Physiology&Behavior,Vol. 23, pp. 291-297. PergamonPress and Brain Research Publ., 1979.Printed in the U.S.A.
Habituation and Prestimulus Inhibition of the Auditory Startle Reflex in Decerebrate Rats J. E. F O X
Department of Physiology, The Medical School, Birmingham B15 2TJ, U.K. R e c e i v e d 14 S e p t e m b e r 1978 FOX, J. E. Habituation and prestimulus inhibition of the auditory startle reflex in decerebrate rats. PHYSIOL. BEHAV. 23(2) 291-297, 1979.--Integrated crags, recorded from muscles of the neck, have been used to study the auditory startle reflex in the decerebrate rat. The reflex had a mean onset latency of 11.6 msec and a mean duration of 90 msec; it was abolished by lesions in the inferior colliculus and, in some animals, lesions in the pontine or caudal mesencephalic reticular formation. It was found that the startle response, in the decerebrate animal, was habituated in a way comparable with that described in the intact animal. The size of the response was also reduced by a single preceding auditory stimulus, whether or not that stimulus was above threshold for eliciting a startle response. It therefore appears that short term habituation and prestimulus inhibition of the startle reflex are not dependent on higher regions of the central nervous system. Habituation
Prestimulus inhibition
Startle
Decerebrate rats
THE auditory startle reflex consists of a characteristic, widespread muscular contraction which is elicited by sudden, moderately intense sounds [18,19]. Several investigators have shown that the reflex exists in decerebrate animals [6, 7; 27] and it appears that the most important relays are through the cochlear nucleus, inferior colliculus and brainstem reticular formation [3, 11, 19, 27]: in addition, some work has suggested the existence of another, more direct pathway into the reticular formation [11,23]. In the intact animal, it has been shown that the reflex is. highly labile: it is subject to habituation and may be modified by preceding visual, cutaneous and weak auditory stimulation [2, 8, 9, 14, 15, 16, 22]. It is not clear, however, whether this lability, which is such a characteristic feature of the response in the intact animal, also exists in the decerebrate animal. Some authors have emphasised the importance of the cerebral cortex and other regions of the higher CNS in mediating habituation of the reflex [5, 6, 25, 26] while others [4] have stressed the possible role of the brainstem reticular formation. More recently, Groves and his colleagues [10,11] suggested that, although the cerebral cortex may be responsible for long term habituation, short term habituation is dependent on labile elements in the reticular core of the brainstem. If this hypothesis is correct, it should be possible to demonstrate short-term habituation of startle in the decerebrate animal. Davis and Gendelman [6] tested this hypothesis, but reported that the acute decerebrate rat does not show habituation of auditory startle, although the reflex is apparently still subject to modification by a single preceding auditory stimulus. However, it is well known that, immediately following decerebration, rats are hyperexcitable and it seems possible that this factor may have influenced their results. The present experiments were therefore carried out to
Brainstem function
further investigate the lability of the startle response in the decerebrate animal, after allowing time for the animals to recover from this period of hyperexcitability. In some animals, additional lesions were made in the mesencephalic or pontine reticular formation and their effects on the reflex were assessed.
METHOD Experiments were carried out on 39 Sprague-Dawley and Wistar swain rats of either sex, weighing 250--400 g. They were anaesthetised with tribromoethyl alcohol (AVERTIN), 25 mg/100 g body weight given IP; the anaesthesia was deepened when necessary with halothane (FLUOTHANE I.C.I.). After making a midline skin incision, holes were drilled, bilaterally, in the parietal and/or frontal bones of the skull and the decerebration performed by suction, the whole of the forebrain being removed. In order to control bleeding, the skull cavity was loosely packed with cotton wool soaked in thrombin (Parke, Davis & Co.). In six animals, the inferior colliculi were also removed by suction. In nine other animals, bilateral lesions were made, in either the mesencephalic reticular formation, or the nucleus reticularis pontis, by placing a stainless steel electrode into the brainstem and passing a DC current of 1.5 mA for 15 sec (electrode tip--negative). • Following surgery, the animals were housed, in groups of two or three, in cages warmed by radiant heat. Fluid balance was maintained by giving 5% dextrose-saline by intragastric or rectal injection and, to prevent infection, the animals were given a single intramuscular or deep subcutaneous injection of penicillin (0.1 rnl TRIPLOPEN, Glaxo). No recording was made until 24 hr had elapsed following decerebration and all recording was completed within the
Copyright © 1979 Brain R e s e a r c h Publications Inc.--0031-9384/79/080291-07501.20/0
292
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FIG. 1. The response to paired stimulation. (A) The top trace (i) shows the emg activity in the posterior neck muscles of a decerebrate rat following two identical 1 kHz 110 dB sounds. The i ~ e d record is shown immediately beneath (ii). Stimulus timing is shown by the open rectangles. (B) ~ h showing the effect of stimulus interval on the size of the response evoked by the second stimulus. Vertical bars show the SE, N=I0. Note that a logarithmic scale has been used on the horizontal axis.
following 24 hr. At the end of this time the animals were killed by a large injection of barbiturate. For recording the emg, flexible stainless steel wires were inserted into the posterior neck muscles; the wire was lacquer coated, except for the last 3 mm which was ~ and bent into a hook shape to ensure good retention in the muscle. Signals were led through a Neurotog ampl/fier system and displayed on a storage oscilloscope; tape records were made (Racal Store 4I)) for subsequent analysis. To measure the amount of emg activity, records were full wave rectified and
integrated: the integrating circuit was reset at 10 msec intervals and a "sample and h o l d " circuit used to display the amount of emg activity in the form of a histogram (see Fig. 1). Experiments were carried out in a soundproof chamber with a loudspeaker fixed to one wall. Background sound level was 30 dB. Auditory stimuli consisted o f a I kHz sine wave, duration 20 msec and intensity 110 dB (except where otherwise stated). Stimulus timing was controlled by a Digitimer. In experiments to determine the response to paired stimu-
HABITUATION OF STARTLE lation, sounds were presented to the animal with stimulus intervals between 0.5 sec and 128.0 sec. An interval of 5 rain was allowed to elapse between the presenation of each pair of stimuli and animals were presented with the paired stimuli in a random order, so that, for example, brief stimulus intervals did not always occur early or late in the animals' testing routines. In the experiments investigating habituation, stimuli were presented at rates of between 1 per 4 sex and 1 per I00 sec. The order in which different rates of stimulation were presented was again random and, in these experiments, an interval of at least 1 hr was allowed to elapse between successive experimental trials. To investigate dishabituation, electrical stimuli were applied to the flank via subcutaneous electrodes. Stimuli consisted of a short burst of 5×2 msec rectangular pulses, the intensity of which varied between 5 mA and 25 mA in different animals. After each experiment, the brains were fixed in Formalin and sections were cut for histology. From these sections the decerebration level was determined: the lowest level was midcollicular (two animals) and in the remainder, the section passed through the superior colliculus; the highest decerebration was through the rostral end of the red nucleus. Since no statistically significant differences were found depending on decerebration level, all results have been pooled. The position of the lesions was also confirmed by histology. RESULTS
General Behaviour At the time of testing (24-48 hr after decerebration) the animals spent most of the time sitting in a crouched position; in addition, they made varying amounts of spontaneous walking and exploratory movements. They also made grooming and chewing movements, but were not seen to eat or drink. In general, it appeared that animals with higher level decerebrations showed greater amounts of spontaneous activity. There was no marked increase in extensor tone, though some animals did show a slight increase in tone in the hindlimbs and tail. Responses .to mild noxious stimulation were present: pin prick led to flexion of the stimulated limb, movements of the whole animal away from the stimulus, and to vocalisation. Righting reflexes, elicited by placing the animal on its side or by suspending from its tail, were also present.
The Startle Response In response to a sudden loud sound, the animals made a very brief, widespread muscular contraction which, on gross observation, appeared similar to that described in the intact animal, viz., pricking up the ears, lifting of the head, and sometimes a clearly visible jump. This report concerns the emg activity recorded in the posterior neck muscles following the presentation of 110 dB, 20 msec, 1 kHz sounds. The response evoked by these stimuli usually consisted 'of a single brief burst of activity with a mean onset latency of 11.6 msec (SE=I.0 msec; N =24) and a mean duration of 90 msec (SE =21 msec). The present work is chiefly concerned with this brief, short latency response. However, other responses were also recorded occasionally. In some animals a longer latency, relatively prolonged increased in activity was seen; this increase in activity was variable in intensity and sometimes persisted for up to 4 sec.
293 In addition to the usual excitation, an inhibition of em8 activity was also occasionally seen. This had a latency of approximately 12 msec and a duration of 7-35 reset. It was only seen when there was a background of spontaneous crag activity and was more clearly seen when relatively weak stimuli were used. These longer latency and inhibitory responses were not studied in detail in the present experimeats.
The Response to Pains of ldentical Auditory Stimuli Pairs of identical sounds were presented to the animals with interstimulus intervals of 0.5-128 sex. At short interstimulus intervals, the response to the second sound was smaller than that elicited by the first of the pair (Fig. IA). Pooled results from 10 animals are shown in Fig. IB, from which it may be seen that the time to 50% recovery was approximately 7 sec.
The Effect of a Weak Prestimulus These experiments were carried out to determine whether a weak auditory prestimulus would modify the response to a subsequent 110 dB sound in a way comparable with that described in the intact animal [14,15]. The prestimulus was set to a level which was subthreshold for eliciting the startle response: it was followed after 500 msec by the 110 dB testing stimulus. The mean size of the response elicited by the testing stimulus was 12% of the control response size (SE=4%, N=5). An example is shown in Fig. 2.
Habituation of the Startle Response and Recovery After an Interval Habituation of the auditory startle response was clearly seen in the present experiments (see Figs. 3 and 4). In general the response size showed an approximately exponential decay with repeated stimulus presentations. In order to determine whether the response recovered after an interval, stimulation was interrupted for a period of 5 min. Animals were tested using stimulus rates between 1 per 4 sec and 1 per 20 sec. Mean response size evoked by the last stimulus before the interval was 10% of the control: mean response size evoked by the first stimulus after the interval was 54% of the control. A paired t-test showed that the difference was statistically significant (p
Habituation and Prestimulus Inhibition of the Auditory Startle Reflex in Decerebrate Rats J. E. F O X
Department of Physiology, The Medical School, Birmingham B15 2TJ, U.K. R e c e i v e d 14 S e p t e m b e r 1978 FOX, J. E. Habituation and prestimulus inhibition of the auditory startle reflex in decerebrate rats. PHYSIOL. BEHAV. 23(2) 291-297, 1979.--Integrated crags, recorded from muscles of the neck, have been used to study the auditory startle reflex in the decerebrate rat. The reflex had a mean onset latency of 11.6 msec and a mean duration of 90 msec; it was abolished by lesions in the inferior colliculus and, in some animals, lesions in the pontine or caudal mesencephalic reticular formation. It was found that the startle response, in the decerebrate animal, was habituated in a way comparable with that described in the intact animal. The size of the response was also reduced by a single preceding auditory stimulus, whether or not that stimulus was above threshold for eliciting a startle response. It therefore appears that short term habituation and prestimulus inhibition of the startle reflex are not dependent on higher regions of the central nervous system. Habituation
Prestimulus inhibition
Startle
Decerebrate rats
THE auditory startle reflex consists of a characteristic, widespread muscular contraction which is elicited by sudden, moderately intense sounds [18,19]. Several investigators have shown that the reflex exists in decerebrate animals [6, 7; 27] and it appears that the most important relays are through the cochlear nucleus, inferior colliculus and brainstem reticular formation [3, 11, 19, 27]: in addition, some work has suggested the existence of another, more direct pathway into the reticular formation [11,23]. In the intact animal, it has been shown that the reflex is. highly labile: it is subject to habituation and may be modified by preceding visual, cutaneous and weak auditory stimulation [2, 8, 9, 14, 15, 16, 22]. It is not clear, however, whether this lability, which is such a characteristic feature of the response in the intact animal, also exists in the decerebrate animal. Some authors have emphasised the importance of the cerebral cortex and other regions of the higher CNS in mediating habituation of the reflex [5, 6, 25, 26] while others [4] have stressed the possible role of the brainstem reticular formation. More recently, Groves and his colleagues [10,11] suggested that, although the cerebral cortex may be responsible for long term habituation, short term habituation is dependent on labile elements in the reticular core of the brainstem. If this hypothesis is correct, it should be possible to demonstrate short-term habituation of startle in the decerebrate animal. Davis and Gendelman [6] tested this hypothesis, but reported that the acute decerebrate rat does not show habituation of auditory startle, although the reflex is apparently still subject to modification by a single preceding auditory stimulus. However, it is well known that, immediately following decerebration, rats are hyperexcitable and it seems possible that this factor may have influenced their results. The present experiments were therefore carried out to
Brainstem function
further investigate the lability of the startle response in the decerebrate animal, after allowing time for the animals to recover from this period of hyperexcitability. In some animals, additional lesions were made in the mesencephalic or pontine reticular formation and their effects on the reflex were assessed.
METHOD Experiments were carried out on 39 Sprague-Dawley and Wistar swain rats of either sex, weighing 250--400 g. They were anaesthetised with tribromoethyl alcohol (AVERTIN), 25 mg/100 g body weight given IP; the anaesthesia was deepened when necessary with halothane (FLUOTHANE I.C.I.). After making a midline skin incision, holes were drilled, bilaterally, in the parietal and/or frontal bones of the skull and the decerebration performed by suction, the whole of the forebrain being removed. In order to control bleeding, the skull cavity was loosely packed with cotton wool soaked in thrombin (Parke, Davis & Co.). In six animals, the inferior colliculi were also removed by suction. In nine other animals, bilateral lesions were made, in either the mesencephalic reticular formation, or the nucleus reticularis pontis, by placing a stainless steel electrode into the brainstem and passing a DC current of 1.5 mA for 15 sec (electrode tip--negative). • Following surgery, the animals were housed, in groups of two or three, in cages warmed by radiant heat. Fluid balance was maintained by giving 5% dextrose-saline by intragastric or rectal injection and, to prevent infection, the animals were given a single intramuscular or deep subcutaneous injection of penicillin (0.1 rnl TRIPLOPEN, Glaxo). No recording was made until 24 hr had elapsed following decerebration and all recording was completed within the
Copyright © 1979 Brain R e s e a r c h Publications Inc.--0031-9384/79/080291-07501.20/0
292
FOX
A i)
I
1 0
I
I
I
I 0.1
,
I 0.2
/,I
I
, I
I
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2.1
I
I 2.2see
B 100
UJ N Z Z
50
|
0.5
i
t,
I
2.0 STIMULUS
64) INTERYAL
32.0
/
|sic)
FIG. 1. The response to paired stimulation. (A) The top trace (i) shows the emg activity in the posterior neck muscles of a decerebrate rat following two identical 1 kHz 110 dB sounds. The i ~ e d record is shown immediately beneath (ii). Stimulus timing is shown by the open rectangles. (B) ~ h showing the effect of stimulus interval on the size of the response evoked by the second stimulus. Vertical bars show the SE, N=I0. Note that a logarithmic scale has been used on the horizontal axis.
following 24 hr. At the end of this time the animals were killed by a large injection of barbiturate. For recording the emg, flexible stainless steel wires were inserted into the posterior neck muscles; the wire was lacquer coated, except for the last 3 mm which was ~ and bent into a hook shape to ensure good retention in the muscle. Signals were led through a Neurotog ampl/fier system and displayed on a storage oscilloscope; tape records were made (Racal Store 4I)) for subsequent analysis. To measure the amount of emg activity, records were full wave rectified and
integrated: the integrating circuit was reset at 10 msec intervals and a "sample and h o l d " circuit used to display the amount of emg activity in the form of a histogram (see Fig. 1). Experiments were carried out in a soundproof chamber with a loudspeaker fixed to one wall. Background sound level was 30 dB. Auditory stimuli consisted o f a I kHz sine wave, duration 20 msec and intensity 110 dB (except where otherwise stated). Stimulus timing was controlled by a Digitimer. In experiments to determine the response to paired stimu-
HABITUATION OF STARTLE lation, sounds were presented to the animal with stimulus intervals between 0.5 sec and 128.0 sec. An interval of 5 rain was allowed to elapse between the presenation of each pair of stimuli and animals were presented with the paired stimuli in a random order, so that, for example, brief stimulus intervals did not always occur early or late in the animals' testing routines. In the experiments investigating habituation, stimuli were presented at rates of between 1 per 4 sex and 1 per I00 sec. The order in which different rates of stimulation were presented was again random and, in these experiments, an interval of at least 1 hr was allowed to elapse between successive experimental trials. To investigate dishabituation, electrical stimuli were applied to the flank via subcutaneous electrodes. Stimuli consisted of a short burst of 5×2 msec rectangular pulses, the intensity of which varied between 5 mA and 25 mA in different animals. After each experiment, the brains were fixed in Formalin and sections were cut for histology. From these sections the decerebration level was determined: the lowest level was midcollicular (two animals) and in the remainder, the section passed through the superior colliculus; the highest decerebration was through the rostral end of the red nucleus. Since no statistically significant differences were found depending on decerebration level, all results have been pooled. The position of the lesions was also confirmed by histology. RESULTS
General Behaviour At the time of testing (24-48 hr after decerebration) the animals spent most of the time sitting in a crouched position; in addition, they made varying amounts of spontaneous walking and exploratory movements. They also made grooming and chewing movements, but were not seen to eat or drink. In general, it appeared that animals with higher level decerebrations showed greater amounts of spontaneous activity. There was no marked increase in extensor tone, though some animals did show a slight increase in tone in the hindlimbs and tail. Responses .to mild noxious stimulation were present: pin prick led to flexion of the stimulated limb, movements of the whole animal away from the stimulus, and to vocalisation. Righting reflexes, elicited by placing the animal on its side or by suspending from its tail, were also present.
The Startle Response In response to a sudden loud sound, the animals made a very brief, widespread muscular contraction which, on gross observation, appeared similar to that described in the intact animal, viz., pricking up the ears, lifting of the head, and sometimes a clearly visible jump. This report concerns the emg activity recorded in the posterior neck muscles following the presentation of 110 dB, 20 msec, 1 kHz sounds. The response evoked by these stimuli usually consisted 'of a single brief burst of activity with a mean onset latency of 11.6 msec (SE=I.0 msec; N =24) and a mean duration of 90 msec (SE =21 msec). The present work is chiefly concerned with this brief, short latency response. However, other responses were also recorded occasionally. In some animals a longer latency, relatively prolonged increased in activity was seen; this increase in activity was variable in intensity and sometimes persisted for up to 4 sec.
293 In addition to the usual excitation, an inhibition of em8 activity was also occasionally seen. This had a latency of approximately 12 msec and a duration of 7-35 reset. It was only seen when there was a background of spontaneous crag activity and was more clearly seen when relatively weak stimuli were used. These longer latency and inhibitory responses were not studied in detail in the present experimeats.
The Response to Pains of ldentical Auditory Stimuli Pairs of identical sounds were presented to the animals with interstimulus intervals of 0.5-128 sex. At short interstimulus intervals, the response to the second sound was smaller than that elicited by the first of the pair (Fig. IA). Pooled results from 10 animals are shown in Fig. IB, from which it may be seen that the time to 50% recovery was approximately 7 sec.
The Effect of a Weak Prestimulus These experiments were carried out to determine whether a weak auditory prestimulus would modify the response to a subsequent 110 dB sound in a way comparable with that described in the intact animal [14,15]. The prestimulus was set to a level which was subthreshold for eliciting the startle response: it was followed after 500 msec by the 110 dB testing stimulus. The mean size of the response elicited by the testing stimulus was 12% of the control response size (SE=4%, N=5). An example is shown in Fig. 2.
Habituation of the Startle Response and Recovery After an Interval Habituation of the auditory startle response was clearly seen in the present experiments (see Figs. 3 and 4). In general the response size showed an approximately exponential decay with repeated stimulus presentations. In order to determine whether the response recovered after an interval, stimulation was interrupted for a period of 5 min. Animals were tested using stimulus rates between 1 per 4 sec and 1 per 20 sec. Mean response size evoked by the last stimulus before the interval was 10% of the control: mean response size evoked by the first stimulus after the interval was 54% of the control. A paired t-test showed that the difference was statistically significant (p
