Behavioural Brain Research 259 (2014) 41–44
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
Ultrasonic vocalizations during intermittent swim stress forecasts resilience in subsequent forced swim and spatial learning tests Robert C. Drugan a,∗ , Timothy A. Warner a,b , Tristan A. Papallo a , Laura L. Castracane a , Nathaniel P. Stafford a a b
Department of Psychology, University of New Hampshire, Durham, NH 03824-3567, USA Department of Neurology, Vanderbilt University, Nashville, TN 37232-8552, USA
h i g h l i g h t s • • • •
Ultrasonic vocalizations (USVs) observed during intermittent swim stress (ISS). USVs during ISS predict reduced immobility in forced swim test. USVs during ISS predict proficiency in spatial learning. USVs during ISS forecast resilience in two subsequent swim-motivated tasks.
a r t i c l e
i n f o
Article history: Received 13 September 2013 Received in revised form 14 October 2013 Accepted 15 October 2013 Available online 26 October 2013
a b s t r a c t The examination of stress resilience has substantially increased in recent years. However, current paradigms require multiple behavioral procedures, which themselves may serve as secondary stressors. Therefore, a novel predictor of stress resilience is needed to advance the field. Ultrasonic vocalizations (USVs) have been observed as a behavioral correlate of stress in various rodent species. It was recently reported that rats that emitted ultrasonic vocalizations during intermittent swim stress (ISS) later showed resilience when tested on an instrumental swim escape test. In the current study, we extend this earlier observation on two additional behavioral endpoints. Rats were subjected to ISS, and USVs were recorded. Twenty-four hours later, behavioral performance was evaluated in either the forced swim test or Morris water maze. Rats that emitted ultrasonic vocalizations were resilient to the effects of ISS as indicated by performance similar to controls on both measures. These results extend the original findings that ISSinduced USVs are associated with resilience and are related to subsequent aversively motivated behavior. Such a non-invasive forecast of stress responsivity will allow future work to utilize USVs to examine the neural correlates of initial stress resistance/resilience, thereby eliminating potential confounds of further behavioral testing. Future studies can utilize USVs to target potentially unappreciated neural systems to provide novel pharmacotherapeutic strategies for treatment-resistant depression. © 2013 Elsevier B.V. All rights reserved.
Past research investigating the etiology of psychiatric disorders, such as depression, has almost exclusively focused on the vulnerable, rather than the resilient brain. Although the process of understanding the neurochemical and biological basis of the resilient phenotype has indeed received increased attention through recent reviews [1,2], the research is limited and has utilized a myriad of behavioral, hormonal, and neurochemical endpoints. The behavioral paradigms that produce a resilient phenotype are designed so that the subject is exposed to some
∗ Corresponding author at: Department of Psychology, Conant Hall, University of New Hampshire 10 Library Way Durham, NH 03824-3567. Tel.: +1 603 862 4570; fax: +1 603 862 4986. E-mail address: [email protected] (R.C. Drugan). 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.10.029
form of prior manipulation or subsequent testing. Animal subjects experience different amounts of testing stress depending on how resilient or vulnerable they are, and this may mask initial correlates of resilience. For example, paradigms, such as environmental enrichment [3], exercise [4], social defeat [5], stressor controllability [6], and behavioral immunization [7] all require prior manipulation or subsequent testing. What is needed is a noninvasive predictor of “native resilience” or initial stress resistance that will be observed in a heterogeneous group of naïve subjects following exposure to an identical single stress experience. Behaviorally, vulnerable animals are those that perform worse than non-stressed controls, while resilient subjects do not differ from non-stressed controls following stressor exposure [8,9]. The behavioral mechanisms that confer resilience may be the result of an active process (i.e., coping or stressor controllability) [6], or
42
R.C. Drugan et al. / Behavioural Brain Research 259 (2014) 41–44
prior experience with controllable stress (i.e., behavioral immunization) [7]. Prior exposure to inescapable stressors (e.g., tailshock and swim stress) produces more heterogeneous stress responsivity than controllable stressors resulting in a bimodal distribution of behaviorally resilient and vulnerable animals [8–11]. A past attempt at predicting resilience to inescapable stress has utilized a pretest procedure that can forecast resilience for weeks [8], although such work is still limited in that the subject experienced some form of manipulation and does not address potential native resilience. Recently, ultrasonic vocalizations (USVs) have been proposed as a potential method of forecasting resilience to intermittent swim stress (ISS) [12]. In the ISS paradigm, a rat is exposed to intermittent 5-s forced cold-water swims, and the emission of USVs during the inter-trial-interval of ISS predicted resilience when subsequently tested on an instrumental swim escape test (SET). Examination of USV emission serves predictive potential similar to pretest procedures as adult rats emit USVs in response to, and anticipation of, a variety of conditions. USVs can be broadly defined into two categories, “50-kHz” characteristic and “22-kHz” characteristic. Fifty-kilohertz vocalizations are emitted during presentation of reward or appetitive stimuli [13], while 22-kHz vocalizations are emitted during presentation of aversive stimuli, such as footshock [14], air puff [15], and acoustic startle [16]. Importantly, investigations within the past decade have indicated 22-kHz USVs may have semantic meaning beyond the interpretation of a behavioral (and potentially affective) correlate to stressor exposure. It is argued that the 22-kHz characteristic vocalizations are a type of safety signal, as the rat emits these USVs when in a position of safety, in both laboratory (i.e., following an auditory safety signal during tailshock [17]) and naturalistic settings (i.e., following termination of predator exposure [18]). Furthermore, Portfors [19], has concluded that 22-kHz vocalizations are emitted in anticipation of inescapable, aversive stimuli (e.g., uncontrollable stress such as shock), further suggesting their predictive validity to animal models utilizing inescapable stressors. The previous examination of USVs during ISS utilized 80 trials of the stressor [12], which significantly increased immobility in the forced swim test (FST) [10], and produced a bimodal distribution of behaviorally resilient and vulnerable subjects to the SET [10,11]. Therefore, the current study was designed to further extend USV emission as a predictor of resilience to ISS effects on additional aversively-motivated tasks of the FST and Morris water maze (MWM). Rats were tested in the FST to assess whether USV-emitting rats would exhibit comparable immobility to naïve controls, or increase immobility as previously noted for ISS exposed rats [10]. Because the desired response during the SET learning trials [12] was to achieve the escape response (lever press), a separate group of rats was tested in the MWM to generalize the phenomenon to another type of aversively-motivated escape learning (i.e., spatial learning to find a hidden escape platform). For either behavioral test (i.e., FST or MWM), USV-emitting rats are expected to perform no differently than controls, while rats that do not emit USVs are predicted to exhibit poorer behavioral outcomes. These criteria are also based on the “threshold” effect of stress that differentially affects ISS rats in the FST and SET by producing resilient and vulnerable cohorts following 80 trials of ISS [10,11], rather than 100 trials of ISS, which produces a more robust deficit and lacks a bimodal distribution [10]. Male SAS Sprague–Dawley rats (Charles River Laboratories, Portage, MI) weighing between 180 and 200 g upon arrival served as subjects for both experiments. Rats were allowed at least one week of acclimation to the vivarium (maintained at a 12-h light/dark cycle with lights on at 0600 h) after arrival. Subjects were housed four per cage prior to behavioral testing and individually housed at the start of experimentation with food and water available ad
libitum. All behavioral procedures were conducted during the first 8 h of the light cycle. On day one, ISS rats were exposed to 80 5-s forced swims in cold water (15 ± 1 ◦ C) presented at a variable 60-s interval, while confined control (CC) rats experienced the same procedure, but in the absence of water. Rats experienced ISS or CC in pairs of two as ISS rats were noted to selectively emit USVs in the presence of a conspecific (unpublished observations). Detailed description of the ISS apparatus and procedure are described elsewhere [10,11]. On day two, rats experienced either FST (experiment 1) or MWM (experiment 2); both apparatuses and procedures are described in detail elsewhere [20]. Briefly, the FST was video-recorded and later scored (by an experimenter blind to group membership) utilizing a serial time-sampling scoring procedure for the following behaviors: immobility, swimming, and climbing as previously defined [10,21]. MWM escape learning was conducted in pairs of two (importantly, only one rat was placed in the maze at a time) by an experimenter blind to group membership. Briefly, learning testing consisted of 18 trials presented in 9 two-trial blocks with a 5 min inter-block-interval. Latency to find a hidden platform was recorded, and if the platform was not located within 60 s, rats were placed on the platform for 10 s, and a latency of 60 s was recorded. USVs were recorded throughout the entire ISS procedure and detected using a prepolarized ¼ inch free-field precision condenser microphone model 377A01 (PCB Piezotronics) coupled with a prepolarized preamplifier model 426B03 (PCB Piezotronics) and connected via a 10-32 coaxial cable to an ICP sensor signal conditioner (PCB Piezotronics). The microphone was positioned just beneath the Plexiglas cylinder containing the ISS rat based on the resting position during the inter-trial-interval (maximum distance 10 cm from cylinder). Preliminary analysis from Drugan et al. [12] and further observations (unpublished results) demonstrated CC subjects do not vocalize, limiting signal acquisition to only the ISS subject. The signal was converted with an analog-to-digital data acquisition hardware device (National Instruments) and transmitted to a computer equipped with Lab View (National Instruments). The data were refined with band-pass filters set to 18 and 32 kHz, based on the frequency range characterizing 22-kHz calls [12,22]. The number of calls, the duration of calls (ms), the frequency of the calls (Hz), and the time at which the calls occurred (h:min:s) were quantified using a custom Lab View program. For both experiments, subsequent division into ISS/Callers and ISS/Non-callers was established. Rats that emitted any amount of USVs were classified as “ISS/Callers” and rats that did not emit any USVs (i.e., zero calls) were characterized as “ISS/Non-callers”. This was conducted following completion of both the experiment and scoring of behavioral data, and was based on whether or not USVs were recorded during ISS exposure. Therefore, in experiment 1, groups consisted of ISS/FST (n = 21) and CC/FST (n = 20; one subject not included due to equipment malfunction). ISS groups were further subdivided based on the emission of ultrasonic vocalizations (ISS/Callers) and no vocalizations (ISS/Non-callers). USV recording determined ISS/Callers, n = 7 and ISS/Non-callers, n = 14. In experiment 2, subjects were randomly divided into two groups, ISS/MWM (n = 11; one outlier was removed following box and whisker plot assessment) and CC/MWM (n = 11; one outlier removed following box and whisker plot assessment), and USV recording determined ISS/Callers, n = 5 and ISS/Non-callers, n = 6. The present analyses were conducted to examine the specific comparisons hypothesized above in order to isolate the effects of ISS/Callers and ISS/Non-callers against CC groups in both experiments. For the FST behaviors, Pearson’s r demonstrated inter-rater-reliability correlations were high: immobility, r(39) = 0.95, p < 0.001; swimming, r(39) = 0.83, p < 0.001; and climbing, r(39) = 0.95, p < 0.001. Analysis of a priori planned comparisons on immobility using the Dunnett test adjusted for unequal
R.C. Drugan et al. / Behavioural Brain Research 259 (2014) 41–44
Fig. 1. Mean (± SEM) counts of immobility, swimming, and climbing in a 5-min FST between ISS/Callers, ISS/Non-callers, and CC groups. *Indicates significantly different from CC (p < 0.05).
group sizes (Myers [23], Section 10.8) was conducted to examine ISS/Callers and ISS/Non-callers against CC groups. As predicted, ISS/Callers were comparable to CC in the amount of immobility [t(38) = 0.54, p = 0.461], while ISS/Non-callers exhibited significantly greater immobility than CC rats [t(38) = 2.04, p = 0.045]. Furthermore, neither ISS/Callers nor ISS/Non-callers differed from CC on swimming or climbing behaviors (Fig. 1). A priori planned comparisons of overall group effects on MWM learning were conducted using the Dunnett test adjusted for unequal group sizes (Myers [23], Section 10.8) in the context of a repeated measures ANOVA with treatment (ISS/Callers, ISS/Non-callers, and CC) as the between subjects factor and trial block as the within subjects factor, respectively. The analysis revealed a significant effect of trial block [F(8,152) = 8.96, p < 0.001] and significant main effect of treatment [F(2,19) = 7.765, p = 0.042]. The trial block x treatment interaction [F(16,152) = 0.57, p = 0.901] was non-significant. Planned comparisons indicated ISS/Callers exhibited comparable overall mean escape latencies to CC [t(19) = 1.20, p = 0.967]. The difference between ISS/Non-callers and CC approached significance [t(19) = 1.93, p = 0.063] (Fig. 2). The present study extends the original observation that USVs emitted during the ISS paradigm possessed predictive value for resilience by demonstrating that these rats did not differ from controls [12]. Comparable performance of ISS/Callers and CC during the FST and MWM confirmed that USV emitting rats did not differ from confined controls. Rats that did not emit USVs were only different from controls on the measure of immobility in the FST, while in the MWM this comparison approached reliable significance. Furthermore, a direct comparison between ISS/Callers and ISS/Non-callers could also provide a compelling argument, but then it becomes rather subjective in terms of which effect (i.e., ISS/Callers vs. CC or ISS/Callers vs. ISS/Non-callers) is more critical
Fig. 2. Mean (± SEM) latency (in s) to locate a hidden platform between ISS/Callers, ISS/Non-callers, and CC groups.
43
to the conclusions being drawn. In the end, the authors decided to pursue the more consistently reliable effect (ISS/Callers vs. CC). This effect can be seen for both FST and MWM procedures, whereas a comparison between ISS/Callers and ISS/Non-callers yields a significant outcome for the MWM, but not the FST (results not reported). The disparity between ISS/Callers and ISS/Non-callers between procedures may be due to two factors: 1) in the FST there is a rather restricted range of responses in the domain of immobility (i.e., between 30 and 40 counts) so that this difference is not easily obtained, compared to that of the MWM where the response times can range between 10 and 60 s, and 2) rather than using a repeated measure design as employed in the MWM, only one observation (i.e., total time immobile) is used in the FST. Nonetheless, the data illustrate a clear lack of separation between ISS/Callers and CC groups in both procedures. In the current study, we provided additional evidence for this new use of an old technique [12]. USVs have been recognized as a correlate of behavior since the 1970s [24], and the more recent examination of USVs has been as an endpoint during or following manipulation of a stressor [14,16]. Vocalizations from both rat pups and adults are characteristic of a thermogenic response, produced by laryngeal braking. In pups, it is the method by which to call to the dam to be returned to the nest. In adult rats, the effect appears to coincide with hypothalamic thermoregulation [25]. Others have demonstrated that 22-kHz range USVs are emitted in anticipation of, and response to, inescapable, aversive stimuli, and also in anticipation to a potential threat [18,19], which may shed light on the predictive nature of the phenomenon observed in the current experiments. Prospective work will examine the physiological, neuroanatomical, and genetic make-up of the callers and non-callers immediately following initial ISS in an effort to determine the key neural and genetic markers of resilience. Ideal examinations would utilize 80 intermittent, inescapable swims, which previous studies have demonstrated to produce individual differences (e.g., bimodal distribution) in that some rats show vulnerability to the aforementioned deficits, while others show resistance to these effects [10,11]. Extension of the initial report [12] demonstrates the ISS/USV phenomenon is not idiosyncratic to instrumental escape learning, and provides additional behavioral endpoints demonstrating this effect. Although resilience, at the behavioral level, was defined as ISS/Callers not differing from controls in the current set of experiments, an alternative explanation of stress resilience is plausible. Variability in stress reactivity could also be partially explained by distinction of two behavioral phenotypes. A more passive phenotype could be characterized by no USVs during ISS, reduced mobility in the FST, and reduced exploration in the MWM. Conversely, a more active phenotype may exhibit a response to threat or stress by emitting USVs during ISS, and greater activity in both the FST and MWM. This alternative explanation may also follow a final common path to resilience by affording greater Darwinian fitness to the active coping subject (i.e., enhanced ability to escape a threat and survive to pass on its genes). Overall, the current results provide validation of ISS-induced USVs as a strategy to evaluate resilience on the behavioral level. In spite of the fact that the current study extends the initial report that ISS-induced USVs forecast resilience on subsequent aversively-motivated tasks [12], the context similarity of water in both stress induction and testing procedures is a limitation. Future studies will evaluate the trans-situational properties of the ISS/USV phenomenon using non-water based endpoints (e.g., social exploration). However, the current findings demonstrate that ISSinduced USVs may now be used to evaluate the neural correlates of resilience that are not confounded by prior manipulations or subsequent testing in preclinical models. Utilization of ISS-induced USVs
44
R.C. Drugan et al. / Behavioural Brain Research 259 (2014) 41–44
affords the opportunity to target potentially unappreciated neural systems and thereby, inform novel pharmacotherapeutic strategies for treatment-resistant depressed patients. Acknowledgements The authors would like to thank Michael J. Carter and Robert Cinq-Mars from the University of New Hampshire for the gracious donation of their time and invaluable contributions to our ultrasonic vocalization-recording set-up. All behavioral procedures were reviewed and approved by the University of New Hampshire Institutional Animal Care and Use Committee (IACUC). This work was supported by the Cole Neuroscience and Behavior Faculty Research Fund and the Undergraduate Research Opportunities Program. The sponsors did not contribute to experimental design; data collection, analysis, or interpretation; writing of the report; or the decision to submit the report for publication. References [1] Charney DS. Psychobiological mechanisms of resilience and vulnerability: implications for successful adaptation to extreme stress. Am J Psychiatry 2004;161:195–216. [2] Feder A, Nestler EJ, Charney DS. Psychobiology and molecular genetics of resilience. Nat Rev Neurosci 2009;10:446–57. [3] Lehmann MI, Herkenham M. Environmental enrichment confers stress resiliency to social defeat through an infralimbic cortex-dependent neuroanatomical pathway. J Neurosci 2011;31:6159–73. [4] Greenwood BN, Foley TE, Day HEW, Campisi J, Hammack SH, Campeau S, Maier SF, Fleshner M. Freewheel running prevents learned helplessness/behavioral depression: role of dorsal raphe serotonergic neurons. J Neurosci 2003;23:2889–98. [5] Krishnan V, Han MH, Graham DL, Berton O, Renthal W, Russo SJ, et al. Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions. Cell 2007;131:391–404. [6] Maier SF, Amat J, Baratta MV, Paul E, Watkins LR. Behavioral control, the medial prefrontal cortex, and resilience. Dialogues Clin Neurosci 2006;8:397–406. [7] Amat J, Paul E, Watkins LR, Maier SF. Activation of the ventral medial prefrontal cortex during an uncontrollable stressor reproduces both the immediate and long-term protective effects of behavioral control. Neuroscience 2008;154:1178–86.
[8] Drugan RC, Skolnick P, Paul SM, Crawley JN. A pretest procedure reliably predicts performance in two animal models of inescapable stress. Pharmacol Biochem Behav 1989;33:649–54. [9] Minor TR, Dess NK, Ben-David E, Chang WC. Individual differences in vulnerability to inescapable shock in rats. J Exp Psychol: Anim Behav Process 1994;20:402–12. [10] Christianson JP, Drugan RC. Intermittent cold water swim stress increases immobility and interferes with escape performance in rat. Behav Brain Res 2005;165:58–62. [11] Levay EA, Govic A, Hazi A, Flannery G, Christianson JP, Drugan RC, Kent S. Endocrine and immunological correlates of behaviorally identified swim stress resilient and vulnerable rats. Brain Behav Immun 2006;20: 488–97. [12] Drugan RC, Christianson JP, Stine WW, Soucy DP. Swim stress-induced ultrasonic vocalizations forecast resilience in rats. Behav Brain Res 2009;202:142–5. [13] Knutson B, Burgdorf J, Panksepp J. Ultrasonic vocalizations as indices of affective states in rats. Psychol Bull 2002;128:961–77. [14] Swiergiel AH, Zhou Y, Dunn AJ. Effects of chronic footshock, restraint and corticotropin-releasing factor on freezing, ultrasonic vocalization and forced swim behavior in rats. Behav Brain Res 2007;183:178–87. [15] Knapp DJ, Pohorecky LA. An air-puff stimulus method for elicitation of ultrasonic vocalizations in rats. J Neurosci Methods 1995;62:1–5. [16] Miczek KA, Weerts EM, Vivian JA, Barros HM. Aggression, anxiety and vocalizations in animals: GABAa and 5-HT anxiolytics. Psychopharmacology 1995;121:38–56. [17] Jelen P, Soltysik S, Zagrodzka J. 22 kHz ultrasonic vocalization in rats is an index of anxiety but not fear: behavioral and pharmacological modulation of affective state. Behav Brain Res 2003;141:63–72. [18] Litvin Y, Blanchard DC, Blanchard RJ. Rat 22 kHz ultrasonic vocalizations as alarm cries. Behav Brain Res 2007;182:166–72. [19] Portfors CV. Types and functions of ultrasonic vocalizations in laboratory rats and mice. J Am Assoc Lab Anim Sci 2007;46:28–34. [20] Warner TA, Libman MK, Wooten KL, Drugan RC. Sex differences associated with intermittent swim stress. Stress 2013;16:655–63. [21] Detke MJ, Rickels M, Lucki I. Active behaviors in the rat forced swimming test differentially produced by serotonergic and noradrenergic antidepressants. Psychopharmacology 1995;121:66–72. [22] Brudzynski SM, Bihari F, Ocipa D, Fu X. Analysis of 22 kHz ultrasonic vocalizations in laboratory rats: long and short calls. Physiol Behav 1993;54: 215–21. [23] Myers JL, Well AD, Lorch Jr RF. Research design and statistical analysis. 3rd ed New York: Routledge; 2010. [24] Barfield RJ, Geyer LA. The ultrasonic postejaculatory vocalization and postejaculatory refractory period of the male rat. J Comp Physiol Psychol 1975;88:723–34. [25] Blumberg MS, Alberts JR. Ultrasonic vocalizations by rat pups in the cold: an acoustic by-product of laryngeal braking. Behav Neurosci 1990;104:808–17.
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
Ultrasonic vocalizations during intermittent swim stress forecasts resilience in subsequent forced swim and spatial learning tests Robert C. Drugan a,∗ , Timothy A. Warner a,b , Tristan A. Papallo a , Laura L. Castracane a , Nathaniel P. Stafford a a b
Department of Psychology, University of New Hampshire, Durham, NH 03824-3567, USA Department of Neurology, Vanderbilt University, Nashville, TN 37232-8552, USA
h i g h l i g h t s • • • •
Ultrasonic vocalizations (USVs) observed during intermittent swim stress (ISS). USVs during ISS predict reduced immobility in forced swim test. USVs during ISS predict proficiency in spatial learning. USVs during ISS forecast resilience in two subsequent swim-motivated tasks.
a r t i c l e
i n f o
Article history: Received 13 September 2013 Received in revised form 14 October 2013 Accepted 15 October 2013 Available online 26 October 2013
a b s t r a c t The examination of stress resilience has substantially increased in recent years. However, current paradigms require multiple behavioral procedures, which themselves may serve as secondary stressors. Therefore, a novel predictor of stress resilience is needed to advance the field. Ultrasonic vocalizations (USVs) have been observed as a behavioral correlate of stress in various rodent species. It was recently reported that rats that emitted ultrasonic vocalizations during intermittent swim stress (ISS) later showed resilience when tested on an instrumental swim escape test. In the current study, we extend this earlier observation on two additional behavioral endpoints. Rats were subjected to ISS, and USVs were recorded. Twenty-four hours later, behavioral performance was evaluated in either the forced swim test or Morris water maze. Rats that emitted ultrasonic vocalizations were resilient to the effects of ISS as indicated by performance similar to controls on both measures. These results extend the original findings that ISSinduced USVs are associated with resilience and are related to subsequent aversively motivated behavior. Such a non-invasive forecast of stress responsivity will allow future work to utilize USVs to examine the neural correlates of initial stress resistance/resilience, thereby eliminating potential confounds of further behavioral testing. Future studies can utilize USVs to target potentially unappreciated neural systems to provide novel pharmacotherapeutic strategies for treatment-resistant depression. © 2013 Elsevier B.V. All rights reserved.
Past research investigating the etiology of psychiatric disorders, such as depression, has almost exclusively focused on the vulnerable, rather than the resilient brain. Although the process of understanding the neurochemical and biological basis of the resilient phenotype has indeed received increased attention through recent reviews [1,2], the research is limited and has utilized a myriad of behavioral, hormonal, and neurochemical endpoints. The behavioral paradigms that produce a resilient phenotype are designed so that the subject is exposed to some
∗ Corresponding author at: Department of Psychology, Conant Hall, University of New Hampshire 10 Library Way Durham, NH 03824-3567. Tel.: +1 603 862 4570; fax: +1 603 862 4986. E-mail address: [email protected] (R.C. Drugan). 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.10.029
form of prior manipulation or subsequent testing. Animal subjects experience different amounts of testing stress depending on how resilient or vulnerable they are, and this may mask initial correlates of resilience. For example, paradigms, such as environmental enrichment [3], exercise [4], social defeat [5], stressor controllability [6], and behavioral immunization [7] all require prior manipulation or subsequent testing. What is needed is a noninvasive predictor of “native resilience” or initial stress resistance that will be observed in a heterogeneous group of naïve subjects following exposure to an identical single stress experience. Behaviorally, vulnerable animals are those that perform worse than non-stressed controls, while resilient subjects do not differ from non-stressed controls following stressor exposure [8,9]. The behavioral mechanisms that confer resilience may be the result of an active process (i.e., coping or stressor controllability) [6], or
42
R.C. Drugan et al. / Behavioural Brain Research 259 (2014) 41–44
prior experience with controllable stress (i.e., behavioral immunization) [7]. Prior exposure to inescapable stressors (e.g., tailshock and swim stress) produces more heterogeneous stress responsivity than controllable stressors resulting in a bimodal distribution of behaviorally resilient and vulnerable animals [8–11]. A past attempt at predicting resilience to inescapable stress has utilized a pretest procedure that can forecast resilience for weeks [8], although such work is still limited in that the subject experienced some form of manipulation and does not address potential native resilience. Recently, ultrasonic vocalizations (USVs) have been proposed as a potential method of forecasting resilience to intermittent swim stress (ISS) [12]. In the ISS paradigm, a rat is exposed to intermittent 5-s forced cold-water swims, and the emission of USVs during the inter-trial-interval of ISS predicted resilience when subsequently tested on an instrumental swim escape test (SET). Examination of USV emission serves predictive potential similar to pretest procedures as adult rats emit USVs in response to, and anticipation of, a variety of conditions. USVs can be broadly defined into two categories, “50-kHz” characteristic and “22-kHz” characteristic. Fifty-kilohertz vocalizations are emitted during presentation of reward or appetitive stimuli [13], while 22-kHz vocalizations are emitted during presentation of aversive stimuli, such as footshock [14], air puff [15], and acoustic startle [16]. Importantly, investigations within the past decade have indicated 22-kHz USVs may have semantic meaning beyond the interpretation of a behavioral (and potentially affective) correlate to stressor exposure. It is argued that the 22-kHz characteristic vocalizations are a type of safety signal, as the rat emits these USVs when in a position of safety, in both laboratory (i.e., following an auditory safety signal during tailshock [17]) and naturalistic settings (i.e., following termination of predator exposure [18]). Furthermore, Portfors [19], has concluded that 22-kHz vocalizations are emitted in anticipation of inescapable, aversive stimuli (e.g., uncontrollable stress such as shock), further suggesting their predictive validity to animal models utilizing inescapable stressors. The previous examination of USVs during ISS utilized 80 trials of the stressor [12], which significantly increased immobility in the forced swim test (FST) [10], and produced a bimodal distribution of behaviorally resilient and vulnerable subjects to the SET [10,11]. Therefore, the current study was designed to further extend USV emission as a predictor of resilience to ISS effects on additional aversively-motivated tasks of the FST and Morris water maze (MWM). Rats were tested in the FST to assess whether USV-emitting rats would exhibit comparable immobility to naïve controls, or increase immobility as previously noted for ISS exposed rats [10]. Because the desired response during the SET learning trials [12] was to achieve the escape response (lever press), a separate group of rats was tested in the MWM to generalize the phenomenon to another type of aversively-motivated escape learning (i.e., spatial learning to find a hidden escape platform). For either behavioral test (i.e., FST or MWM), USV-emitting rats are expected to perform no differently than controls, while rats that do not emit USVs are predicted to exhibit poorer behavioral outcomes. These criteria are also based on the “threshold” effect of stress that differentially affects ISS rats in the FST and SET by producing resilient and vulnerable cohorts following 80 trials of ISS [10,11], rather than 100 trials of ISS, which produces a more robust deficit and lacks a bimodal distribution [10]. Male SAS Sprague–Dawley rats (Charles River Laboratories, Portage, MI) weighing between 180 and 200 g upon arrival served as subjects for both experiments. Rats were allowed at least one week of acclimation to the vivarium (maintained at a 12-h light/dark cycle with lights on at 0600 h) after arrival. Subjects were housed four per cage prior to behavioral testing and individually housed at the start of experimentation with food and water available ad
libitum. All behavioral procedures were conducted during the first 8 h of the light cycle. On day one, ISS rats were exposed to 80 5-s forced swims in cold water (15 ± 1 ◦ C) presented at a variable 60-s interval, while confined control (CC) rats experienced the same procedure, but in the absence of water. Rats experienced ISS or CC in pairs of two as ISS rats were noted to selectively emit USVs in the presence of a conspecific (unpublished observations). Detailed description of the ISS apparatus and procedure are described elsewhere [10,11]. On day two, rats experienced either FST (experiment 1) or MWM (experiment 2); both apparatuses and procedures are described in detail elsewhere [20]. Briefly, the FST was video-recorded and later scored (by an experimenter blind to group membership) utilizing a serial time-sampling scoring procedure for the following behaviors: immobility, swimming, and climbing as previously defined [10,21]. MWM escape learning was conducted in pairs of two (importantly, only one rat was placed in the maze at a time) by an experimenter blind to group membership. Briefly, learning testing consisted of 18 trials presented in 9 two-trial blocks with a 5 min inter-block-interval. Latency to find a hidden platform was recorded, and if the platform was not located within 60 s, rats were placed on the platform for 10 s, and a latency of 60 s was recorded. USVs were recorded throughout the entire ISS procedure and detected using a prepolarized ¼ inch free-field precision condenser microphone model 377A01 (PCB Piezotronics) coupled with a prepolarized preamplifier model 426B03 (PCB Piezotronics) and connected via a 10-32 coaxial cable to an ICP sensor signal conditioner (PCB Piezotronics). The microphone was positioned just beneath the Plexiglas cylinder containing the ISS rat based on the resting position during the inter-trial-interval (maximum distance 10 cm from cylinder). Preliminary analysis from Drugan et al. [12] and further observations (unpublished results) demonstrated CC subjects do not vocalize, limiting signal acquisition to only the ISS subject. The signal was converted with an analog-to-digital data acquisition hardware device (National Instruments) and transmitted to a computer equipped with Lab View (National Instruments). The data were refined with band-pass filters set to 18 and 32 kHz, based on the frequency range characterizing 22-kHz calls [12,22]. The number of calls, the duration of calls (ms), the frequency of the calls (Hz), and the time at which the calls occurred (h:min:s) were quantified using a custom Lab View program. For both experiments, subsequent division into ISS/Callers and ISS/Non-callers was established. Rats that emitted any amount of USVs were classified as “ISS/Callers” and rats that did not emit any USVs (i.e., zero calls) were characterized as “ISS/Non-callers”. This was conducted following completion of both the experiment and scoring of behavioral data, and was based on whether or not USVs were recorded during ISS exposure. Therefore, in experiment 1, groups consisted of ISS/FST (n = 21) and CC/FST (n = 20; one subject not included due to equipment malfunction). ISS groups were further subdivided based on the emission of ultrasonic vocalizations (ISS/Callers) and no vocalizations (ISS/Non-callers). USV recording determined ISS/Callers, n = 7 and ISS/Non-callers, n = 14. In experiment 2, subjects were randomly divided into two groups, ISS/MWM (n = 11; one outlier was removed following box and whisker plot assessment) and CC/MWM (n = 11; one outlier removed following box and whisker plot assessment), and USV recording determined ISS/Callers, n = 5 and ISS/Non-callers, n = 6. The present analyses were conducted to examine the specific comparisons hypothesized above in order to isolate the effects of ISS/Callers and ISS/Non-callers against CC groups in both experiments. For the FST behaviors, Pearson’s r demonstrated inter-rater-reliability correlations were high: immobility, r(39) = 0.95, p < 0.001; swimming, r(39) = 0.83, p < 0.001; and climbing, r(39) = 0.95, p < 0.001. Analysis of a priori planned comparisons on immobility using the Dunnett test adjusted for unequal
R.C. Drugan et al. / Behavioural Brain Research 259 (2014) 41–44
Fig. 1. Mean (± SEM) counts of immobility, swimming, and climbing in a 5-min FST between ISS/Callers, ISS/Non-callers, and CC groups. *Indicates significantly different from CC (p < 0.05).
group sizes (Myers [23], Section 10.8) was conducted to examine ISS/Callers and ISS/Non-callers against CC groups. As predicted, ISS/Callers were comparable to CC in the amount of immobility [t(38) = 0.54, p = 0.461], while ISS/Non-callers exhibited significantly greater immobility than CC rats [t(38) = 2.04, p = 0.045]. Furthermore, neither ISS/Callers nor ISS/Non-callers differed from CC on swimming or climbing behaviors (Fig. 1). A priori planned comparisons of overall group effects on MWM learning were conducted using the Dunnett test adjusted for unequal group sizes (Myers [23], Section 10.8) in the context of a repeated measures ANOVA with treatment (ISS/Callers, ISS/Non-callers, and CC) as the between subjects factor and trial block as the within subjects factor, respectively. The analysis revealed a significant effect of trial block [F(8,152) = 8.96, p < 0.001] and significant main effect of treatment [F(2,19) = 7.765, p = 0.042]. The trial block x treatment interaction [F(16,152) = 0.57, p = 0.901] was non-significant. Planned comparisons indicated ISS/Callers exhibited comparable overall mean escape latencies to CC [t(19) = 1.20, p = 0.967]. The difference between ISS/Non-callers and CC approached significance [t(19) = 1.93, p = 0.063] (Fig. 2). The present study extends the original observation that USVs emitted during the ISS paradigm possessed predictive value for resilience by demonstrating that these rats did not differ from controls [12]. Comparable performance of ISS/Callers and CC during the FST and MWM confirmed that USV emitting rats did not differ from confined controls. Rats that did not emit USVs were only different from controls on the measure of immobility in the FST, while in the MWM this comparison approached reliable significance. Furthermore, a direct comparison between ISS/Callers and ISS/Non-callers could also provide a compelling argument, but then it becomes rather subjective in terms of which effect (i.e., ISS/Callers vs. CC or ISS/Callers vs. ISS/Non-callers) is more critical
Fig. 2. Mean (± SEM) latency (in s) to locate a hidden platform between ISS/Callers, ISS/Non-callers, and CC groups.
43
to the conclusions being drawn. In the end, the authors decided to pursue the more consistently reliable effect (ISS/Callers vs. CC). This effect can be seen for both FST and MWM procedures, whereas a comparison between ISS/Callers and ISS/Non-callers yields a significant outcome for the MWM, but not the FST (results not reported). The disparity between ISS/Callers and ISS/Non-callers between procedures may be due to two factors: 1) in the FST there is a rather restricted range of responses in the domain of immobility (i.e., between 30 and 40 counts) so that this difference is not easily obtained, compared to that of the MWM where the response times can range between 10 and 60 s, and 2) rather than using a repeated measure design as employed in the MWM, only one observation (i.e., total time immobile) is used in the FST. Nonetheless, the data illustrate a clear lack of separation between ISS/Callers and CC groups in both procedures. In the current study, we provided additional evidence for this new use of an old technique [12]. USVs have been recognized as a correlate of behavior since the 1970s [24], and the more recent examination of USVs has been as an endpoint during or following manipulation of a stressor [14,16]. Vocalizations from both rat pups and adults are characteristic of a thermogenic response, produced by laryngeal braking. In pups, it is the method by which to call to the dam to be returned to the nest. In adult rats, the effect appears to coincide with hypothalamic thermoregulation [25]. Others have demonstrated that 22-kHz range USVs are emitted in anticipation of, and response to, inescapable, aversive stimuli, and also in anticipation to a potential threat [18,19], which may shed light on the predictive nature of the phenomenon observed in the current experiments. Prospective work will examine the physiological, neuroanatomical, and genetic make-up of the callers and non-callers immediately following initial ISS in an effort to determine the key neural and genetic markers of resilience. Ideal examinations would utilize 80 intermittent, inescapable swims, which previous studies have demonstrated to produce individual differences (e.g., bimodal distribution) in that some rats show vulnerability to the aforementioned deficits, while others show resistance to these effects [10,11]. Extension of the initial report [12] demonstrates the ISS/USV phenomenon is not idiosyncratic to instrumental escape learning, and provides additional behavioral endpoints demonstrating this effect. Although resilience, at the behavioral level, was defined as ISS/Callers not differing from controls in the current set of experiments, an alternative explanation of stress resilience is plausible. Variability in stress reactivity could also be partially explained by distinction of two behavioral phenotypes. A more passive phenotype could be characterized by no USVs during ISS, reduced mobility in the FST, and reduced exploration in the MWM. Conversely, a more active phenotype may exhibit a response to threat or stress by emitting USVs during ISS, and greater activity in both the FST and MWM. This alternative explanation may also follow a final common path to resilience by affording greater Darwinian fitness to the active coping subject (i.e., enhanced ability to escape a threat and survive to pass on its genes). Overall, the current results provide validation of ISS-induced USVs as a strategy to evaluate resilience on the behavioral level. In spite of the fact that the current study extends the initial report that ISS-induced USVs forecast resilience on subsequent aversively-motivated tasks [12], the context similarity of water in both stress induction and testing procedures is a limitation. Future studies will evaluate the trans-situational properties of the ISS/USV phenomenon using non-water based endpoints (e.g., social exploration). However, the current findings demonstrate that ISSinduced USVs may now be used to evaluate the neural correlates of resilience that are not confounded by prior manipulations or subsequent testing in preclinical models. Utilization of ISS-induced USVs
44
R.C. Drugan et al. / Behavioural Brain Research 259 (2014) 41–44
affords the opportunity to target potentially unappreciated neural systems and thereby, inform novel pharmacotherapeutic strategies for treatment-resistant depressed patients. Acknowledgements The authors would like to thank Michael J. Carter and Robert Cinq-Mars from the University of New Hampshire for the gracious donation of their time and invaluable contributions to our ultrasonic vocalization-recording set-up. All behavioral procedures were reviewed and approved by the University of New Hampshire Institutional Animal Care and Use Committee (IACUC). This work was supported by the Cole Neuroscience and Behavior Faculty Research Fund and the Undergraduate Research Opportunities Program. The sponsors did not contribute to experimental design; data collection, analysis, or interpretation; writing of the report; or the decision to submit the report for publication. References [1] Charney DS. Psychobiological mechanisms of resilience and vulnerability: implications for successful adaptation to extreme stress. Am J Psychiatry 2004;161:195–216. [2] Feder A, Nestler EJ, Charney DS. Psychobiology and molecular genetics of resilience. Nat Rev Neurosci 2009;10:446–57. [3] Lehmann MI, Herkenham M. Environmental enrichment confers stress resiliency to social defeat through an infralimbic cortex-dependent neuroanatomical pathway. J Neurosci 2011;31:6159–73. [4] Greenwood BN, Foley TE, Day HEW, Campisi J, Hammack SH, Campeau S, Maier SF, Fleshner M. Freewheel running prevents learned helplessness/behavioral depression: role of dorsal raphe serotonergic neurons. J Neurosci 2003;23:2889–98. [5] Krishnan V, Han MH, Graham DL, Berton O, Renthal W, Russo SJ, et al. Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions. Cell 2007;131:391–404. [6] Maier SF, Amat J, Baratta MV, Paul E, Watkins LR. Behavioral control, the medial prefrontal cortex, and resilience. Dialogues Clin Neurosci 2006;8:397–406. [7] Amat J, Paul E, Watkins LR, Maier SF. Activation of the ventral medial prefrontal cortex during an uncontrollable stressor reproduces both the immediate and long-term protective effects of behavioral control. Neuroscience 2008;154:1178–86.
[8] Drugan RC, Skolnick P, Paul SM, Crawley JN. A pretest procedure reliably predicts performance in two animal models of inescapable stress. Pharmacol Biochem Behav 1989;33:649–54. [9] Minor TR, Dess NK, Ben-David E, Chang WC. Individual differences in vulnerability to inescapable shock in rats. J Exp Psychol: Anim Behav Process 1994;20:402–12. [10] Christianson JP, Drugan RC. Intermittent cold water swim stress increases immobility and interferes with escape performance in rat. Behav Brain Res 2005;165:58–62. [11] Levay EA, Govic A, Hazi A, Flannery G, Christianson JP, Drugan RC, Kent S. Endocrine and immunological correlates of behaviorally identified swim stress resilient and vulnerable rats. Brain Behav Immun 2006;20: 488–97. [12] Drugan RC, Christianson JP, Stine WW, Soucy DP. Swim stress-induced ultrasonic vocalizations forecast resilience in rats. Behav Brain Res 2009;202:142–5. [13] Knutson B, Burgdorf J, Panksepp J. Ultrasonic vocalizations as indices of affective states in rats. Psychol Bull 2002;128:961–77. [14] Swiergiel AH, Zhou Y, Dunn AJ. Effects of chronic footshock, restraint and corticotropin-releasing factor on freezing, ultrasonic vocalization and forced swim behavior in rats. Behav Brain Res 2007;183:178–87. [15] Knapp DJ, Pohorecky LA. An air-puff stimulus method for elicitation of ultrasonic vocalizations in rats. J Neurosci Methods 1995;62:1–5. [16] Miczek KA, Weerts EM, Vivian JA, Barros HM. Aggression, anxiety and vocalizations in animals: GABAa and 5-HT anxiolytics. Psychopharmacology 1995;121:38–56. [17] Jelen P, Soltysik S, Zagrodzka J. 22 kHz ultrasonic vocalization in rats is an index of anxiety but not fear: behavioral and pharmacological modulation of affective state. Behav Brain Res 2003;141:63–72. [18] Litvin Y, Blanchard DC, Blanchard RJ. Rat 22 kHz ultrasonic vocalizations as alarm cries. Behav Brain Res 2007;182:166–72. [19] Portfors CV. Types and functions of ultrasonic vocalizations in laboratory rats and mice. J Am Assoc Lab Anim Sci 2007;46:28–34. [20] Warner TA, Libman MK, Wooten KL, Drugan RC. Sex differences associated with intermittent swim stress. Stress 2013;16:655–63. [21] Detke MJ, Rickels M, Lucki I. Active behaviors in the rat forced swimming test differentially produced by serotonergic and noradrenergic antidepressants. Psychopharmacology 1995;121:66–72. [22] Brudzynski SM, Bihari F, Ocipa D, Fu X. Analysis of 22 kHz ultrasonic vocalizations in laboratory rats: long and short calls. Physiol Behav 1993;54: 215–21. [23] Myers JL, Well AD, Lorch Jr RF. Research design and statistical analysis. 3rd ed New York: Routledge; 2010. [24] Barfield RJ, Geyer LA. The ultrasonic postejaculatory vocalization and postejaculatory refractory period of the male rat. J Comp Physiol Psychol 1975;88:723–34. [25] Blumberg MS, Alberts JR. Ultrasonic vocalizations by rat pups in the cold: an acoustic by-product of laryngeal braking. Behav Neurosci 1990;104:808–17.
