High Illumination Levels Potentiate the Acoustic ... - Semantic Scholar

Behavioral Neuroscience 2003, Vol. 117, No. 6, 1458 –1462

Copyright 2003 by the American Psychological Association, Inc. 0735-7044/03/$12.00 DOI: 10.1037/0735-7044.117.6.1458

High Illumination Levels Potentiate the Acoustic Startle Response in Preweanling Rats Marianne Weber, Nicola Watts, and Rick Richardson University of New South Wales Fear potentiation of the acoustic startle response (FPS) by aversive conditioned stimuli does not emerge in rats until Postnatal Day (P)23 (see P. S. Hunt & B. A. Campbell, 1997). However, the present study found that when presented with an unconditioned fear-eliciting stimulus, rats younger than P23 display FPS. Specifically, high illumination levels were found to enhance startle amplitudes in rats aged 18 and 25 days, but not 14 days. Furthermore, the light-enhanced startle observed in P18 rats was prevented by a systemic injection of the noradrenergic beta-receptor antagonist propranolol. These data suggest that conditioned and unconditioned FPS have different ontogenetic trajectories, and thereby provide support for the idea that learned and unlearned fear are subserved by dissociable neural systems.

land, Scott, & Yeomans, 1995; Lee, Lopez, Meloni, & Davis, 1996; Lingenhohl & Friauf, 1994; Wagner, Pilz, & Fendt, 2000). The PnC is the “sensorimotor interface” of the ASR (Lingenhohl & Friauf, 1994) and is the site at which fear modulation of the ASR occurs (Fendt, 1998; Fendt, Koch, & Schnitzler, 1996b; Frankland & Yeomans, 1995; Hitchcock & Davis, 1991; Rosen, Hitchcock, Sananes, Miserendino, & Davis, 1991; Yeomans & Pollard, 1993). Both learned and unlearned fear are thought to potentiate the ASR through activation of the basolateral amygdaloid complex (BLC; Walker & Davis, 1997b). However, conditioned cues potentiate the ASR via projections from the BLC to the central nucleus of the amygdala (Ce), which in turn directly (and indirectly) innervates the PnC (Fendt et al., 1996a; Frankland & Yeomans, 1995; Hitchcock & Davis, 1991; Rosen et al., 1991; Yeomans & Pollard, 1993). In contrast, a neuronal inactivation study by Walker and Davis (1997b) demonstrated that LES requires a projection from the BLC to the bed nucleus of the stria terminalis (BST), and then from the BST to the PnC. Thus, conditioned FPS is dependent on BLC–Ce–PnC circuitry, whereas unconditioned FPS requires a BLC–BST–PnC projection route. As an alternative to lesion studies in adult rats, a method of investigating the potential differences in the neural pathways mediating the expression of learned versus unlearned FPS is to study the ontogenetic profile of these responses. That is, developing rats can be viewed as a “naturally lesioned” preparation because their sensory, motor, and central nervous systems do not fully mature until well after birth (Fanselow & Rudy, 1998). Consequently, the response repertoire of rats varies greatly with age (Alberts, 1984; Rudy & Cheatle, 1979), and therefore, data from studies investigating fear responses during development may provide an alternative view on how the fear system is organized in the brain. Indeed, it has been found that conditioned fear responses develop in a sequential manner, with conditioned FPS emerging relatively late (see Hunt & Campbell, 1997). That is, various studies have found that conditioned FPS does not emerge in rats until Postnatal Day (P) 23 (Hunt, 1999; Hunt, Richardson, & Campbell, 1994; Richardson, Paxinos, & Lee, 2000; Richardson & Vishney, 2000). The failure to observe conditioned FPS prior to P23 is not due to a failure of rats to form and retain an associative

The acoustic startle reflex (ASR) is a fast, sequential contraction of head, body, and limb muscles after a rapid-onset acoustic stimulus. The ASR is hypothesized to prevent injuries resulting from attack, by initiating a flight/fight response and shielding off blows to the head (Koch, 1999; Yeomans, Li, Scott, & Frankland, 2002). Fear potentiation of the ASR occurs when an acoustic startle stimulus is presented in the presence of a fear-eliciting stimulus. That is, startle amplitude is greater in the presence of a fear-eliciting stimulus than in its absence. In the laboratory, fear potentiated startle (FPS) occurs in the presence of a cue, such as a tone or light, that has previously been paired with shock, and has been widely used as an index of learned fear (Davis, Falls, Campeau, & Kim, 1993; Koch, 1999). However, Walker and Davis have also shown that the ASR can be enhanced when a startle probe occurs in the presence of an unconditioned fear-eliciting stimulus, namely, a bright light (Walker & Davis, 1997a, 1997b, 2002). The interpretation offered for the light-enhanced startle (LES) effect is that high illumination levels are naturally aversive to rats (possibly because it leaves them more vulnerable to predators) and are therefore likely to produce a state of fear that is innate, or unlearned. The neural mechanisms mediating FPS in the presence of learned versus unlearned fear-eliciting stimuli are thought to be dissociable (Davis & Shi, 1999). The neural circuitry underlying the startle reflex itself involves both direct and indirect projections from the cochlear root neurons to the caudal pontine reticular nucleus (PnC) in the brainstem, which in turn projects to areas of the spinal cord containing cranial and spinal motoneurons (Frank-

Marianne Weber, Nicola Watts, and Rick Richardson, School of Psychology, University of New South Wales, Sydney, New South Wales, Australia. This research was supported by a grant from the Australian Research Council and the University of New South Wales to Rick Richardson. We would like to acknowledge the assistance of A. Sophie Parnas in Experiment 1. Correspondence concerning this article should be addressed to Rick Richardson, School of Psychology, University of New South Wales, Sydney, New South Wales 2052, Australia. E-mail: [email protected] 1458

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memory because the same rats exhibit other learned fear responses (such as avoidance and freezing) to the conditioned stimuli (Richardson & Fan, 2002; Richardson et al., 2000; Richardson, Tronson, Bailey, & Parnas, 2002). In addition, rats display a basic startle response from as early as P12 (Parisi & Ison, 1979), and by P16, can express an enhancement of startle amplitudes after administration of strychnine or corticotropin releasing hormone (Weber & Richardson, 2001). It is likely, therefore, that the late onset of conditioned FPS is attributable to a failure of fear to increase activity in the primary startle pathway by activation of the amygdala. However, the ontogenetic emergence of FPS in rats has only been examined using conditioned fear cues. It is possible that the BLC–Ce–PnC pathway has a different ontogenetic trajectory than the BLC–BST–PnC pathway. If this were the case, then LES may be observed either earlier, or later, than FPS to a conditioned cue (at P23), which would provide further evidence that conditioned and unconditioned FPS are subserved by dissociable neural systems. Thus, this study aimed to establish the ontogenetic trajectory of LES in rats.

Experiment 1 To test the effects of high illumination levels on the ASR of preweanling rats, separate groups of 14- (approximate time of eye-opening), 18-, and 25-day-old rats received startle probes in both the presence and the absence of high light levels.

Method Subjects. Experimentally naive, male and female Sprague–Dawley rats obtained from the breeding colony in the School of Psychology at the University of New South Wales were used. In each of the P18 and P25 groups, 10 rats were used, whereas there were 14 rats in the P14 group. No more than 2 rats were used from a single litter for any group. All rats were treated in accordance with the principles of laboratory animal use published by the American Psychological Association, and all procedures were approved by the Animal Care and Ethics Committee at the University of New South Wales. Apparatus. Startle was assessed in a set of two identical cages in which the front wall, rear wall, and ceiling were made of Plexiglas, and the side walls and floor of stainless steel bars. The cages measured 13 cm long ⫻ 9 cm wide ⫻ 9 cm high, with 1 cm between each bar on the grid floor. Each startle cage was suspended from a piece of clear Plexiglas (3.0 mm thick) to which a sheet of piezoelectric film had been laminated. Movements within the startle cages caused flexion in the Plexiglas that produced a voltage in the piezoelectric film, which was proportional to the intensity of movement in the chamber; that is, larger movements produced larger voltages. A custom-built unit amplified and digitized (at a 1-kHz rate) the voltage produced by the piezoelectric film for a 250-ms period after onset of the startle stimulus. The peak response during this period was used as the measure of the subject’s startle response. Each cage was located within a sound- and light-attenuating wooden chamber, and a ventilation fan provided a 60-dB ambient noise level in each chamber. Illumination levels were provided by two 20-W fluorescent bulbs that were placed on the floor of each sound-attenuating chamber, 18 cm beneath the startle cage, as well as a single 60-W incandescent bulb located 15 cm above the startle cage. In addition, the fluorescent ceiling lights of the test room were on. When the lights were on, the illumination level in each chamber was approximately 630 cd/m2, measured from the center of the cage with a CS-100 Minolta Chrome Meter. Procedure. A within-subjects design was used whereby each rat was tested twice: once in a dark– dark session and once in a dark–light session;

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test order was counterbalanced. Each session was separated by 24 ⫾ 4 hr. The dark– dark session comprised two phases separated by 5 min. In Phase 1, rats were taken from the litter and placed in the startle cage with all lights off. After a 2-min adaptation period, the rats received thirty 100-ms white-noise startle pulses of either 90, 95, or 105 dB (10 of each) in a random order with an interstimulus interval of 30 s. Rats were then removed from the startle cage and placed in a bucket containing bedding for 5 min while the cage was cleaned. The rats were then placed back in the cage for the second dark test phase, which was identical to the first. The dark–light test session also comprised two phases, in which the dark phase was as described above. After the 5-min between-phase period, however, the rats were placed back into the startle cages for the same series of 30 startle pulses, but with all the lights on. Data analysis. Startle amplitudes were averaged across each phase, and a percent change score was then calculated from the first phase to the second for each session. Percent change in startle amplitude was calculated with the formula [(P2 – P1) ⫼ P1] ⫻ 100, where P1 was Phase 1 and P2 was Phase 2. Each rat had two percentage change scores, one for the dark– dark session and one for the dark–light session. These scores were analyzed statistically with a repeated measures analysis of variance (ANOVA), as well as individual paired-samples t tests.

Results and Discussion Data from 1 P25 rat were excluded as a result of equipment failure. In addition, 3 P14 rats were statistical outliers in the dark– dark test condition, with scores 10, 13, and 16 SD away from the group mean; data from these rats were also excluded. A repeated measures ANOVA confirmed that there were no significant differences in startle amplitudes in the first dark test phase of each day across age groups: within-subjects, F(1, 27) ⫽ 2.84, p ⫽ .10; Interaction Age ⫻ Test Phase, F ⬍ 1, nor was there a difference in startle amplitudes between the three age groups in the dark phases: between groups, F(2, 27) ⫽ 1.33, p ⫽ .28. Percent change in startle amplitudes was higher in the dark-tolight condition than in the dark-to-dark condition for both the P18 and P25 age groups, but not in rats aged P14 (see Figure 1). A repeated measures ANOVA yielded a significant within-subjects

Figure 1. Mean (⫾ SEM) percent change in startle amplitudes on darkto-dark test conditions and dark-to-light test conditions in rats aged 14, 18, and 25 days. P ⫽ postnatal day.


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effect of test condition, F(1, 27) ⫽ 16.66, p ⬍ .01; however, there was no interaction between age and test condition, F(2, 27) ⫽ 2.31, p ⫽ .12. There was a significant between-group difference, F(2, 27) ⫽ 4.40, p ⫽ .02, where mean percent increase in startle amplitudes was higher at P25 than at P14 ( p ⬍ .05, Tukey’s honestly significant difference test). The P18 rats were intermediate to the other two groups. Paired samples t tests at each age revealed that percent change in startle amplitudes was significantly higher in the dark–light condition than the dark– dark condition for P25 rats: within-subjects, t(8) ⫽ 2.3, p ⬍ .05, and P18 rats: within-subjects, t(9) ⫽ 3.4, p ⬍ .01, but not P14 rats: withinsubjects, t ⬍ 1. These results demonstrate that the ASR is potentiated with high illumination levels in rats aged P18 and P25, but not P14. Thus, when rats are presented with a fear-eliciting stimulus that does not require the neural mechanisms involved in associative learning, the ASR can be potentiated at an age younger than P23 (when conditioned FPS emerges). The failure to observe significant levels of LES in 14-day-old rats is possibly due to individual differences in visual system maturation. That is, because the eyes open around this time, all rats may not have had a completely functional visual system at the time of test. Indeed, it was the case that about half the P14 rats exhibited an increase in startle amplitude from the dark to the light, whereas half did not (although it should be noted that all rats tested had open eyes). It may be the case, therefore, that an unconditioned fear-eliciting stimulus that is reliant on an earlier developing sensory modality (e.g., olfaction) may produce an enhancement of the ASR at this age. The finding that high illumination levels potentiate the ASR in 18-day-old rats suggests that FPS that is elicited by unconditioned stimuli has an ontogenic trajectory different from FPS that is elicited by conditioned cues, and thus supports the view that different neural systems subserve learned versus unlearned fear. However, an important concern is whether the LES effect observed at P18 is the result of a fear state. In support of the interpretation that high illumination levels produce a state of fear, LES is disrupted when adult rats are injected with anxiolytics such as buspirone (Walker & Davis, 1997a), the benzodiazepine chlordiazepoxide (De Jongh, Groenink, Van Der Gugten, & Olivier, 2002; Walker & Davis, 2002), the noradrenergic beta-receptor antagonist propranolol (Walker & Davis, 2002), or the 5-HT1A receptor agonist flesinoxan (De Jongh et al., 2002). Thus, to determine whether the LES response observed at P18 in Experiment 1 was due to a state of fear, Experiment 2 tested P18 rats for an LES response following systemic injections of propranolol.

the startle cage and immediately injected with either saline or propranolol, and then placed in a bucket for 5 min. Each rat was then placed back in the startle cage for a second phase, which was identical to the first except that the lights were turned on. Data analysis. Startle amplitudes were averaged across each phase, and a percent change score was then calculated from the first phase to the second. These scores were analyzed statistically with an independent samples t test, as well as individual paired-samples t tests.

Results and Discussion Rats that received injections of saline displayed greater startle amplitudes in the light phase than in the dark phase, whereas rats that received injections of propranolol did not (see Figure 2). That is, within-subjects t tests revealed that startle amplitudes of the saline group were significantly greater when tested in the light compared with in the dark, t(7) ⫽ 4.0, p ⬍ .01, whereas there were no differences in startle amplitude between the two test phases for the propranolol group, t(7) ⫽ 1.74, p ⫽ .13. In addition, an independent-samples t test revealed that saline rats had significantly greater percent change in startle amplitude from the dark to the light phase than rats that received propranolol, t(14) ⫽ 3.72, p ⬍ .01. Further, there were no group differences in startle amplitude in the dark phase, t(14) ⬍ 1. It should be noted that the disruption of LES by propranolol is not likely to be due to a disruption of the startle reflex itself. That is, although the propranolol group displayed a mean decrease in startle amplitude from the dark phase to the light, this decrease was not statistically significant, and indeed was comparable to the mean decrease observed in the dark– dark session of P18 rats in Experiment 1 (i.e., ⫺14% in Experiment 1, ⫺19% in Experiment 2). In addition, although no systematic measurements were taken, rats that received propranolol did not display any obvious changes in behavior. The finding that LES can be disrupted by propranolol suggests that potentiation of the ASR by high illumination levels in rats aged 18 days is likely to be due to a state of fear, as has been found in adult rats (De Jongh et al., 2002; Walker & Davis, 1997a, 2002). It should be noted that the mean increase in startle response of P18

Experiment 2 Method Subjects. Sixteen experimentally naive, male and female Sprague– Dawley rats were allocated to one of two groups: a 3 ml/kg saline control group and a propranolol (20 mg/kg ip, No. P-0884, Sigma-Aldrich Chemicals, St. Louis, MO) group. Each rat was only used once, and no more than 2 subjects from a single litter were used in either condition. Apparatus. Startle was assessed as described in Experiment 1. Procedure. Rats were tested for startle in two phases that were separated by 5 min. In the first phase, rats were presented with 30 white-noise startle pulses (see Experiment 1) in the dark. Rats were then removed from

Figure 2. Mean (⫾ SEM) percent change in startle amplitude from dark-to-light test sessions in 18-day-old rats that received either saline or 20 mg/kg propranolol prior to the light phase.


rats in the saline group of this experiment was slightly lower than that observed in Experiment 1 (i.e., 28% vs. 50%). The reason for this is unclear, although slight procedural differences may be involved (e.g., Experiment 1 had 2 days of test and no injections, whereas Experiment 2 had only 1 day of test and ip injections). Nevertheless, P18 rats in both experiments showed a significant increase in startle amplitude when tested in the light.

General Discussion The present study found that the ASR is potentiated by high illumination levels in rats aged 18 and 25 days, but not 14 days, and further, that the increase in startle observed on P18 is prevented by a systemic injection of propranolol. These data suggest that FPS to an unlearned fear-eliciting visual stimulus emerges sometime between P14 and P18. Sheets, Dean, and Reiter (1988) also reported that the ASR can be potentiated with environmental stimuli before P23. That is, they demonstrated that a continuous low-level (75 dB) background white noise produces a “sensitized” startle response in rats as early as P16, a finding that has also been observed in adult rats (Davis, 1974). It is unclear, however, whether sensitization of the ASR by background noise is a fear-related effect. On one hand, excitotoxic lesions of the amygdala, including the BLC, have no effect on sensitization of the ASR by background noise in adult rats (Schanbacher, Koch, Pilz, & Schnitzler, 1996), but on the other hand, Kellogg, Sullivan, Bitran, and Ison (1991) demonstrated that noise enhancement of startle is disrupted by the benzodiazepine receptor agonist diazepam. There are a number of findings, however, that suggest LES is a fear-related response. Firstly, LES in adult rats has been found to critically depend upon the integrity of the BLC (Walker & Davis, 1997b). Secondly, various anxiolytic drugs have been found to attenuate LES (De Jongh et al., 2002; Walker & Davis, 1997a, 2002). Finally, high illumination has been found, in some cases, to affect other models of anxious behavior in rats, such as performance on the elevated plus-maze (e.g., Bert, Fink, Huston, & Voits, 2002; Bertoglio & Carobrez, 2002; Cardenas, Lamprea, & Morato, 2001), and time spent exploring the lighted compartment of a dark–light box (e.g., Chaouloff, Durand, & Mormede, 1997). Given then that the LES effect is likely to result from an unconditioned state of fear, the finding that LES emerges between P14 and P18 is interesting in light of previous research showing that FPS to conditioned fear-eliciting cues is not observed prior to P23 (Hunt, 1999; Hunt et al., 1994; Richardson et al., 2000; Richardson & Vishney, 2000). That is, it appears that learned and unlearned FPS emerge at different stages of development, with unlearned cues potentiating the ASR earlier than conditioned cues. This finding supports the view, based on the adult neuronal inactivation study of Walker and Davis (1997b), that learned and unlearned FPS are subserved by dissociable neural systems. Because the LES effect is likely to reflect a state of fear that is not dependant on specific stimulus contingencies, it may be useful as an animal model of anxiety disorders, such as generalized anxiety disorder, which involve less defined fear-eliciting cues (Davis & Shi, 1999). Although it can be hypothesized that bright lights and background noise elicit a state of fear, it would be of interest whether an unconditioned fear-eliciting stimulus such as predator odor

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potentiates the ASR, and whether anxiolytic compounds prevent such a response. It has been well established that adult rats display fear responses, such as freezing, in the presence of predator odor (Dielenberg & McGregor, 2001; Fendt, Endres, & Apfelbach, 2003; Vazdarjanova, Cahill, & McGaugh, 2001; Wallace & Rosen, 2000, 2001; Wiedenmayer & Barr, 1998), and it has been reported that predator odor-induced defensive behavior is reduced by anxiolytic compounds such as midazolam (Dielenberg, Arnold, & McGregor, 1999). To our knowledge, however, it is not known whether predator odor potentiates the ASR. Indeed, it would be interesting to determine whether rats younger than P23 exhibit an enhanced ASR to predator odor, because it has been demonstrated that predator odor elicits defensive behaviors in rats as young as P14 (Wiedenmayer & Barr, 1998). In addition, it would be of interest whether fear responses other than FPS, such as freezing and changes in heart rate, also have an earlier ontogenetic trajectory to unconditioned compared with conditioned stimuli. In conclusion, the results of the present study provide evidence that conditioned and unconditioned FPS have different ontogenetic trajectories. Specifically, it appears that a potentiated startle response that relies on associative learning processes emerges later in development than a potentiated startle response that does not. This finding complements the results of the adult neuronal inactivation study of Walker and Davis (1997b), which suggests that different neural pathways govern learned versus unlearned fear.

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Received May 19, 2003 Revision received July 3, 2003 Accepted July 7, 2003 䡲