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Potentiation of poststartle activity by conditioned fear: Effects of anxiolytic and anxiogenic drugs
BIOL PSYCHIATRY 1991~29:683-686
683
BRIEF REPORTS
Potentiation of Poststartle Activity by Conditioned Fear: Effects of Anxiolytic and Anxiogenic Drugs Uba S. Munonyedi, Hiremagalur J. Hrishikeshavan, Ravindranath S. Shanbhogue, and Kshama Devi
This study was aimed at developing r simple novel technique for evaluati:g poststarfle behavior. Poststartle behavior is a global behavioral response displayed by an organism following a startle episode. It is generally be!ieved that a sudden stimulus such as loud noise will prompt a person to bend quickly, jerk his head forward, blink his eyes, and in various ways display the "startle reflex." The startle reflex is a primary, innate, and involuntary reaction (Landis and Hunt 1939), and has been viewed as one of the predisposing factors for emotional reactions, such as fear and or anxiety (Kubie 1941). Elevated startle response is associated with conditioned fear (Brown et al 1951). The augmentation of the startle reflex in presence of a cue (potentiation of startle) that has been previously paired with a shock is termed the fear potentiated startle effect (Davis and Astrachan 1978). This indicates that the startle response is increased by a central state of fear (McAllister and McAllister 1971). Potentiation of the startle episode is a strategy employed to elicit measurable effects of startle and relevant behavior, and has been used as a sensitive index of fear and anxiety (Davis et al 1979). The startle reflex, characterized by its short latency and fast motor act, is known to be sensitive to anxiolytics and anxiogenics; but the fear-potentiated startle para-
Address reprint requests to H.J. Hrishikeshavan, Ph.D., Drug Research and Development Centre, Pharmacology Department, Kasturba Medical College, Manipal-576 119, Kamataka South, India. Received December 6, 1989; revised July 6, 1990.
© 1991 Society of Biological Psychiatry
digm does not address the behavior as a consequence of startle. This behavior, namely poststra-tle behavior, is significantly influenced or modified in a subject experiencing fear or anxiety. Poststartle behavior, as a paradigm of fear or anxiety, has not been adequately exangned. In this study, potentiation of poststartle behavior was accomplished by pairing a conditioned stimulus (CS), namely light, with an unconditioned stimulus (US), namely nolle, with behavior evaluated on a ten-point rating scale (Table 1). This study also examines the effect of anxiolytic and anxiogenic agents on poststartle activity.
Methods Male rats (Wistar strain), weighing about 200 g and 13-15 weeks of age, were employed. The vivarium and the behavioral laboratory was maintained on a reversed light/dark cycle (off-0600-1800 hr, o n - - 1 8 0 0 - ( g ~ hr) and was noise attenuated. Training and testing were done between 0800 and 1600 hr (during the dark phase). The experiments were performed in a Plexiglas cage having a grid floor and a perforated roof for providing acoustic stimulus. This apparatus was placed inside a sound-attenuated chamber, with a 0-W red light source, fixed 30 ¢m above the startle apparatus. The dim light provided an uniform illumination of 10 lux to the interior of the box. The sound-attenuated outer chamber had a hinged transparent door for visual observation. The CS, in the form of a visual cue (40
0006-3223/91/$03.50
~4
Brief Reports
BIOL PSYCHIATRY 1991 ;29:683-686
Table 1. Ten-Point Rating Scale Rating
time of testing. The rats were tested 48 hr after training in the potentiated poststartle paradigm. Potentiation of poststartle response was obtained by pairing the acoustic stimulus with light. In the subsequent trial, noise was presented alone in the absence of light. The block of 20 trials were run in an alternated sequence of light-noise (LN) and noise-alone (NA) trials. Although the CS alone (LA) was an integral part of the LN trial, the response to LA could be separated from the response to LN Fdling. In other words, behavior was rated twice in a LN trial: the first rating referred to LA and the second referred to LN. The poststartle activity was rated using a ten-point behavioral rating scale (Table 1). For testing anxiogenic and anxiolytic agents in this paradigm, it was essential to rule out any sedative or other untoward effects. Behavior, viz ambulation, rearing, and preening, were ~ corded for 2 min in a novel arena (Figure 1). Muscle tone and coordination were also evaluated.
Response
0 !+ 2+ 3+ 4+ 5+
No response. Pinnareflex/head or limb movement. Jerk,involvingthe whole body. Jerk and forelimb lifted from cage floor. Jerk and jumping with help of hindlimb. Jerk and jumping with hindlimb with change in position or sharp turning. Jerk and sharp forward propulsion/running. Reducedlocomotor activity and defensive burying/crouching. Crouchingand tremulousness. Freezing/vocalisation.
6+ 7+ 8+ 9+
lux), was presented through a 100-W bulb placed 30 cm from the cage. The US was given in the form of footshocks (0.8 mA), by means of a shock generator (Inco, Ambala, India). Subjects were given 20 light-shock pairings (0.5 sec of CS immediately followed by 0.5 sec of US), at intervals of 60 sec. Training concluded when CS alone elicited pinna reflex (inclusion criterion). Rats exhibiting motor incoordination, unsteady gait, failure in visual placeme;~.t test, jumping behavior, or aggression were excluded from the study. The novel startle eliciting stimulus (98 dB noise) was applied through an electric bell, placed directly above the cage, at the
Results and Discussion The relationship between the LN and NA trials, eaid the effects of drug treatments on these two trial types, were analyzed by the Mann-Whitney "U" test. The results indicate that a con-
'i
41"
fl.
O
A B
IF
-=
Aller lesting Before Testing
a
Ambulations
r p
Rearings Preenings
e
p < O-OS p <001
i
t
,,
BABABA a
r
Snlin e
B ABA p
o
BA
r
Clonidine 10ug/k,j
p
BABABA a
r
CI onidine 40ug/kg
BABABA p
a
r
BABABA p
Xylazln e 2.5rng/kg
a
r
p
Ruspir one 5mg/kg
Figure 1. Analysis of normal behavioral profile (ambulations, rearings, and preenings) in a novel environment before and after testing.
II iii
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5
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Light
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Saline
0 1
0 25
0 5
0.75
YOHIMBINE
(n, glk9 i.p.)
Figure 2. Anxiogenic effect of yohimbine--dose-dependent response.
LA -- Lighl -Alone ¢
LI'I -- Li-.lht- Noi,.~ IIA -41 t
i-h
Hoise - Alone p ".a 0 0 1
-h
T
!
w
o
I
i. o
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....
L A LN Saline
NA
LA LN NA Clonldine t0uq/k 9
T LA LN NA Clonidine {.0ug/kg
I T
LA LN NA Xylazine 2-5rnglkq
Figure 3. Blockade of potentiated poststau,'tle effect by various treatments.
LA
LN NA 13uspirone 5rng/kg
LA
LN NA
Diazepam 2mg/k 9
686
Brief Reports
BIOL PS~ CHIATRY 1991:29:6~3-686
ditioning stimulus potentiated poststartle behavior (Figure 2). The effect of various doses of yohimbi~e Can anxiogenic agent), O. 125-1 mg/kg, was evaluated (Figure 2). Yohimbine s~g~ific~tly potentiated poststartle activity in the LN trial. Sensitivity of this paradigm to various putative anxiolytic treatments was also demonstrated. Clonidine (10 and 40 I~g/kg), diazepam (2 mg/kg), buspirone (5 mg/kg), and xylazine (2.5 mg/kg) blocked potentiation of poststartle activity (Figure 3). Startle elicited by acoustic stimuli has been widely studied without much data, emphasis, or design to examine poststartle behavior. The startle reflex itself is a rapid, involuntary generalized motor response, which cannot be mimicked by voluntary motor behavior (May 1950). The state of fear and level of anxiety of a subject modulate the amplitude and latency of the startle reflex. Under this emotional state, poststartle behavior may be related to escape behavior. The postural changes an animal assumes to escape an impending danger is purposeful and depends on the state of fear. It is possible that startle may serve as a priming system for these behavioral changcs, and the startle itself may be primed by the central state ,~f tear. Perhaps, if we consider startle as a basic and generalized motor process, it may be relevant to measure behavior immediately following startle, i.e., poststartle behavior. The results were consistent with reports elsewhere, employing the fear-potentiated startle paradigm, on the modulating effects of
yohimbine and putat~-,c z~iclytics. This novel method may offer an altemative to the currently available paradigms that utilize startle as a parameter for understanding the biology and pharmacology of anxiety. This work was supported by Indian Council of Medical Research (ICMR), New Delhi, India, grant No. 513-5(3)/87 BMS il 8700600.
References Brown JS, Kalish HI, Faber IE (1951): Conditioned fear as revealed by magnitude of startle response to auditory stimulus. J Exp Psycho141:317-328. Davis M, Astrachan DI (1978): Conditioned fear and startle magnitude: Effects of different footshock or backshock intensities used in training. J Exp Psychol Anita Behav Processes 4:95-103. Davit M, Redmond DE Jr, Baraban JM (1979): Noradrenergic agonists and antagonists: Effect on conditioned fear as measured by the Potentiated Startle Paradigm. Psychopharmacology 65:111118. Kubie LS (1941): The ontogeny of anxiety. Psychoanal Rer 28(1):78-85. Landis C, Hunt WA (1939): The Startle Pattern. New York: Farrar and Rinehart. May R (1950): The Meaning of Anxiety: Anxiety Interpreted Biologically. New York: Ronald Press, pp 46-85. McAllister WR, McAllister DE (1971): Behavioural measurement of conditioned fear. In Brush FIR (ed), Aversive Conditioned Learning. New York: Academic, pp 105-179.
683
BRIEF REPORTS
Potentiation of Poststartle Activity by Conditioned Fear: Effects of Anxiolytic and Anxiogenic Drugs Uba S. Munonyedi, Hiremagalur J. Hrishikeshavan, Ravindranath S. Shanbhogue, and Kshama Devi
This study was aimed at developing r simple novel technique for evaluati:g poststarfle behavior. Poststartle behavior is a global behavioral response displayed by an organism following a startle episode. It is generally be!ieved that a sudden stimulus such as loud noise will prompt a person to bend quickly, jerk his head forward, blink his eyes, and in various ways display the "startle reflex." The startle reflex is a primary, innate, and involuntary reaction (Landis and Hunt 1939), and has been viewed as one of the predisposing factors for emotional reactions, such as fear and or anxiety (Kubie 1941). Elevated startle response is associated with conditioned fear (Brown et al 1951). The augmentation of the startle reflex in presence of a cue (potentiation of startle) that has been previously paired with a shock is termed the fear potentiated startle effect (Davis and Astrachan 1978). This indicates that the startle response is increased by a central state of fear (McAllister and McAllister 1971). Potentiation of the startle episode is a strategy employed to elicit measurable effects of startle and relevant behavior, and has been used as a sensitive index of fear and anxiety (Davis et al 1979). The startle reflex, characterized by its short latency and fast motor act, is known to be sensitive to anxiolytics and anxiogenics; but the fear-potentiated startle para-
Address reprint requests to H.J. Hrishikeshavan, Ph.D., Drug Research and Development Centre, Pharmacology Department, Kasturba Medical College, Manipal-576 119, Kamataka South, India. Received December 6, 1989; revised July 6, 1990.
© 1991 Society of Biological Psychiatry
digm does not address the behavior as a consequence of startle. This behavior, namely poststra-tle behavior, is significantly influenced or modified in a subject experiencing fear or anxiety. Poststartle behavior, as a paradigm of fear or anxiety, has not been adequately exangned. In this study, potentiation of poststartle behavior was accomplished by pairing a conditioned stimulus (CS), namely light, with an unconditioned stimulus (US), namely nolle, with behavior evaluated on a ten-point rating scale (Table 1). This study also examines the effect of anxiolytic and anxiogenic agents on poststartle activity.
Methods Male rats (Wistar strain), weighing about 200 g and 13-15 weeks of age, were employed. The vivarium and the behavioral laboratory was maintained on a reversed light/dark cycle (off-0600-1800 hr, o n - - 1 8 0 0 - ( g ~ hr) and was noise attenuated. Training and testing were done between 0800 and 1600 hr (during the dark phase). The experiments were performed in a Plexiglas cage having a grid floor and a perforated roof for providing acoustic stimulus. This apparatus was placed inside a sound-attenuated chamber, with a 0-W red light source, fixed 30 ¢m above the startle apparatus. The dim light provided an uniform illumination of 10 lux to the interior of the box. The sound-attenuated outer chamber had a hinged transparent door for visual observation. The CS, in the form of a visual cue (40
0006-3223/91/$03.50
~4
Brief Reports
BIOL PSYCHIATRY 1991 ;29:683-686
Table 1. Ten-Point Rating Scale Rating
time of testing. The rats were tested 48 hr after training in the potentiated poststartle paradigm. Potentiation of poststartle response was obtained by pairing the acoustic stimulus with light. In the subsequent trial, noise was presented alone in the absence of light. The block of 20 trials were run in an alternated sequence of light-noise (LN) and noise-alone (NA) trials. Although the CS alone (LA) was an integral part of the LN trial, the response to LA could be separated from the response to LN Fdling. In other words, behavior was rated twice in a LN trial: the first rating referred to LA and the second referred to LN. The poststartle activity was rated using a ten-point behavioral rating scale (Table 1). For testing anxiogenic and anxiolytic agents in this paradigm, it was essential to rule out any sedative or other untoward effects. Behavior, viz ambulation, rearing, and preening, were ~ corded for 2 min in a novel arena (Figure 1). Muscle tone and coordination were also evaluated.
Response
0 !+ 2+ 3+ 4+ 5+
No response. Pinnareflex/head or limb movement. Jerk,involvingthe whole body. Jerk and forelimb lifted from cage floor. Jerk and jumping with help of hindlimb. Jerk and jumping with hindlimb with change in position or sharp turning. Jerk and sharp forward propulsion/running. Reducedlocomotor activity and defensive burying/crouching. Crouchingand tremulousness. Freezing/vocalisation.
6+ 7+ 8+ 9+
lux), was presented through a 100-W bulb placed 30 cm from the cage. The US was given in the form of footshocks (0.8 mA), by means of a shock generator (Inco, Ambala, India). Subjects were given 20 light-shock pairings (0.5 sec of CS immediately followed by 0.5 sec of US), at intervals of 60 sec. Training concluded when CS alone elicited pinna reflex (inclusion criterion). Rats exhibiting motor incoordination, unsteady gait, failure in visual placeme;~.t test, jumping behavior, or aggression were excluded from the study. The novel startle eliciting stimulus (98 dB noise) was applied through an electric bell, placed directly above the cage, at the
Results and Discussion The relationship between the LN and NA trials, eaid the effects of drug treatments on these two trial types, were analyzed by the Mann-Whitney "U" test. The results indicate that a con-
'i
41"
fl.
O
A B
IF
-=
Aller lesting Before Testing
a
Ambulations
r p
Rearings Preenings
e
p < O-OS p <001
i
t
,,
BABABA a
r
Snlin e
B ABA p
o
BA
r
Clonidine 10ug/k,j
p
BABABA a
r
CI onidine 40ug/kg
BABABA p
a
r
BABABA p
Xylazln e 2.5rng/kg
a
r
p
Ruspir one 5mg/kg
Figure 1. Analysis of normal behavioral profile (ambulations, rearings, and preenings) in a novel environment before and after testing.
II iii
6X"-.-. . . -. --A" ~ z_
.....
5
•
x
~
/
/
Light
,, < oos
//~//--..~
P<001
~4 z w
Alone INOiSe igh! AI oneN°J'~e
t=
:/~~[//
3
"I
- ~_ ~7i~ .
.
.
.
.
.
.
~.
.
.
.
.
.
.
I- ~.
.
.
.
.
~".
.
.
.
.
.
.
I
i
~
!
t
Saline
0 1
0 25
0 5
0.75
YOHIMBINE
(n, glk9 i.p.)
Figure 2. Anxiogenic effect of yohimbine--dose-dependent response.
LA -- Lighl -Alone ¢
LI'I -- Li-.lht- Noi,.~ IIA -41 t
i-h
Hoise - Alone p ".a 0 0 1
-h
T
!
w
o
I
i. o
I
e~
m
!
!; JL
....
L A LN Saline
NA
LA LN NA Clonldine t0uq/k 9
T LA LN NA Clonidine {.0ug/kg
I T
LA LN NA Xylazine 2-5rnglkq
Figure 3. Blockade of potentiated poststau,'tle effect by various treatments.
LA
LN NA 13uspirone 5rng/kg
LA
LN NA
Diazepam 2mg/k 9
686
Brief Reports
BIOL PS~ CHIATRY 1991:29:6~3-686
ditioning stimulus potentiated poststartle behavior (Figure 2). The effect of various doses of yohimbi~e Can anxiogenic agent), O. 125-1 mg/kg, was evaluated (Figure 2). Yohimbine s~g~ific~tly potentiated poststartle activity in the LN trial. Sensitivity of this paradigm to various putative anxiolytic treatments was also demonstrated. Clonidine (10 and 40 I~g/kg), diazepam (2 mg/kg), buspirone (5 mg/kg), and xylazine (2.5 mg/kg) blocked potentiation of poststartle activity (Figure 3). Startle elicited by acoustic stimuli has been widely studied without much data, emphasis, or design to examine poststartle behavior. The startle reflex itself is a rapid, involuntary generalized motor response, which cannot be mimicked by voluntary motor behavior (May 1950). The state of fear and level of anxiety of a subject modulate the amplitude and latency of the startle reflex. Under this emotional state, poststartle behavior may be related to escape behavior. The postural changes an animal assumes to escape an impending danger is purposeful and depends on the state of fear. It is possible that startle may serve as a priming system for these behavioral changcs, and the startle itself may be primed by the central state ,~f tear. Perhaps, if we consider startle as a basic and generalized motor process, it may be relevant to measure behavior immediately following startle, i.e., poststartle behavior. The results were consistent with reports elsewhere, employing the fear-potentiated startle paradigm, on the modulating effects of
yohimbine and putat~-,c z~iclytics. This novel method may offer an altemative to the currently available paradigms that utilize startle as a parameter for understanding the biology and pharmacology of anxiety. This work was supported by Indian Council of Medical Research (ICMR), New Delhi, India, grant No. 513-5(3)/87 BMS il 8700600.
References Brown JS, Kalish HI, Faber IE (1951): Conditioned fear as revealed by magnitude of startle response to auditory stimulus. J Exp Psycho141:317-328. Davis M, Astrachan DI (1978): Conditioned fear and startle magnitude: Effects of different footshock or backshock intensities used in training. J Exp Psychol Anita Behav Processes 4:95-103. Davit M, Redmond DE Jr, Baraban JM (1979): Noradrenergic agonists and antagonists: Effect on conditioned fear as measured by the Potentiated Startle Paradigm. Psychopharmacology 65:111118. Kubie LS (1941): The ontogeny of anxiety. Psychoanal Rer 28(1):78-85. Landis C, Hunt WA (1939): The Startle Pattern. New York: Farrar and Rinehart. May R (1950): The Meaning of Anxiety: Anxiety Interpreted Biologically. New York: Ronald Press, pp 46-85. McAllister WR, McAllister DE (1971): Behavioural measurement of conditioned fear. In Brush FIR (ed), Aversive Conditioned Learning. New York: Academic, pp 105-179.