These results indicate that a minimally immobilizing acoustic startle paradigm produces whole-body startle responses in squirrel monkeys that habituate across repeated stimulus presentations and are directly proportional to stimulus intensity. These data are similar to findings from humans and rodents (Lang and Davis, 2006
), and replicate and extend recent findings in rhesus monkeys (Davis et al., 2008
) and capuchins (Linn and Javitt, 2001
). Our paradigm is unique among monkey startle paradigms in that it is characterized by the absence of any direct restraint or positional restriction of the test subject beyond that imposed by the testing chamber. Because immobilization restraint alters parameters that acoustic startle likewise probes (e.g., emotionality), use of restraint in startle paradigms necessitates extensive acclimation prior to experimental initiation to avoid such confounds. Our low-restraint paradigm therefore provides an expeditious alternative to more time-consuming immobilization-based startle paradigms.
These experiments also examined whether standard broad-band noise bursts and species-specific alarm vocalizations (“yaps”) produce differential startle responses. Yaps are typically elicited in response to threatening circumstances (Newman, 1985
), leading us to hypothesize that yaps might produce enhanced responsivity due to their increased biological salience as compared to simple, non-biologically relevant noise bursts. Although monkey subjects exhibited habituation and graded responses to both types of acoustic startle stimuli, we indeed observed significant effects of stimulus type. Yap stimuli elicited larger initial whole-body startle responses which subsequently habituated more quickly than standard noise burst stimuli. Yaps did so despite violating the requirement that startle stimuli have near-instantaneous rise-times. It is tempting to speculate that the efficacy of yaps in this context reflects their specific biological salience. An inductive test of this hypothesis could examine the impact of variations in yap stimulus parameters on startle-like responses. It would also be of interest to determine whether startle-like squirrel monkey responses to yaps, like those to noise bursts, are inhibited or facilitated by prepulses, attenuated by anxiolytics, and so represent a useful alternative probe of fear system function. Similar studies might be possible in macaques employing their “shrill bark” alarm calls (cf. Romanski, Averbeck, and Diltz, 2005
). It would also be of interest to determine whether previously neutral stimuli diverging from classical startle stimuli in rise-time, intensity, and bandwidth might, through conditioning, come to produce startle-like responses in monkeys.
There is considerable pharmacological evidence indicating that activation of the HPA axis increases startle responsivity as reviewed above, but few previous rodent (Anisman et al., 2001
; Engelmann et al., 1996
; Glowa et al., 1992
), and no known primate, studies have examined the stress-activating role of startle stimuli on the HPA axis. In the present studies, ACTH and cortisol concentrations were significantly elevated above baseline levels after startle stimuli presentation in squirrel monkeys. Following the 17.5 min habituation to repeated stimuli experiment (experiment 1), monkeys exhibited an average percentage increase of 202% and 56% above baseline levels, respectively, for ACTH and cortisol. Monkeys likewise exhibited an average percentage increase of 67% and 97% above baseline levels for ACTH and cortisol, respectively, following the 33.5 min experiment examining the relationship between stimulus intensity and response amplitude (experiment 2). The differences in percentage increases between ACTH and cortisol activation within each study (i.e., percentage increase is higher for ACTH vs. cortisol in experiment 1 whereas the reverse was observed in experiment 2) likely reflect the temporal dynamics of the HPA axis. Because the adrenal response to stress temporally follows that of the pituitary, it is likely that the 17.5 min period was better suited for capturing maximal pituitary activation, whereas the 33.5 min period was better suited for detecting the onset of adrenal activation (Parker, Buckmaster, Sundlass, Schatzberg, and Lyons, 2006
It should be noted that while our experimental goal was to compare baseline and post-test cortisol levels within
experiments, it is evident from that there are pronounced differences in baseline as well as post-test cortisol values between
experiments. This observed difference in cortisol levels between studies is likely due to circannual changes in circulating “total” (bound + unbound) cortisol (Schiml, Mendoza, Saltzman, Lyons, and Mason, 1999
). Our cortisol assay measures “total” cortisol levels, and as experiments 1 and 2 were conducted during different times of the year, this fact likely accounts for the observed differences in “total” cortisol levels between experiments 1 and 2. In should be noted that basal and post-test blood samples were collected during tight time periods within experiments, and therefore cortisol measurements within experiments are unlikely to be confounded with circannual cortisol rhythms. Moreover, stress responses are relatively stable across the year, with cortisol levels post-stress generally related to baseline values (Coe and Levine, 1995
), indicating minimal circannual influences on the magnitude of HPA axis activation.
In these experiments, monkeys did not exhibit differential neuroendocrine responses to standard acoustic startle vs. biologically salient stimuli. This is in contrast to the somatic findings reviewed above. It is possible that HPA axis activity was not temporally sensitive enough to manifest such differences, unlike those afforded by other relatively rapid biological measurement techniques such as electrocardiography. Several studies have shown differential cardiac responses to biologically salient vs. non-biologically salient acoustic stimuli when presented to great apes, dolphins, and birds (Berntson and Boysen, 1989
; Miksis, Grund, Nowacek, Solow, Connor, and Tyack, 2001
; Ryden, 1980
). In other studies, heart rate has been used to differentiate between startle, defensive, and orienting responses to standard acoustic startle stimuli (Berntson and Boysen, 1984
; Graham, 1979
). Future studies using electrocardiography combining these two approaches would be valuable to examine whether monkeys exhibit differential cardiac response signatures to biologically salient vs. non-biologically salient acoustic startle stimuli.
This study has several limitations. The monkeys studied in these experiments were juvenile animals, so we do not know whether these results generalize across lifespan development, or whether gender differences emerge following pubertal changes in circulating gonadal steroids as has been reported for rodents and humans (Aasen, Kolli, and Kumari, 2005
; Lehmann, Pryce, and Feldon, 1999
; Toufexis, Myers, and Davis, 2006
). A second limitation of these studies is that we cannot determine from the available data the extent to which post-test HPA axis responses are due to exposure to the startle stimuli vs. exposure to the startle apparatus. Group separation and subsequent placement in a novel environment have been shown to activate the HPA axis in squirrel monkeys (Coe, Franklin, Smith, and Levine, 1982
). Follow up studies will require inclusion of an additional experimental condition to determine the extent to which exposure to the startle apparatus per se induces HPA axis activation to address this unanswered question. Finally, our studies did not validate pre-pulse inhibition or fear conditioning aspects of the startle response, as have other monkey startle paradigms (Linn and Javitt, 2001
; Winslow et al., 2002
). Investigation of fear conditioning and extinction is a particularly attractive direction for future monkey research as abnormal fear memory responses are thought to be a core characteristic of anxiety disorders. Though a goal of our investigation was to evaluate an expeditious startle assessment paradigm that could be more easily embedded in multi-element testing sequences, we cannot conclusively state that these monkeys exhibited lower HPA axis-indexed stress responses than if they had been head-restrained. A direct test of this possibility may be warranted. Measurement-related stress is often ignored in human studies despite the potential for interactions with trait fear and anxiety (Eatough, Shirtcliff, Hanson, and Pollak, 2009
). It is self-evident that reducing uncontrolled measurement-related stress responses should be a goal of translational studies in this area.
Development of monkey models which examine differences in fear reactivity and fear recovery, as has been done in rodents (Bush, Sotres-Bayon, and LeDoux, 2007
; Imanaka, Morinobu, Toki, and Yamawaki, 2006
), will provide tractable means by which to model and manipulate fear memory formation and extinction, two core features of stress vulnerability and resilience (Yehuda, Flory, Southwick, and Charney, 2006
). We are well-positioned to examine these two core features as they pertain to resilience, having recently developed a squirrel monkey model of early life stress inoculation-induced resilience. In our laboratory, monkeys exposed to early life stress inoculation protocols subsequently exhibit diminished anxiety, attenuated stress-induced HPA axis activation, greater prefrontal inhibition of behavior, and larger ventromedial prefrontal cortical volumes compared to non-inoculated control monkeys (Katz, Liu, Schaer, Parker, Ottet, Epps, Buckmaster, Bammer, Moseley, Schatzberg, Eliez, and Lyons, 2009
; Levine and Mody, 2003
; Lyons, Martel, Levine, Risch, and Schatzberg, 1999
; Parker, Buckmaster, Justus, Schatzberg, and Lyons, 2005
; Parker, Buckmaster, Schatzberg, and Lyons, 2004
; Parker et al., 2006
). Future studies will examine whether stress inoculated versus non-inoculated monkeys exhibit differential startle responses, and higher resistance to form, and faster rates to extinguish, fear memories.