how the behavioral and biological outcomes of stress can be counter-intuitive. These studies explore the outer extremes of stress volume, and follow-up exploring intermediary values is clearly necessary. Furthermore, while we controlled for several factors, several procedural differences did remain. For example, while the MVS and HVS exposures occur over a relatively similar timeframe (90 and 114 minutes, respectively), this necessitates that the intervals between shocks are vastly different (six minutes and one minute, respectively). The length of individual shocks is also different between procedures. These aspects of shock undoubtedly impact the subsequent behavior of the animal (in fact, see [65-67]) and should also be parametrically studied. It is important to note that this study reports effects on male rats exclusively. This non-trivial limitation was necessary, as the LH model has historically used males due to the intriguing, but confounding, resiliency in female rats [68]. Therefore, these findings cannot be extended to female rats, and potential sex differences must be explored further. Finally, this is the first study to demonstrate tailshock-to-footshock SEFL using the 15-shock design. Therefore, this study provides further evidence that SEFL is not simply a generalization of the footshock event carrying over to the single-trial conditioning session.

The effects of stress exposure are variable in severity and quality [69, 70]. Treatment efficacy for patients suffering from stress-induced psychiatric disease continues to remain similarly variable, with only a small percent of the population seeing a persistent quelling of symptoms [71, 72]. One hypothesis is that the observed variability in treatment effectiveness may correlate with variability in stress exposure; the quality, severity, and chronicity of the experienced trauma may have a direct impact on symptoms expressed and the probability of a positive treatment outcome. While parametric study of trauma exposure is impossible in a clinical population, animal models provide us with the necessary tools to interrogate the effects of stress dimensions through direct, controlled comparison. Surprisingly, the experiments described in this manuscript are some of the first to directly study the effects of stress volume using appropriate controls. Therefore, despite an enormous literature devoted to the effects of stress, we are one of the first to provide evidence that the dimensions of the stressor used can directly impact the subsequent behavioral and biological profiles. The eventual goal of this (and future) effortful, parametric work on the effects of various stress dimensions is to provide insight on how information regarding the type of trauma experienced can help inform predictions on disease development and treatment.

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20 May 2022

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Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: The manuscript reports a series of experiments examining how “shock volume” impacts glucose-mediated fear learning. In the manuscript, the authors contrast three groups: High-volume shock (HVS; 100 shocks), moderate-volume shock (MVS; 15 shocks) and no-shock controls (NS). In the first experiment there was a double dissociation between shock volume and contextual fear and weight gain. MVS increased contextual fear and had no impact on weight gain, whereas HVS had no impact on fear but decreased weight gain. Both HVS and MVS increased expression of auditory fear conditioning. Next, rats that consumed a glucose solution also showed enhanced contextual fear relative to NS, and glucosprivated rats did not show enhanced contextual fear following MVS. Thus, the authors show bi-directional control of this the impact of MVS on contextual fear. Finally, the authors report dissociable effects of glutamate receptor expression in the BLA and DH.

Overall, this is an excellent series of experiments. As mentioned earlier, it is particularly thoughtful to see effects manipulated in both directions. I have one major comment, and several more minor comments.

Major: My main concern is related to the cued fear conditioning experiment. The was a nice addition; it allowed the authors to examine if the MVS vs. HVS dissociation was general to fear expression or specific to the expression of contextual fear learning. In this experiment, while MVS increased contextual fear, and HVS did not (relative to NS), expression of cued fear was increased for both MVS and HVS.

The concern I have is that the authors describe the tone-shock association as not requiring the hippocampus. While it is certainly true that there are many reports that delay fear conditioning is not impacted by pre- or post-training damage to the hippocampus, there is additional research that suggests this may depend on the “strength” of the association. Indeed, Quinn et al. (2008) demonstrate that when the tone-shock association is weak, then expression of auditory fear conditioning does depend on the hippocampus. Given that in the current paper there was only one pairing of the tone and shock, it seems reasonable that this would be considered a weak association.

I’m sure the authors are aware of this, and likely have some thoughts. I don’t think this needs to be addressed with additional experiments. But I wonder if the authors could address this issue. Although it would be a strong argument if indeed this tone-shock association were hippocampal independent, I do think it is still a compelling dissociation.

Minor:

1) Pg. 7. The experiments were run in only male rats. However, the authors provide suitable logic for this in the general discussion.

2) All the figures were “fuzzy”. This might have been an issue with my own printer, or maybe with the pre-processing by the journal. I only mention it just so the authors are aware in case it is related to the resolution they were saved as.

3) Pg. 8 and 9. In general the reader could use some more details regarding the procedures of the experiment. As just one example, the authors note that rats were pre-exposed to a novel context, but they don’t mention duration of exposure.

4) Pg. 11. The authors report an ANOVA on post-shock freezing. But I think they are referring to the retrieval test?

5) Pg. 11. The results for figure 1 might be re-ordered. For the reader, it might be more logical to discuss panel A then B then C then D. I thought this was also true for the way the data is described for Figure 2.

6) I like the additional analysis of shock reactivity and baseline freezing. I think the authors can decide – but would it be worth it to include in the figures?

7) For brevity, when an F value is less than 1, I think the authors could report (F < 1) as opposed to the full value (e.g, F = 0.006837).

8) Figure 2 panel C. Trials on the X-axis. This this 1-minute time bins?

Reviewer #2: The present manuscript (PONE-D-22-09431) examines the impact of different doses of tailshock (15 vs 100 shock procedures) on subsequent fear conditioning and glutamate receptor protein expression. The authors state that the 15-shock procedure is analogous to the stress-enhanced fear learning model and the 100-shock procedure is analogous to the learned helplessness paradigm. The authors present a reasonable hypothesis that the two procedures generate different molecular and behavioral sequelae due to differences in metabolic demand.

The hypothesis would be strengthened if the authors provide a comparison of post-stress metabolic measures between the two stress types (moderate vs high-volume stress). Giving 2-deoxy-D-glucose is not the same as directly measuring energy homeostasis. Furthermore, do the authors have any evidence that the glucose manipulations that alter the behavioral phenotype also alter their hippocampal receptor subunit findings?

One issue with framing the present results in the context of stress-enhanced fear learning (SEFL) versus learned helplessness (LH) is that the authors don’t accurately define what learned helplessness actually is. LH refers to stressor outcomes that depend on the uncontrollability of the stressor. That is, to qualify as a LH effect an outcome must follow exposure to inescapable (IS), but not physically identical escapable (ES), shocks or other aversive events. To ignore the controllability issue is to make LH effects synonymous with generic stress effects, which they are not. There are plenty of outcomes (e.g., neurochemical, behavioral) of tail shock that are not sensitive to the dimension of controllability. Thus, there can be many reasons for poor shuttlebox escape responding, only one of which is learned helplessness.

Related to above, the authors imply that the gold standard for LH experiments is to deliver 100 x 1.0 mA shocks for an average length of 8 seconds (800 seconds of total shock). The majority of published LH studies (those that include inescapable and escapable groups) use considerably less shock volume. Mean wheel-turn escape times across 100 shocks are around 3-5 seconds (300-500 seconds of total shock). If the authors frame their high-volume stress treatment as a typical learned helplessness design, then they should provide references demonstrating that their shock parameters produce effects that are selective to the uncontrollability of the stressor.

The claim that these results represent “the first study to demonstrate tailstock-to-footshock” stress-enhanced fear learning ignores a number of published papers showing that tail shock enhances fear learning (both cued and contextual). As an example, uncontrollable tail shock leads to enhanced footshock-elicited freezing in a shuttle box 24 hr later (plenty of studies from the Maier laboratory), a conditional response attributable to contextual cues of the shuttle box apparatus (Fanselow, 1980). In fact, the majority of studies show that uncontrollable tail shock increases, rather than interferes with, contextual fear.

The abstract states that weight gain was impacted only in high-volume stress animals. Was this a consistent finding throughout the studies? It appears that weight gain was depressed in both high and moderate groups compared to No Stress in Figure 3. And in Figure 4B the % weight change for the MVS-Veh group is similar to that of the HVS group in Figure 1D.

**********

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10 Jun 2022

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The above funding statement is correct. As such there are no changes necessary. For financial disclosures, however, we would like to add “Financial Disclosures-MSF is a board member of Neurovation Inc. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.” The acknowledgment and financial disclosure sections have been removed, per request, to avoid redundancy.

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It appears we failed to note the github link somewhere within the submission, our apologies. It is available at https://github.com/mconoscenti/shock_volume. This has also been noted in the revised cover letter.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

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The raw Western Blot data has been uploaded to Github as well, https://github.com/mconoscenti/shock_volume/tree/mconoscenti-westernblot-raw.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: The manuscript reports a series of experiments examining how “shock volume” impacts glucose-mediated fear learning. In the manuscript, the authors contrast three groups: High-volume shock (HVS; 100 shocks), moderate-volume shock (MVS; 15 shocks) and no-shock controls (NS). In the first experiment there was a double dissociation between shock volume and contextual fear and weight gain. MVS increased contextual fear and had no impact on weight gain, whereas HVS had no impact on fear but decreased weight gain. Both HVS and MVS increased expression of auditory fear conditioning. Next, rats that consumed a glucose solution also showed enhanced contextual fear relative to NS, and glucosprivated rats did not show enhanced contextual fear following MVS. Thus, the authors show bi-directional control of this the impact of MVS on contextual fear. Finally, the authors report dissociable effects of glutamate receptor expression in the BLA and DH.

Overall, this is an excellent series of experiments. As mentioned earlier, it is particularly thoughtful to see effects manipulated in both directions. I have one major comment, and several more minor comments.

Major: My main concern is related to the cued fear conditioning experiment. The was a nice addition; it allowed the authors to examine if the MVS vs. HVS dissociation was general to fear expression or specific to the expression of contextual fear learning. In this experiment, while MVS increased contextual fear, and HVS did not (relative to NS), expression of cued fear was increased for both MVS and HVS.

The concern I have is that the authors describe the tone-shock association as not requiring the hippocampus. While it is certainly true that there are many reports that delay fear conditioning is not impacted by pre- or post-training damage to the hippocampus, there is additional research that suggests this may depend on the “strength” of the association. Indeed, Quinn et al. (2008) demonstrate that when the tone-shock association is weak, then expression of auditory fear conditioning does depend on the hippocampus. Given that in the current paper there was only one pairing of the tone and shock, it seems reasonable that this would be considered a weak association.

I’m sure the authors are aware of this, and likely have some thoughts. I don’t think this needs to be addressed with additional experiments. But I wonder if the authors could address this issue. Although it would be a strong argument if indeed this tone-shock association were hippocampal independent, I do think it is still a compelling dissociation.

This really is an interesting worth acknowledgement in the paper. It is true that out single-trial conditioning would reasonably fall within a procedure that produces a weak association. Indeed, that’s why the procedure is chosen! A weak association allows for very clear SEFL effects. However, in a sufficiently stressed animal, a single trial no longer produces a weak association. In the 100 shock group, however, since they are not exhibiting high levels of contextual freezing, it becomes a much larger puzzle to determine if our single-trial conditioning should be considered a procedure that nourishes a weak or strong association.

In an effort to remain succinct within the manuscript, we have added a note regarding the Quinn et al (2008) series of experiments, but have not gone into the weeds regarding this issue. Note that in the Quinn study hippocampal lesion effects were typically seen after, not during CS presentation, the data we presented were all during CS presentation. We have also softened claims suggesting that this experiment comprehensively tests this hypothesis.

Minor:

1) Pg. 7. The experiments were run in only male rats. However, the authors provide suitable logic for this in the general discussion.

Thank you for noting this important point.

2) All the figures were “fuzzy”. This might have been an issue with my own printer, or maybe with the pre-processing by the journal. I only mention it just so the authors are aware in case it is related to the resolution they were saved as.

Thank you for pointing this out. The files were uploaded at high resolution, but we will be sure that the figures look clear during the preprint process.

3) Pg. 8 and 9. In general the reader could use some more details regarding the procedures of the experiment. As just one example, the authors note that rats were pre-exposed to a novel context, but they don’t mention duration of exposure.

Information regarding preexposure has been added. Other clarifying language regarding procedures and reorganization of the method section has been performed to better spell out all procedures performed.

4) Pg. 11. The authors report an ANOVA on post-shock freezing. But I think they are referring to the retrieval test?

Yes, that is correct. Wording has been changed to accurately reflect this.

5) Pg. 11. The results for figure 1 might be re-ordered. For the reader, it might be more logical to discuss panel A then B then C then D. I thought this was also true for the way the data is described for Figure 2.

This point is appreciated. We have reordered the results of B & D to read in the appropriate order. We have kept the discussion of A & C together, however, to avoid seeming unnecessarily redundant by discussing the results of 1-day and 1-week separately where they are nearly identical.

6) I like the additional analysis of shock reactivity and baseline freezing. I think the authors can decide – but would it be worth it to include in the figures?

These data were included in initial internal drafts. However, because much of the baseline freezing data was zero, it felt unnecessary to provide graphs that were essentially empty.

7) For brevity, when an F value is less than 1, I think the authors could report (F < 1) as opposed to the full value (e.g, F = 0.006837).

Where applicable, the full reporting has been changes to simply “F < 1”.

8) Figure 2 panel C. Trials on the X-axis. This this 1-minute time bins?

The x-axis denotes the three preexposure trials. Hopefully, with the additional information regarding our preexposure procedure, this becomes clearer to the reader. We have also included further description in the figure legend.

Reviewer #2: The present manuscript (PONE-D-22-09431) examines the impact of different doses of tailshock (15 vs 100 shock procedures) on subsequent fear conditioning and glutamate receptor protein expression. The authors state that the 15-shock procedure is analogous to the stress-enhanced fear learning model and the 100-shock procedure is analogous to the learned helplessness paradigm. The authors present a reasonable hypothesis that the two procedures generate different molecular and behavioral sequelae due to differences in metabolic demand.

The hypothesis would be strengthened if the authors provide a comparison of post-stress metabolic measures between the two stress types (moderate vs high-volume stress). Giving 2-deoxy-D-glucose is not the same as directly measuring energy homeostasis. Furthermore, do the authors have any evidence that the glucose manipulations that alter the behavioral phenotype also alter their hippocampal receptor subunit findings?

Post-stress metabolic measures would indeed help illuminate the potential mechanism of these effects. Indeed, we have previously reported that HVS greatly challenges metabolic homeostasis (Conoscenti, et al. 2019). However, these types of studies are extensive and we felt were beyond the purview of the current study. However, getting into the physiological mechanisms of the reported effects is an excellent future direction that has been added to the discussion section. We did indeed attempt to examine the effects of glucose on the receptor subunit findings in the same animals that underwent behavioral testing. We have found, however, that subsequent testing (ie. fear conditioning) shrouds the stress effects, and the data were therefore inconclusive.

One issue with framing the present results in the context of stress-enhanced fear learning (SEFL) versus learned helplessness (LH) is that the authors don’t accurately define what learned helplessness actually is. LH refers to stressor outcomes that depend on the uncontrollability of the stressor. That is, to qualify as a LH effect an outcome must follow exposure to inescapable (IS), but not physically identical escapable (ES), shocks or other aversive events. To ignore the controllability issue is to make LH effects synonymous with generic stress effects, which they are not. There are plenty of outcomes (e.g., neurochemical, behavioral) of tail shock that are not sensitive to the dimension of controllability. Thus, there can be many reasons for poor shuttlebox escape responding, only one of which is learned helplessness.

We initially reference learned helplessness not as a means to tie the current results with the phenomena, but merely to provide a justification for the stress procedure used. We agree that it is impossible to determine LH without the escapable group. However, we also know that the effect is somewhat unique in that it requires a significant exposure to (uncontrollable and unpredictable) stress. Whether that be 300-800 seconds of total shock, this is still far above the level of shock required for many other behavioral effects (such as SEFL). It therefore provides us with an excellent procedural comparison to more moderate stressors that do not produce these unique shuttle-escape deficit effects. To be clear, here we are looking at the effects that two types of stress have on behavior and are not exploring how control can reduce the effects of stress. Wording stressing this effect has been added to the introduction.

Additionally, we had previously attempted to separate LH from HVS as quickly as possible, while still acknowledging it as a source of inspiration. To extend this end, we have further refined this section of the introduction to further clarify that we are making no statements about LH per se, but rather interested in the 100 shock procedure, originally tied to LH, that produces an array of unique effects.

Related to above, the authors imply that the gold standard for LH experiments is to deliver 100 x 1.0 mA shocks for an average length of 8 seconds (800 seconds of total shock). The majority of published LH studies (those that include inescapable and escapable groups) use considerably less shock volume. Mean wheel-turn escape times across 100 shocks are around 3-5 seconds (300-500 seconds of total shock). If the authors frame their high-volume stress treatment as a typical learned helplessness design, then they should provide references demonstrating that their shock parameters produce effects that are selective to the uncontrollability of the stressor.

This point is noted and appreciated. The description of canonical shock duration has been adjusted to a range to reflect this point.

The claim that these results represent “the first study to demonstrate tailstock-to-footshock” stress-enhanced fear learning ignores a number of published papers showing that tail shock enhances fear learning (both cued and contextual). As an example, uncontrollable tail shock leads to enhanced footshock-elicited freezing in a shuttle box 24 hr later (plenty of studies from the Maier laboratory), a conditional response attributable to contextual cues of the shuttle box apparatus (Fanselow, 1980). In fact, the majority of studies show that uncontrollable tail shock increases, rather than interferes with, contextual fear.

The reviewer is absolutely right on this point. We were really intending this statement to be specific to the canonical SEFL design (15 shock stressor followed by 1-trial conditioning) in order to address previous critiques of the model. This has been reworded to clarify this claim.

The abstract states that weight gain was impacted only in high-volume stress animals. Was this a consistent finding throughout the studies? It appears that weight gain was depressed in both high and moderate groups compared to No Stress in Figure 3. And in Figure 4B the % weight change for the MVS-Veh group is similar to that of the HVS group in Figure 1D.

This finding was consistent across all studies (ie. see figure 5), except for the study where the glucose manipulation was present (though the general trend here is consistent). We did find it interesting in the case of the glucose study, and our best guess was that the preexposure to glucose provides its own (albeit opposite) challenge to energy homeostasis which may be somewhat hormetic to future challenges. This may be enough to obfuscate any stress effects on weight. This experiment also required several cohorts due to its size. Therefore it’s also possible that the variability between cohorts was enough to mitigate the statistical power between stress groups. As for Figure 4B, the control group suggests that this cohort may have generally been slower at gaining weight.

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5 Jul 2022

19 Jul 2022

The authors still use the term “learned helplessness” in a way that is synonymous with “stress”. The term is not appropriate for simply any behavioral or physiological consequence of uncontrollable tailshock. Rather, it is appropriate for behavioral changes that are specifically mediated by the inescapable/uncontrollable nature of the shock, and not the shock itself. The distinction is important since the motivation of the present experiments (Intro), the hypothesis (line 171), and experimental design (line 208) are framed within the context of LH.

Statements such as “Rats exposed to LH experience a total of about 800 seconds of 1 mA shock (100 shocks at an average of 8 seconds each)” and “HVS and MVS parameters were chosen to mimic previously published work on LH…” are not supported by any reference that shows that these parameters produce learned helplessness. Certainly they produce stress effects, but not necessarily LH.

- We have further clarified this point. As stated before, it is not our intention to draw conclusions regarding learned helplessness, or draw a misleading premise. Specifically, we have added language in the into to reinforce the point that the HVS stress is simply a stressor that is parametrically similar to the LH-inducing stressor. We have also added clarifying language to the hypothesis line and experimental design to avoid any further confusion on this point.

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16 Aug 2022

Dissociable consequences of moderate and high volume stress are mediated by the differential energetic demands of stress.

PONE-D-22-09431R2

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23 Aug 2022

PONE-D-22-09431R2

Dissociable consequences of moderate and high volume stress are mediated by the differential energetic demands of stress.

Dear Dr. Conoscenti:

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