Behavioral Neuroscience
1994, Vol. 108, No. 1,69-80
Copyright 1994 by the American Psychological Association, Inc.
0735-7044/94/S3.00
Delayed Development of Fear-Potentiated Startle in Rats
Pamela S. Hunt, Rick Richardson, and Byron A. Campbell
The developmental emergence of fear-potentiated startle was examined in rats ranging in age from
16 to 75 days. In Experiment 1, a pure tone served as the conditioned stimulus (CS) and an acoustic
startle pulse served as the unconditioned stimulus (US) for fear conditioning. Fear-potentiated
startle by the tone CS was observed in rats 23 days of age and older but not in rats 16 days of age. In
Experiment 2, a light served as the CS. Rats 30 days of age and older showed fear-potentiated
startle, whereas 23-day-old rats did not. The final experiment demonstrated that another behavioral
index of fear, stimulus-elicited freezing, was observed earlier in development than fear-potentiated
startle, confirming the effectiveness of the training procedure for conditioning fear. The results
suggest that fear-potentiated startle is a relatively late-emerging response system, parallelling the
development of conditioned autonomic changes (e.g., heart rate) rather than that of freezing.
Fear-potentiated startle is a commonly used measure of fear
conditioning (Brown, Kalish, & Farber, 1951; Davis & Astra-
chan, 1978; Hamm, Greenwald, Bradley, Cuthbert, & Lang,
1991; Lang, Bradley, & Cuthbert, 1990; Leaton & Borszcz,
1985). For example, when rats are presented with a startle-
eliciting stimulus, typically a brief, intense auditory stimulus,
they respond behaviorally with a whole-body jerk, referred to
as the startle reflex. When the startle stimulus is preceded by a
cue that evokes fear, such as a light that has been paired with
shock, the startle response is greater than when the startle
stimulus is presented alone. This enhancement in startle
responding is termed fear-potentiated startle, and it has been
extensively used as a tool for examining the pharmacological
and neuroanatomical bases of conditioned fear (Cassella &
Davis, 1985; Davis, 1979, 1984, 1986, 1992a, 1992b; Davis,
Hitchcock, & Rosen, 1992; Hitchcock & Davis, 1986, 1987,
1991; Rosen & Davis, 1988; Sananes & Davis, 1992). The
purpose of the following experiments was to investigate the
emergence of fear-potentiated startle during development,
using auditory and visual stimuli as conditioned stimuli (CSs).
Two disparate lines of evidence converge to make the
ontogenetic study of fear-potentiated startle particularly inter-
esting. First, Wecker and Ison (1986) showed that the magni-
tude of the startle response is highly correlated with the type of
behavior occurring just prior to the elicitation of startle. In
their study, adult rats were shown to exhibit a greater startle
response when they were immobile prior to the presentation of
a startle-eliciting noise burst than when they were active. The
reduction in startle response magnitude during activity was
particularly marked when the animals were engaged in consum-
Pamela S. Hunt, Rick Richardson, and Byron A. Campbell, Depart-
ment of Psychology, Princeton University.
This research was supported by National Institute of Mental Health
Grants MH01562 and MH49496 to Byron A. Campbell and National
Institutes of Child Health and Human Development Postdoctoral
Grant HD07694 to Pamela S. Hunt.
Correspondence concerning this article should be addressed to
Pamela S. Hunt, Department of Psychology, Princeton University,
Green Hall, Princeton, New Jersey 08544-1010. Electronic mail may be
sent to pshunt@pucc.princeton.edu.
ing (eating or drinking), grooming, or sniffing behavior. This
relation between activity and startle response magnitude has
also been implicated in fear-potentiated startle. According to
Leaton and Borszcz (1985), a CS that has been paired with an
aversive stimulus elicits the species-specific response of freez-
ing in the laboratory rat and that when that CS precedes a
startle stimulus, the resulting immobility potentiates the startle
response (see also Leaton & Cranney, 1990).
Although we could find no research on the correspondence
between freezing and startle responding during development,
several researchers have demonstrated that infant, as well as
adult, rats show a decrease in activity in the presence of a CS
that has been paired with shock (Campbell & Ampuero, 1985;
Coulter, Collier, & Campbell, 1976; Mellon, Kraemer, &
Spear, 1991; Moye & Rudy, 1985, 1987). For example, Moye
and Rudy (1987) paired a tone CS with shock for preweanlings
rats of different ages; when the tone was presented 24 hr later,
it produced a substantial decrease in general activity in rats 15
days of age and older. It is a reasonable prediction then that
animals as young as 15-16 days of age would show the
potentiated startle effect given their disposition to respond
with inactivity (freezing) to a tone that had been paired with
shock.
A second line of evidence, however, suggests that potenti-
ated startle may not be observed until much later in develop-
ment. A number of investigators, working with other species
such as the rabbit and cat, have shown that reflex sensitivity is
altered by stimulation of the amygdala, particularly the central
nucleus, from which concomitant changes in heart rate (HR)
are also evoked. Whalen and Kapp (1991) reported that the
amplitude of the rabbit's nictitating membrane reflex (NMR)
was enhanced by stimulation of the central nucleus of the
amygdala that also evoked bradycardia but that it was inhibited
by stimulation of other regions of the amygdala that concomi-
tantly evoked tachycardia. From these results, Whalen and
Kapp (1991) proposed that heart rate change and reflex
modification covaried, with heart rate decreases accompanying
facilitation and heart rate increases accompanying inhibition
of the magnitude of the response (see also Gary Bobo &
Bonvallet, 1975; Gebber & Klevans, 1972; Kapp, Whalen,
69
70 P. HUNT, R. RICHARDSON, AND B. CAMPBELL
Supple, & Pascoe, 1992; Marks, Frysinger, Trelease, & Harper,
1983; Pascoe, Bradley, & Spyer, 1989; Schlor, Stumpf, & Stock,
1984). Although none of these researchers explicitly postu-
lated a causal relation between HR changes and alterations in
reflex amplitude, the findings that the two measures occur
together suggest that they are somehow related.
Conditioned changes in HR have been documented in
young rats when auditory and visual stimuli are paired with an
aversive stimulus such as shock (Campbell & Ampuero, 1985)
or a startle pulse (Richardson, Wang, & Campbell, 1993).
However, conditioned heart rate responses emerge much later
in development than behavioral measures of fear such as
freezing or conditioned suppression of locomotion. Specifi-
cally, although there are numerous demonstrations of stimulus-
elicited freezing to auditory and visual CSs as early as 15-17
days postnatal (Campbell & Ampuero, 1985; Coulter et al.,
1976; Mellon et al., 1991; Moye & Rudy, 1985, 1987), condi-
tioned changes in heart rate to auditory and visual stimuli are
not observed until approximately 21 and 28 days of age,
respectively (Campbell & Ampuero, 1985). Thus, if facilitation
of the startle reflex is correlated with stimulus-elicited changes
in heart rate (in this case attributable to conditioning),
potentiated startle would not be expected to occur until about
21 days of age to auditory stimuli that had previously been
paired with shock and not until 28 days of age to visual stimuli.
These two lines of evidence lead to quite different predic-
tions concerning the age at which fear-potentiated startle
should first emerge during the course of development. If it is
conditioned immobility (freezing) that is associated with en-
hanced startle magnitude, then fear-potentiated startle to
auditory stimuli should emerge about 15 days of age and
around 17 days of age to visual stimuli. Conversely, if condi-
tioned changes in heart rate are critical predictors of startle
modulation, then fear-potentiated startle should not be ob-
served until approximately 21 and 28 days of age to auditory
and visual stimuli, respectively.
Experiments 1A and IB
In the fear-potentiated startle paradigm, fear is typically
conditioned off-line with shock as the unconditioned stimulus
(US; Brown et al., 1951; Davis, 1984, 1992a, 1992b). However,
Leaton and Cranney (1990) demonstrated that the startle
pulse itself could be used as a US to establish fear to a visual
CS. Using the startle pulse as a US has one particular
advantage for conditioning fear to the CS over off-line shock in
that it allows the experimenter to observe the acquisition of
fear-potentiated startle trial by trial. Also, Richardson et al.
(1993) demonstrated the effectiveness of the startle pulse as an
aversive US for HR conditioning. With repeated pairings of a
tone CS with the startle pulse, animals 21 days of age and older
showed reliable conditioned bradycardia to the tone. How-
ever, 16- and 17-day-olds did not exhibit conditioned cardiac
changes in this situation. These results are strikingly similar to
those reported by Campbell and Ampuero (1985) with shock
as the US.
The purpose of Experiment 1A was to study the ontogeny of
fear-potentiated startle using an 80-dB, 1600-Hz tone as the
CS and a 130-dB burst of white noise as the US. Three age
groups of rats—16, 23, and 75 days—were selected to investi-
gate the emergence of fear-potentiated startle during develop-
ment.
Experiment 1A: Method
Subjects. The subjects were experimentally naive Sprague-Dawley-
derived rats, bred and reared in the vivarium of the psychology
department of Princeton University. On Day 2 after birth (day of
birth = Day 0), litters were culled to 8 pups. Animals were either 16
(± 1), 23 (± 1), or 75 (± 15) days old at the time of testing. Purina Rat
Chow and water were available in the home cage ad libitum. Both male
and female rats were included in the 16- and 23-day-old groups
(ns = 10), whereas only males were tested at 75 days of age (n = 10-
11). Animals tested at 16 and 23 days of age were maintained with their
dams and littermates in 48.0-cm x 25.5-cm x 20.5-cm polycarbonate
cages. No more than 2 rats from any given litter were assigned to each
treatment group. Rats tested as adults were weaned when 23-26 days
old and housed in groups of five with same-sex conspecifics in hanging
wire cages. Adult animals were housed singly and handled daily 3 days
prior to training. The vivarium was maintained on a 16:8 light-dark
cycle, with light onset at 7 a.m.
Apparatus, Rats were trained and tested in a 16.3-cm x 8.5-cm x
8.5-cm plastic and stainless steel restraining cage (Coulbourn Instru-
ments, Allentown, PA, Model EOS-15). The restraining cage was
placed on a Coulbourn Instruments transducer platform (Models
E45-11 and E45-15) that was placed in an Industrial Acoustics (Lodi,
NJ) sound-attenuating chamber (IAC). The IAC contained a wall-
mounted 7.5-W houselight, a floor-mounted JBL speaker (Model
2426H) for producing the startle stimulus, and a ceiling-mounted
Jensen speaker (Model Jl 176) for producing the CS.
The auditory CS used in this research consisted of a 10-s, 1600-Hz,
pulsating (2 pulses per second with a rise-fall time of 250 ms) pure
tone of 80 dB as measured on the C scale of a Simpson (Elgin, IL)
sound level meter (Model 886). The US was an acoustic startle
stimulus consisting of a 100-ms, 130-dB burst of white noise, with an
instantaneous rise-fall time.
The startle response was recorded using a Coulbourn Instruments
Startle Response System. The system consisted of two transducer
platforms that produced electrical signals proportional to the force
applied to them. One platform with a 1-lb (0.454-kg) maximum force
limit was used for the 16- and 23-day-old rats and one with a 5-lb
(2.27-kg) maximum was used for the adult rats. The analog output of
each platform was converted to a digital score for each millisecond of a
200-ms period that began with the onset of the startle stimulus. From
these data, we determined the peak startle response. The sensitivity of
the startle recording equipment was adjusted to produce peak force
outputs in the middle of the platform's sensitivity for each age. The
range for both platforms was 0-255 mV, and the average peak outputs
on the first 3 trials of the training session for the three ages were 132
mV for the 16-day-old rats, 126 mV for the 23-day-old rats, and 167
mV for the 75-day-old rats. Although the peak outputs of the adult rats
were somewhat higher than that of the younger rats, they were still
within a range that permitted assessment of the fear-potentiated
startle response.
Procedure. Rats were removed from their home cages, randomly
assigned to either a paired or explicitly unpaired group, weighed, and
placed in the restraining cage, which was then positioned on the
transducer platform inside the IAC chamber. Rats were trained and
tested individually. All subjects were given a 15-min period of
adaptation prior to the onset of training.
Training consisted of 4 CS-alone habituation trials, followed by
either 20 paired or 20 unpaired trials. Animals in the paired group
200
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ONTOGENY OF FEAR-POTENTIATED STARTLE
16-day o lds 23-day o lds 75-day olds
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NOISE ALONE
TONE-NOISE
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PAIRED UNPAIRED PAIRED UNPAIRED PAIRED UNPAIRED
CONDITIONING TREATMENT
Figure 1. Mean (and standard error of the mean) startle responses (in millivolts) of 16-, 23-, and
75-day-old subjects in Experiment 1A. Rats were given paired or explicitly unpaired presentations of a
tone conditioned stimulus (CS) with the startle pulse during fear conditioning and were tested for startle
responding immediately after acquisition. For testing, animals were given the startle pulse presented
alone (noise-alone trials) or preceded by the 10-s tone CS (tone-noise trials).
were given 20 CS-US presentations, in which the offset of the 10-s tone
CS was paired with the 100-ms, 130-dB startle stimulus. The intertrial
intervals (ITIs) ranged from 60 to 120 s. Unpaired animals received
the same number of CSs and USs but in an explicitly unpaired fashion.
The order of CS and US presentations was random, with the stipulation
that not more than 3 of a given trial type could occur in succession, and
the interstimulus intervals (ISIs) ranged from 30 to 60 s.
Immediately following completion of the training sequence, all of
the animals were given 6 additional test trials with 60-120-s ITIs. On 3
of these trials, the startle pulse was presented alone (noise-alone
trials), and on the other 3 trials the startle pulse was preceded by the
10-s tone CS (tone-noise trials). The order of test trials was randomly
determined. Fear-potentiated startle was assessed by comparing the
magnitude of the startle response on the tone-noise trials with that on
the noise-alone trials.
Statistical analyses. The startle responses (analog-to-digital output,
in millivolts) for each rat were averaged over blocks of two training
trials for the acquisition phase. These data were analyzed separately
for subjects of each age using a 2 (group) x 10 (trial block) mixed
analysis of variance (ANOVA). The responses to the startle pulse
during test for each subject were averaged over the three trials of each
type (noise alone or tone-noise) and were analyzed separately for each
age using a 2 (group) x 2 (trial type) mixed ANOVA. When
appropriate, post hoc comparisons were made using Newman-Keuls
tests (p = .05), and planned comparisons were conducted between
groups with t tests (p = .05).
Experiment 1A: Results
Test data. Analyses of the data obtained during the test
phase revealed that 23-day-oIds and adults exhibited fear-
potentiated startle, whereas 16-day-olds did not. For the two
older groups in the paired condition, the magnitude of the
startle response was greater when the startle stimulus was
preceded by the fear-eliciting CS than when it was presented
alone. By contrast, the 16-day-old subjects responded equiva-
lently to the startle pulse, regardless of training experience and
test trial type. The mean startle response amplitudes for the
test trials for the paired and unpaired groups are presented in
Figure 1.
The 2 (group) x 2 (trial type) mixed ANOVA conducted on
the test data for the 16-day-olds revealed no significant effects
(all Fs < 1.0). The ANOVA conducted on the data from the
23-day-olds yielded a significant main effect of trial type, F(l,
18) = 7.58, p < .05. Planned comparisons confirmed that for
the paired group, responding to the startle pulse was greater
on the tone-noise trials than on the noise-alone trials. For the
unpaired group, responding was the same on both trial types.
The ANOVA conducted on the adult test data revealed a
significant main effect of trial type, F(l, 19) = 21.32, p < .01,
as well as a significant Group x Trial Type interaction, F(l,
19) = 6.73, p < .05. Although the response to the startle
stimulus was the same for the unpaired rats on both test trial
types, the paired rats responded more on the tone-noise trials
than on the noise-alone trials.
Acquisition data. The mean startle response amplitudes
during the acquisition phase are presented in Figure 2. A 2
(group) x 10 (trial block) mixed ANOVA conducted on the
data obtained from the 16-day-olds yielded a significant main
effect of trial block, F(9, 162) = 4.90, p < .01. The main effect
of group and the Group x Trial Block interaction were both
nonsignificant (Fs < 1.0). The startle responses of both groups
showed equivalent habituation over the course of the training
session.
For the 23-day-olds, the analysis revealed significant main
effects of group, F(l, 18) = 14.07,p < .01, and trial block, F(9,
162) = 6.05,p < .01, but the Group x Trial Block interaction
72 P. HUNT, R. RICHARDSON, AND B. CAMPBELL
200 16-day o lds _ 23-day olds 75-day olds
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BLOCKS OF 2 TRIALS
Figure 2. Mean startle responses (in millivolts) of 16-, 23-, and 75-day-old subjects during the acquisition
phase of Experiment 1 A. Subjects were given either paired or explicitly unpaired presentations of a tone
conditioned stimulus with the startle pulse unconditioned stimulus. Startle responses were averaged over
blocks of 2 training trials.
was nonsignificant, F(9, 162) = 1.14,p > .05. Startle response
amplitude habituated over the course of training for both
groups, but the paired group showed consistently higher levels
of responding than the unpaired group. The apparent differ-
ence between the initial levels of responding (Trial Block 1)
was nonsignificant, F(l, 18) = 3.43,p > .05, whereas on Trial
Block 10 the two groups were significantly different, F(l, 18) =
10.77,p < .01.
An analysis conducted on the acquisition data obtained from
the adult subjects yielded significant main effects of group,
F(l, 19) = 7.19,p < .05, and trial block, F(9,171) = 3.33,p <
.01, as well as a significant interaction between group and trial
block, F(9, 111) = 2.76,p < .05. All rats began training with a
high level of responding to the startle stimulus, and this
response habituated across the training episode in the un-
paired group. For the paired group, responding was main-
tained at a high level throughout the training procedure.
Experiment 1A: Discussion
The results of this experiment suggest that fear-potentiated
startle does not emerge ontogenetically until approximately 23
days of age, at least when an auditory stimulus serves as the CS
and the startle pulse serves as the US for fear conditioning.
After 20 pairings of the tone CS with the startle pulse US,
23-day-olds and adults showed exaggerated startle responses
on the tone-noise test trials compared with the level of
responding on the noise-alone trials. By contrast, there were
no differences in startle response amplitude of the 16-day-old
subjects on the tw
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