Pain, 49 (1992) 187-197 0 1992 Elsevier Science

187 Publishers

B.V. A11 rights reserved

0304-3959/92/$05.00

PAIN 02002

Possible startle response contamination of the spinal nociceptive withdrawal reflex Robert

Dowman

Department of Psychology, Clarkson Uniuersity, Potsdam, hJY 13699-5825 (USA) (Received

9 May 1991, revision

received

9 August

1991, accepted

19 September

1991)

Summary The objective of this study was to examine the possibility that the spinal nociceptive withdrawal reflex, otherwise known as the RI11 reflex, is contaminated by the startle response, which is a non-pain-related supraspinal response. Startle response contamination of the RI11 reflex would seriously compromise the RIIIs ability to measure spinal nociceptive processes in man, since a change in the startle response affecting EMG amplitude in the RI11 latency range would be erroneously interpreted as a change in a spinal nociceptive process. EMG responses evoked by electrical stimulation of the sural nerve were recorded from the orbicularis oculi, neck, biceps, and biceps femoris muscles in 31 healthy human volunteers. The startle response was elicited under conditions often used to record the RI11 reflex. Procedures are described that will completely eliminate the startle response. Comparisons between subjects that did and did not elicit a startle response revealed that the startle does not appear to significantly contaminate the biceps femoris RI11 reflex, at least when performing group comparisons. There are, however, situations not dealt with in this study in which the startle might significantly contaminate the RI11 reflex, such as patients with pre-existing negative emotional states, experimental procedures that induce fear and/or anxiety, and single case studies. It is important, therefore, that investigators using the RI11 reflex be cognizant of the startle response and take appropriate precautions to monitor and if necessary eliminate the startle before attributing a change in the RI11 reflex to a spinal nociceptive process. Key words: Pain; RI11 reflex; Withdrawal reflex; Startle response

Introduction The nociceptive withdrawal reflex, otherwise known as the RI11 reflex, has shown considerable promise as a means for studying spinal nociceptive processes in man (Wilier 1977, 1985; Willer et al. 1984; Chan and Dallaire 1989; Price 1989; Dowman 1991). However, very little work has been done investigating possible supraspinal contamination of the RI11 reflex. Indeed, the interneurons and motoneurons in the RI11 reflex pathway are known to be influenced by a number of supraspinal processes that are not related to pain (Bal-

Correspondence to: Robert Dowman, Department of Psychology, Clarkson University, Potsdam, NY 13699-5825, USA. E-mail: [email protected].

dissera et al. 1981). These non-pain-related supraspinal processes must be considered when interpreting a change in the RI11 reflex; otherwise a change in these supraspinal processes affecting transmission in the RI11 reflex pathway could be erroneously interpreted as a change in a spinal nociceptive process. The present study focused on one of these possible supraspinal contaminants, the startle response. The startle response is elicited by any intense stimulus that has a rapid onset (Landis and Hunt 1939; Davis 1984). The startle response consists of short latency activity in the orbicularis oculi muscle (which generates an eye blink), as well as activity in a number of flexor muscles (Landis and Hunt 1939; Davis 19841, including those often used to record the RIII. The expected latency of the startle response in the limb flexor muscles overlaps with that of the RI11 reflex (Durkovic and Nord 1988).

It is possible, therefore, that stimuli used to evoke the RI11 reflex also elicit a startle response which could overlap with and thereby contaminate the RI11 reflex. Although no previous study has directly addressed this issue, work by Bromm and his colleagues suggests that the startle response might contaminate the RI11 reflex. Bromm and Treede (1980) reported reflex activity in the RI11 latency range elicited at non-painful and painful stimulus levels. The activity at the non-painful stimulus levels is very unlikely to be the RI11 rctlcx (Wilier 1977; Roby-Brami and Bussel 1987). This activity could, however, bc a startle response, as a stimulus need not be noxious to elicit the startle (Davis 1984; Durkovic and Nord 198X). Bromm and Scharein ( 1982) reported ocular potentials elicited by painful stimulation of the finger tip that were very suggestive of startle (see Davis 1984). These ocular potentials, which would have measured the eye-blink component c\f the startle response (Peters 1967). had a very early onset latency, and its amplitude was positively related to stimulus intensity and decreased with repeated stimulus prescntation. Thus, the results of these two studies suggest that the startle can be elicited by stimuli used to evoke the RI11 reflex and might, therefore, contaminate the RI11 reflex. Results of other studies, however, suggest that the startle does not contaminate the RI11 reflex. Willcr, for example, has reported that the RI11 threshold is very close to the pain threshold and that no activity is obtained at non-painful stimulus levels (e.g., Wilier 1977; Willcr et al. 1979, 1984). These data demonstrate that startle was not elicited at non-painful stimulus levels. However, startle response amplitude is well known to be positively related to stimulus intensity (Davis 1984) and hence may have been present at painful stimulus levels. Dowman (19911, using stimulus conditions similar to those used by Wilier (1977). rcported little or no EMG activity in the RI11 latency range at non-painful stimulus levels and no ocular potentials at non-painful or painful stimulus levels. These data suggest that the startle response was not elicited by the painful or non-painful stimuli and did not, therefore, contaminate the RI11 reflex. The most obvious explanation for why startle may have been elicited in the Bromm studies, but not in the Willer or Dowman studies, is the interval between the evoking stimuli. The 70-40 set interstimulus interval (ISI) used in the Bromm studies is considerably longer than the 4-6 set IS1 used in the Wilier and the Dowman studies. The startle response is known to decrease with decreasing IS1 (Davis 1984). which may explain why there was evidence of startle in the Bromm studies but not in the Willer or Dowman studies. The objective of the present study was to determine whether the startle response is a possible contaminant of the RI11 reflex. Three experiments were performed.

The objective of cxperimcnt I was to characterize the startle response elicited by sural nerve stimulation. The second experiment determined whether the startle response could be elicited under conditions often used to record the RIII. Experiment 2 also determined whether startle is elicited under conditions that include a Z-40 set IS1 but not under conditions that include a 4-6 see ISI, as suggested by the evidence presented above. The that the results of experiment 9 clearly demonstrate startle response can be elicited under both conditions. The protocol used by Dowman (19911, in which an RI11 was reported in the absence of the eye-blink component of the startlc, differed somewhat from those used in the Bromm and Willer studies discussed above. Hence. a third experiment was conducted to replicate exactly the Dowman ( 1991) study. The results of the third experiment demonstrate that repeated stimulation at different innocuous levels prior to recording the RI11 completely eliminates the startle and with it any possible startle contamination of the RI11 reflex.

General methods

Thirty-one

healthy adults (J femaler and 27 mnles, 18-3X years ot

age) participated written

in the study. Each received

explanation

of the procedures

and all signed an informed

consent document

The suhjt’cts were comfortably the study.

Data

a thorough

oral and

and ohjectivch of the study. before

seated in a recliner

from cnch subject wcrr

participating.

chair throughout

obtained

during

one 3-h

\ession (usually mid to late nftcrnoon).

All recordings

and stimulations

gold cup electrodes. achieve electrode

were accomplished

The skin was &greased

impedances

stimulation

evoke the RI11 and startle

of the hural nerve. The electrical

(I

tion consiQed of a S-pulse train frequency) the

right

administered rural

nerve

msec pulse duration,

through 7 electrodes at the

ankle.

The

straps. Stimulus intensity was controlled 1991). In each experiment.

given in pseudorandom

order.

stimula-

250 !II

ankle

was restrainal

and monitored

X different

by computer

stimulus levels were

with the constraint

tude of each stimulus on a V-point scale, where and 0 = the maximum

The startle response was recorded measure startle: the orhicularis Orhicularis inferior

oculi EMG

that all levels be

4 cm superior including

from electrodes

IW4).

placed over the

placed over the 7th cervical vertebrae

measured

the paravertebral

of the balanced

record

tolerable.

from muscles typically used to

portion of the muscle and at the lateral canthus. both of the

right eye. Electrodes

part

magni-

I = sensory thresh-

oculi. neck, and biceps (Davis

~3s recorded

hy

brace with velcro

given within X consecutive trials. Subjects rated the perceived old. 5 = pain threshold.

pulp

placed 2 cm apart over

securing the foot and lower hmb to an L-shaped (Dowman

IO

of less than 5000 f2.

l‘he stimulus used to simultaneously HXS electrical

using q-mm

and gently abraded

pain-related

Dowman

IWO).

EMCi

from

and troprzius

stcrnovertehral

somatoscnsory

Thus.

muscles. The

reference

evoked

the configuration

(C7) and

musclcb in the neck electrode

potentials

\itc

i\

usctl to

(Turville

used to record

sponse\ from the neck muscles also enabled evaluation

region.

(‘7

startle

and I-C-

of the contri-

189

bution

of the startle

this reference.

response

Biceps EMG

cm apart over the center

to pain-related

was obtained

SEPs recorded

from 2 electrodes

using

placed 3

from a pair of electrodes

placed 3

cm apart over the right biceps femoris muscle. from each muscle was amplified

bandpass of I-300

Hz t-6

dB points),

512 msec. For each channel,

recording

from

epoch.

obtained

each

The

data

absolute

for each channel,

50000X,

point

recorded

the evoking stimulus

in the

post-stimulus

of the subtracted

which is equivalent

with a

at 500 Hz for

value of EMG

preceding

value

filtered

and sampled

the average

from the 1.28-see period immediately was subtracted

EMG

EMG

to full-wave

was

rectifica-

tion.

Data analysis Startle rectified

was identified amplitude

amplitude

of the

in each

subject

of a baseline EMG EMG

segment

by comparing

evoking

stimulus,

which

the mean

segment to the mean rectified

which

encompassed

response. The baseline segment was 27-53 of the

the

msec following

was well

after

the

startle

the onset

offset

of

the

evoking stimulus and always preceded

the startle response onset. In

each muscle,

segment

the onset of the EMG

response was determined iment 1. The duration of the

startle

defining

the startle

from the onset latencies obtained

in exper-

of these segments was based on the duration

response

in these

muscles

reported

review, see Davis 1984). The startle EMG

by others

(for

segments (measured

from

the onset of the evoking stimulus) were as follows: orbicularis

oculi,

61-141

msec; neck,

response between

89-139

was defined

msec; biceps,

for each

subject

test with

a probability

level

because we are only interested is larger

than the baseline

cause startle intensity

89-139

msec. A startle

as a significant

difference

the means of the baseline and startle segments in 3 of the 4

highest stimulus levels. Significance

t

response

(Davis

inspected

to ensure

spurious background

was determined

of 0.05.

A

segment.

This criterion

is positively

EMG,

test was used was chosen be-

related

the highest stimulus

For each subject the EMG that

using a l-tailed

l-tailed

in cases in which the startle segment

amplitude

19841, hence

likely to elicit startle.

0.13 k 0.10 (level 1) and 6.17 + 0.54

of the biceps muscle of the non-dominant

arm. The RI11 reflex was recorded EMG

ranged between (level 8).

significant

differences

to stimulus

levels are most was also visually

were

not

due

to

etc.

Experiment 1. The startle nerve stimulation

response

elicited

by sural

The objective of this experiment was to characterize the startle response elicited by sural nerve stimulation. The startle response is known to decline rapidly with repeated stimulus presentations, especially when short ISIS are used (Davis 19841. Thus, stimulus conditions optimal for eliciting the startle response will include a small number of stimulus presentations at a long ISI. Methods

Ten subjects (1 female and 9 males, 18-24 years of age) were given 3 stimulus presentations (trials) at each of the 8 stimulus levels with a random 60-90 set IS1 (mean: 75 set). Five of 8 stimulus levels were non-painful, for which the mean magnitude ratings for all subjects were less than 5.0. The remaining 3 levels were rated as painful. Mean (+ S.E.M.) stimulus currents ranged between 0.54 * 0.11 mA (level 1) and 9.64 f 1.10 mA (level 81, and mean magnitude ratings

Results and discussion

The grand averages of EMG recorded from each muscle are shown in Fig. 1. Stimulus time-locked activity was obtained from the orbicularis oculi, neck and biceps muscles. The onset of the grand average orbicularis oculi, neck and biceps responses were 69, 93 and 97 msec, respectively. This pattern of activity, in which EMG in the orbicularis oculi muscle preceded that in the more caudal neck and biceps muscles, is characteristic of the startle response (Landis and Hunt 1939; Davis 1984). This pattern results from the motor output for all muscles involved in the startle originating from the nucleus reticularis pontis caudalis, which is located in the medulla (Davis 1984). Furthermore, the onset latencies of the EMG responses obtained here are comparable to those elicited by acoustic stimulation, allowing for the extra time required for activity in the sural nerve to reach the medullary reticular nuclei. For example, the onset of the orbicularis oculi acoustic startle response ranges between 20-40 msec (Davis 19841, which is comparable to the 69-msec onset observed here, given that activity initiated in the fastest conducting afferents of the sural nerve reaches the lower neck region in approximately 32 msec (Vogel et al. 1986). The number of subjects exhibiting startle in the orbicularis oculi, neck and/or biceps muscles, as determined by the statistical and visual criteria described in Methods, is shown in Table I. The majority of subjects (8/101 exhibited startle in at least one muscle. The startle response onset latencies for subjects exhibiting startle are also shown in Table I. These values are comparable to the onset latencies of the grand average responses shown in Fig. 1. The incidence of startle in these subjects is consistent with that reported for the acoustic startle, in which individual differences in susceptibility to startle have been reported by others (Hoffman 1984; Rossignol 1975). Furthermore, the higher incidence of startle in the orbicularis oculi muscle compared to the biceps muscle has also been reported for the acoustic startle (Gogan 1970). The onset of reflex activity in the grand average of the biceps femoris muscle (63 msec) was earlier than that in the other muscles. This early response corresponds to the RI1 reflex, which reflects activity in the low-threshold cutaneous flexor reflex pathways (Willer 1977; Baldissera et al. 19811. EMG activity was seen in the RI11 latency range (100-200 msec) in 3 of the stimulus levels that were rated non-painful (i.e., for which the mean magnitude ratings were less than 5.0). Clearly, the activity at this latency range evoked at non-painful stimulus levels cannot be the RI11 reflex (Wilier 1977; Roby-Brami and Bussel 19871. The onset

190

latency of the acoustic startle response in lower limb muscles, such as the gastrocnemius, is about 120 msec (Rossignol 1975). Given the longer conduction time required for activity initiated in the sural nerve to

TABLE

I

INCIDENCE OF STARTLE IN THE ORBICULARIS OCULI (Orb. oculi), NECK AND BICEPS MUSCLES FOR THE EXPERIMENT I SUBJECTS Startle was identified using the statistical and visual criteria described in Methods. Symbols: - = startle not present; the number is the onset latency (in msec) of the startle response in that muscle.

18 15

12 9 6 3 0

Subject

Orb. oculi

Neck

1 2 3 4 5 6 7 8 9 10

6.5 67 67 67 _

95 87 _

61 61

103 19 _

67 67

97 81

_

Biceps 101 97 109 _ _ 99 91 _ _ _

reach the medullary reticular nuclei compared to auditory stimuli (approximately 32 msec, see above), the onset of the startle response in the biceps femoris muscle elicited by sural nerve stimulation should occur at around 150 msec. The startle response could, therefore, account for at least part of the activity in the RI11 latency range elicited at non-painful stimulus levels. Furthermore, the well known positive relationship between startle response amplitude and stimulus intensity (Davis 1984) implies that the startle response was elicited at painful stimulus levels as well. Hence, the startle response can contaminate the RI11 reflex, at least under the conditions presented in this experiment.

6 Experiment 2. Contribution the RI11 reflex

Fig. 1. The grand average of rectified EMG activity obtained at each stimulus level under conditions optimal for eliciting the startle response. For each subject, the rectified EMG obtained at each level was averaged over the stimuli presented at that level. The grand average is the average of rectified EMG recorded at that stimulus level for all subjects, Abbreviations: ORB, orbicularis oculi EMG; C7, neck muscle EMG; B, biceps muscle EMG; and BFI, biceps femoris EMG recorded from the muscle ipsilateral to the evoking stimulus.

of the startle response

to

The stimulus conditions used in experiment 1, in which the startle response appears to have contaminated the RI11 reflex, differ considerably from the conditions often used to record the RI11 reflex. Indeed, the larger number of stimulus presentations and shorter ISIS typically used to record the RI11 (e.g., Willer 1977; Bromm and Treede 1980) would be expected significantly to reduce, if not eliminate, the startle (Davis 1984). Thus, the objective of experiment 2 was to determine whether the startle response could be elicited under conditions (i.e., ISI, prior stimulus exposure, number of stimulus presentations) often used to record the RI11 reflex. Experiment 2 also determined whether startle is more likely to be elicited under conditions that include a 20-40 set IS1 (e.g., Bromm and Treede 1980) than under conditions that include a 4-6 set IS1 (e.g., Willer 19771, as suggested by evidence discussed in the Introduction.

191

Methods Eleven subjects (2 females and 9 males, H-21 years of age) were presented with stimulus conditions described by Bromm and Treede (1980). Each subject was initially presented with 5 trials per stimulus level and a random 20-40 set ISI (mean: 30 set). These practice trials familiarized the subjects with both the range of stimulus intensities to be expected and the magnitude rating procedure. Following the practice trials, the subjects were given 10 trials per stimulus level with the random 20-40 set ISI. Four of 8 stimulus levels were rated as non-painful, for which the mean magnitude I

ratings were less than 5.0. The remaining 4 stimulus levels were rated as painful. Mean ( rt S.E.M.) stimulus currents ranged between 1.2 f 0.09 mA (level 1) and 17.06 f 1.68 mA (level 81, and mean magnitude ratings ranged between 1.5 k 0.34 (level 1) and 7.25 _+0.39 (level 8). Twenty-one subjects were presented with stimulus conditions comparabIe to those described by Wilfer (1977). Two different groups of subjects were used. Group 1 was comprised of the 11 subjects given the 20-40 set ISI described above. Immediately following those blocks of stimulation, the subjects were preII

30 1

ORB 0

15

12 9 66 3 0 12 9 6

6

3

3

0

0

6-

0’ 18

24 I‘

BFI

BFI

Fig. 2. I: grand average of rectified EMG activity obtained from subjects in experiment 2 presented with the 20-40 set ISI. II: grand average of rectified EMG activity obtained from subjects in experiment 2 (group 1) receiving the 4-6 see ISI. III: grand average of rectified EMG obtained from subjects in experiment 2 (group 2) receiving the 4-6 set 1%. For each subject, the rectified EMG obtained at each level was averaged over the stimuli presented at that level. The grand average is the average of rectified EMG recorded at that stimulus level for all subjects. For subjects in I and II, there was considerable variability in the orbicularis oculi background EMG. This variabiiity was reduced by subtracting the baseline EMG segment amplitude (see Methods) from each data point in the post-stimulus recording epoch. Abbreviations: ORB, orbicularis oculi EMG; C7, neck muscle EMG; B,biceps muscle EMG; and BFI, biceps femoris EMG recorded from the muscle ipsilateral to the evoking stimulus.

192

sented with 2 additional stimulus blocks, where 10 trials per stimulus level and a random 4-6 set ISI (mean: 5 set) were given in each block. Group 2 was comprised of the 10 subjects who took part in experiment 1. Immediately following completion of experiment 1, the subjects’ pain thresholds and pain tolerance levels were determined using the method of limits (see Dowman 1991). The subjects were then presented with 8 different stimulus levels ranging between sensory threshold and pain tolerance. Thirty trials were given at each stimulus level, with a random 4-6 set ISI (mean: 5 set>. For the 21 subjects receiving the 4-6 set ISI, 4 of the stimulus levels were rated as non-painful, for which the mean magnitude rating was less than 5.0. The remaining 4 stimulus levels were rated as painful. Mean (5 S.E.M.) stimulus currents ranged between 1.15 + 0.12 mA (level 1) and 16.90 k 1.02 mA (level 8), and mean magnitude ratings ranged between 1.59 + 0.24 (level 1) and 7.95 + 0.18 (level 8).

TABLE

Results and discussion

Stimulus time-locked EMG activity was seen in the orbicularis oculi, neck and biceps muscles under stimulus conditions often used to record the RI11 reflex, including those that use a 4-6 set IS1 (Fig. 2). The onset latencies of the grand-average responses obtained under these conditions were similar to those seen in experiment 1. The incidence of startle, as defined by the statistical and visual criteria described in Methods, is shown in Table II. Six of 11 subjects receiving the 20-40 set IS1 exhibited startle in at least one of the muscles. A similar proportion (14/21) of the subjects receiving the 4-6 set IS1 also exhibited startle. The startle response onset latencies for each muscle are also given in Table II. These values are comparable to those obtained in experiment 1. There are a number of other features of the orbicularis oculi, neck and biceps responses observed in this experiment besides onset latency that are consistent

11

(A) INCIDENCE OF STARTLE IN THE ORBICULARIS OCULI BLOCK FOR SUBJECTS IN EXPERIMENT 2, GROUP 1 Startle was identified using the statistical and visual criteria latency (in msec) of startle in that muscle. Subject

20-40

set IS1

described

oculi),

in Methods.

NECK

AND

Symbols:

BICEPS

- = startle

Neck

Biceps

block 1

Orb. oculi

Neck

Biceps

the number

STIMULUS

is the onset

block 2 Neck

Biceps

_

12 13

61 _

_ _

7s

_ _

14 15 I6 17

67 65 _

_ 81 _ _

57 _ _ _

109 7’) x7 _

_ 87 _ _

57 71 hY _

1X 19

71 69

_ 97

97 _

_ _

65

_

20 21

51 _

91

91

(B) INCIDENCE OF STARTLE EXPERIMENT 2, GROUP 2

97 _ IN THE

ORBICULARIS

Startle was identified using the statistical and visual criteria the startle response (in msec) in that muscle. 4-6 set IS1 Neck

Biceps

1 2 3 4 5

67 h3 69 69

_ _ _ _ _

_ _ _ _ _

6 7 8

75 75 73

87 83 _

93 93 _

9

69

79

105

I0

not present:

Orb. oculi

_

Orb. oculi

IN EACH

Stimulus

11

Subject

MUSCLES

4-6 set IS1 Stimulus

Orb. oculi

(Orb.

_

_ OCULI

described

(Orb.

oculi),

in Methods.

75 _

Symbols:

79 x7

x7

_ _ _

_ NECK

109

AND

BICEPS

- = startle

7’)

MUSCLES

not present;

FOR

the number

8’)

SUBJECTS

IN

is the onset of

193

with startle. First, there was considerable individual variability in susceptibility to startle, as is the case with the acoustic startle (Rossignol 1975; Hoffman 1984). Second, the higher incidence of startle in the orbicularis oculi muscle as compared to the biceps muscle observed here has also been reported by others (Gogan 19701. Third, the changes in these responses with repeated stimulation and IS1 are similar to what has been reported for the acoustic startle. It is well known that the startle response decreases rapidly with repeated stimulus presentations and with decreasing ISIS (Davis 1984; Ornitz and Gutherie 19891, as is clearly evident in the responses obtained from the group 2 subjects (cf., Figs. 1 and 2111). (The subjects in experiment 2, group 1, received a number of stimulus presentations prior to the first startle recording, hence the rapid decline in startle amplitude is not evident.) Although startle amplitude declines rapidly with repeated stimulus presentations, the response has been shown to persist (Davis 1984; Ornitz and Gutherie 1989), as was often the case for the responses obtained from the subjects in experiment 2 (see Tables I and II). Thus, the responses recorded from the orbicularis oculi, neck and biceps muscles clearly reflect the startle response and demonstrate that the startle can be elicited under conditions often used to record the RIII. The grand-average response in the biceps femoris muscle obtained in experiment 2 is similar to that reported previously (Dowman 1991). The presence of startle in the orbicularis oculi, neck and biceps muscles presents the possibility that the biceps femoris RI11 reflex was contaminated by the startle response. This question is addressed in experiment 3 below. Note that there was considerable EMG activity following the startle segments in experiments 1 and 2 (see Figs. 1 and 2). The rather long latency of this activity suggests that it is voluntary. These voluntary responses were most prominent at painful stimulus levels and hence most likely correspond to facial grimacing and other pain-related voluntary responses that have been reported by others (Craig and Patrick 1985; Watkins 1989).

Experiment

3. Elimination

of the startle response

The results of experiment 2 demonstrated that the startle response can be elicited under stimulus conditions often used to record the RIII, including those that use a 4-6 set ISI. These results differ from the Dowman (1991) study described in the Introduction, in which the .absence of ocular potentials indicated that the startle response was not elicited. Although the Dowman study used stimulus conditions very similar to those employed by Willer (1977), additional procedures used in the Dowman study may account for the ab-

sence of startle. In particular, subjects were given different levels of innocuous stimulation prior to recording the RI11 reflex in order to obtain a sural nerve compound action potential (CAP) recruitment curve. Davis and Wagner (1969) have shown that this type of procedure facilitates habituation of the acoustic startle. The additional innocuous stimulation may, therefore, account for the absence of startle in the Dowman study. Thus, the objective of experiment 3 was to determine whether the startle response could be elicited under stimulus conditions and procedures identical to those used by Dowman (1991). The RI11 reflex was also compared between subjects in experiments 2 and 3 that did and did not elicit startle to help estimate the contribution of startle to the RIII. Methods

Ten subjects (1 female and 9 males, 19-38 years of age) participated in the experiment. The procedure was comprised of 3 stages: obtaining a sural nerve CAP recruitment curve; finding the subject’s pain threshold and pain tolerance levels; and recording the EMG responses to painful and non-painful sural nerve stimulation. A description of the methods used to obtain the sural nerve CAP recruitment curve and the pain threshold and tolerance levels are given elsewhere (Dowman 1991). Once the sural nerve CAP recruitment curve and the pain threshold and pain tolerance levels were determined, the subjects were presented with 8 different stimulus levels with a 4-6 set ISI. Four of the stimulus levels were rated as non-painful, for which the mean magnitude rating was less than 5.0. The remaining 4 stimulus levels were rated as painful. Mean ( f S.E.M.) stimulus currents ranged between 1.45 + 0.20 mA (level 1) and 16.20 + 1.46 mA (level 81, and mean magnitude ratings ranged between 1.53 + 0.17 (level 1) and 7.09 + 0.17 (level 81. Individual trials on which orbicularis oculi EMG was greater than 40 I.LV or biceps EMG was greater than 20 PV were rejected by computer, as was done by Dowman (1991). It should be noted, however, that relatively few trials were rejected because of artifact (typically lo-15%). Subjects were given 2 different blocks of stimulation. The first block was comprised of 80 artifact-free trials (10 trials/stimulus level) to provide the subjects with practice at rating the perceived magnitude of the stimuli. The second block was comprised of a total of 240 artifact-free trials (30 trials/stimulus level). Results and discussion

The grand average of EMG responses recorded from the orbicularis oculi, neck, biceps, and the biceps femoris muscles are shown in Fig. 3. There was no

194

l-10

11-40

TIME (ms)

TIME (ms)

Fig. 3. Grand average of rectified EMG activity obtained at each stimulus level for the orbicularis oculi (ORB), neck (C7), biceps (B) and ipsilateral biceps femoris (BFI) muscles obtained for subjects in experiment 3. For each subject, rectified EMG obtained at each level was averaged over the stimuli presented at that level for each stimulus block. The grand average is the rectified EMG recorded at that stimulus level and block averaged across all subjects. Stimulus block 1 (trials l-10) is shown in the left panels. Stimulus block 2 (trials 11-40) is shown in the right panels,

stimulus time-locked activity at the orbicularis oculi, neck or biceps muscles. The statistical and visual criteria described in Methods did not identify startle in any subject in the neck or biceps muscles. Startle was identified in only 1 subject at the orbicularis oculi muscle in stimulus block 2, but not in stimulus block 1. Startle amplitude in this subject was extremely small, being less than 0.5 PV greater than the baseline segment amplitude. Thus, the startle response was negligible in this group of subjects. It is extremely unlikely, therefore, that the startle response contaminated the RI11 reflex in these subjects. Comparisons were made between subjects in experiments 2 and 3 that did and did not exhibit startle to help estimate the contribution of the startle response to the RI11 reflex. Subjects that exhibited startle in at least one of the orbicularis oculi, neck or biceps muscles (startle group) were compared to those that did not (no-startle group). This analysis was restricted to EMG obtained at the 4-6 set IS1 to avoid possible ISI effects on the startle (Davis 1984) and flexor reflex (Meinck et al. 1985). For subjects who received 2 stimulus blocks (i.e., subjects in experiment 3 and in group 1 of experiment 2), data from both stimulus blocks were combined, with the exception of subject no. 20 (experiment 2, group l), who exhibited startle only during the second stimulus block (see Table IIA). Only data from the second stimulus block was used in that subject. There were 14 subjects in the startle

group, all from experiment 2. There were 17 subjects in the no-startle group (7 subjects from experiment 2 and 10 subjects from experiment 3). Startle amplitude was evaluated using the orbicularis oculi response, as is usually done for the acoustic startle (e.g., Gogan 1970; Ornitz and Gutherie 1989). Two biceps femoris EMG segments were evaluated within the RI11 interval (100-200 msec); 101-149 msec and 151-199 msec. The amplitudes of these 2 segments were computed as the average amplitude of rectified EMG within each segment. As discussed in experiment 1, the expected onset of the startle response in the biceps femoris is about 150 msec. Thus, if present, startle should only be seen in the 151-199 msec segment. The repeated measures analysis of variance comparing the startle and no-startle groups is shown in Table III. There were no differences in stimulus current between the groups. There was, however, a significant group by stimulus level interaction for the magnitude ratings. Post-hoc t tests revealed a difference in magnitude ratings between the groups at stimulus levels 1 and 2 (P < O.OS),but not at the other stimulus levels (P > 0.05). Orbicularis oculi EMG amplitudes obtained from the startle and no-startle groups are shown in Fig. 4. There was a significant group-by-stimulus-level interaction (Table III), where the startle group (stimulus level

TABLE

111

2 (group: startle vs. no-startleJX8 VARIANCE, WITH REPEATED OND FACTOR Abbreviations:

L = stimulus

df A: stimulus intensity 1, 29 G 7,203 L 7, 203 GxL B: magnitude rating I. 29 G 7, 203 L 7, 203 GXL C: startle amplitude I. 29 G 7, 203 L 7,203 GxL D: biceps femoris (101-149 msec amplitude) I, 29 G 7, 203 L 7. 203 GxL E: biceps femoris (151-199 msec amplitude) 1. 29 G 7. 203 L 7, 203 GxL

(stimulus level) ANALYSIS OF MEASURES ON THE SEC-

level: G = group. F

P

0.81 165.59 0.33

> 0.10

I.24 347.5 1 5.75

> t1.10

< 0.001 > 0.10

< 0.001 < 0.001

25.89 3.91 4.14

< 0.00 1

2.40 33.94 0.83

> 0.10

2.03 27.65 0.79

> 0.10 < 0.001 > 0.10

< 0.001 < 0.001

< 0.001 > 0.10

19.5

BFI 151-199

ms

activity at non-painful stimulus levels (levels l-4) and the amplitude of this activity increased with increasing painful stimulus levels (levels 5-8; see Table III). There is no segmental reflex activity in the RI11 latency range evoked by non-painful sural nerve stimulation (Wilier 1977; Roby-Brami and Bussel 1987). Hence, any activity observed in the 151-199 msec biceps femoris EMG segment at non-painful stimulus levels cannot be due to the RI11 reflex and could be attributed to startle. The lack of activity observed here (Fig. 4), therefore, clearly demonstrates that startle was not elicited in the biceps femoris at non-painful stimulus levels. At painful stimulus levels the startle and RI11 responses will overlap, making direct observation of the startle in the biceps femoris impossible. However, the amplitude of the 151-199 msec biceps femoris EMG segment should be larger in the startle group, in which the activity will be comprised of RI11 and startle, than in the no-startle group, where the activity will be comprised only of RIII. Therefore, the lack of difference between the groups for either of the biceps femoris EMG segments (Table III) implies that the startle was not elicited in this muscle at painful stimulus levels either.

General discussion Fig. 4. Mean (+ S.E.M.) startle response amplitude (measured from the orbicularis oculi (ORB) muscle; mean ( f S.E.M.) 101-149 msec ipsilateral biceps femoris EMG segment amplitude (BFI: 101-149 msec), and mean ( f S.E.M.) 1.51-199 msec ipsilateral biceps femoris EMG segment amplitude (BFI: 151-199 msec) for subjects in the startle (0) and the no-startle groups (0 1. In the startle group, startle amplitude was positively related to stimulus intensity, whereas startle amplitude was zero for subjects in the no-startle group. There were no differences in either biceps femoris EMG segment amplitude between subjects with and without startle.

simple main effect F (7, 203) = 7.93, P < 0.001) but not the no-startle group (stimulus level simple main effect F (7, 203) = 0.12, P > 0.10) showed a stimulus level effect in orbicularis oculi amplitude. Trend analysis revealed a linear increase in orbicularis oculi amplitude with increasing stimulus level in the startle group (F (1, 13) = 12.40, P < 0.01). Startle amplitude is well known to be positively related to stimulus intensity (Davis 1984), hence these data, along with the evidence described in experiments 1 and 2, strongly suggest that this activity is the startle response. The lack of a startle response at any of the stimulus levels in the no-startle group also demonstrate that the statistical and visual criteria accurately determined whether startle was present. The activity in the 101-149 msec and 151-199 msec biceps femoris EMG segments shown in Fig. 4 are very similar to what has been reported for the RI11 (e.g., Wilier 1977; Dowman 1991). There was little or no

The evidence presented here clearly demonstrates that the startle response can be elicited under conditions often used to record the RI11 reflex. The onset latencies of activity in the orbicularis oculi, neck and biceps muscles, the individual variability in the susceptibility to startle, and the effects of ISI, repeated stimulation and stimulus intensity observed here are all consistent with the startle response (Rossignol 1975; Davis 1984; Hoffman 1984). However, the lack of difference in RI11 amplitude between subjects who did and did not exhibit startle implies that the startle response does not make a significant contribution to the biceps femoris RI11 reflex, at least when group comparisons are made. It is important to note, however, that there are situations in which startle contamination of the RI11 might be significant that were not evaluated in this study. For example, negative emotional states, such as fear and anxiety, have been shown to potentiate the startle response (Davis 1984; Lang et al. 1990). These negative emotional states may be expected in some pain patients (Craig 1984) and under some experimental conditions (e.g., Wilier 1980). It remains to be determined, therefore, whether the larger startle response expected in those situations will result in significant contamination of the RI11 reflex. In addition, startle response contamination of the RI11 reflex may become important when studying single cases. The lack

196

of a difference in RI11 amplitude between subjects who did and did not elicit startle in experiment 3 may be due to only a small number of subjects exhibiting startle in the biceps femoris muscle. Indeed, evidence presented here (Tables I and II) and elsewhere (Gogan 1970; Rossignol 1975) demonstrates that the startle response is much more prominent in the orbicularis oculi muscle than the limb flexors. Thus, the startle response may have been elicited in the biceps femoris muscle in only a small proportion of subjects in the startle group, as was the case with the biceps muscle. Although this level of contamination does not appear to be important when looking at group effects, it may be important when studying changes in RI11 amplitude in a single case over time if that individual is one of the few who exhibit startle in the biceps femoris. The procedures used in experiment 3 completely eliminated the startle response from the orbicularis oculi, neck and biceps muscles. It is highly unlikely, therefore, that the biceps femoris EMG was contaminated by the startle in experiment 3. Available evidence suggests that exposure to the different levels of innocuous sural nerve stimulation associated with the sural nerve CAP recruitment curve was most likely responsible for eliminating the startle reflex. For example, it has been shown that pre-exposure to gradually increasing stimulus intensities facilitates habituation of the startle response (Davis and Wagner 1969). The artifact rejection procedure used in experiment 3 was most likely responsible for eliminating the pain-related voluntary responses observed in experiments 1 and 2. Thus, the results of this study suggest that the startle response does not significantly contaminate the biceps femoris RI11 reflex under stimulus conditions typically used to record the RIII. However, as noted above, there may be situations encountered with clinical or experimental use of the RI11 not examined here where startle contamination may be significant. The most conservative approach in those situations, therefore, would be to take steps to eliminate the startle (e.g., the repeated innocuous stimulation used in experiment 3) and/or to monitor activity in other muscles, such as the orbicularis oculi, to ensure that changes in the RI11 reflex cannot be attributed to alterations of the startle response.

References Baldissera.

F.. Hulthorn.

neuronal

lllert.

M..

Integration

Brooks (Ed.).

Bromm,

B. and Scharein,

E..

MD,

Response

in spinal

Handbook

ogy. Sect. I. The Nervous System. Vol. 2, Motor

of Physiol-

Control.

Ameri-

1981, pp. 509-5YS.

plasticity

of pain

evoked

reactions in man, Physiol. Behav., 2X (19X2) 109- 116. Bromm.

B. and Trcede.

R.-D..

Withdrawal

reflex.

reaction and pain ratings due to electrical

skin resistance

stimuli in man. Pain, Y

(1080) 33Y-354. C‘han. C.W.Y. correlated

and Dallaire,

M.. Subjective pain sensation is linearly

with

reflex

flexion

in man.

Brain

Res..

47Y (198’))

145~1.50. Craig. K.D..

Emotional

(Eds.). Textbook

aspects of pain. In: P.D. Wall and R. Melzack

of Pain. Churchill

Livingstone,

New York.

19X4.

pp. 153-161. C‘raig, K.D.

and Patrick,

C.J.. Facial expression during induced pain.

J. Pers. Sot. Psychol.. 4X (IYSS) 10X0-IOYI. Davis,

M.. The mammalian

Neural

Mechanisms

York,

of

startle

response.

In: R.C.

Startle

Behavior.

Plenum

Eaton

(Ed.).

Press,

New

lYX4. pp. 2X7-351.

Davis. M. and Wagner. incremental

AR..

sequence

Ilabituation

of stimulus

of startle respome

intensities.

J. Comp.

under Physiol.

Psychol.. 67 (1969) 4X6-492. Dowman.

R..

Supraspinal

and spinal

correlates

of nociception

in

man, Pain, 45 (1991) 269-28. Durkovic.

R.G. and Nerd,

S., Exteroceptive

responses to shocking experiences,

reflexes in man: startling

Sot. Neurosci.

Ahst., I4 (1988)

1303. Gogan.

P.. The

startle

and orienting

their characteristics

reactions

and habituation.

in man. a study of

Brain Res..

IX (lY70)

I l7-

135. Hoffman.

H.S..

startle.

In:

Behavior, Landis.

Methodological R.C.

Eaton

factors in the behavioral

(Ed.).

Neural

Plenum Press, New York.

C. and Hunt,

W.A..

hart, New York,

193’).

Lang, P.J., Bradley,

M.M.

analysis of

Mechanisms

of

Startle

1984, pp. 267-284.

The Startle and Cuthbert.

Pattern, B.N.,

Farrar Emotion.

and Rineattention.

and the startle reflex, Psychol. Rev.. 97 (1990) 377-395. Meinck,

H.-M.,

Kuster.

reflex - influence Electroenceph. Ornitz,

E.M.

S.. Benecke.

R. and Conrad,

of stimulus parameters

Clin. Neurophysiol.,

and Guthrie,

61 (1985)

D.. Long-term

B., The tlexor

on the reflex response, 287-20X.

habituation

and sensitiza-

tion of the acoustic startle response in the normal Psychophysiology.

26 (19X9) 166

J. EEG Price,

Technol.,

D.D.,

by eye movements,

Nociceptive

reflexes

and pain.

In: CR.

in Pain Research

12, Raven Press, New York,

1989, pp. 353-359.

A. and Bussel. B., Long-latency

after flexor reflex afferent

Turville,

S., Startle

stimulation,

responses recorded

Clin. Neurophysiol..

K. and Dowman,

for pain-related P., Ruber,

P. and Klein.

Watkins.

L.R..

concepts

meeting, R., The

Clin. Neurophysiol., Algesiometry and

Loeser (Eds.).

future

in laboratory

249-26.5.

in the leg of man, of reference

Elec-

electrode

latency

1990.

difference

vs. central

sites

in man, Ameri-

St. Louis, MO.

In:

of the

factors.

Elec-

animals and man: current C.R.

in Pain Research

Issues in Pain Measurement.

spinal reflexes in man

Brain, 110 (19X7) 707-725.

65 (1986) 269-275.

directions.

Advances

and Vol.

evoked potentials

tibia1 and sural nerve SEP: peripheral troenceph.

Chapman

and Therapy,

39 (1975) 389-397.

R., Evaluation

somatosensory

can Pain Society, 9th Annual Vogel.

Am.

1 (1967) 27-40.

J.D. Loeser (Eds.), Advances Roby-Brami,

adult human,

173.

Peters. J.F., Surface electric fields generated

troenceph.

I would like to thank Mr. J. Rescigno for aid in data collection, Ms. L. Goshko for aid in data analysis and preparing the figures, and Dr. R.G. Durkovic for providing a number of extremely useful comments and suggestions. This work was supported in part by a grant from the National Institutes of Health (NINDS No. NS-28797).

and

can Physiological Society. Bethesda,

Rossignol.

Acknowledgements

H.

systems. In: V.B.

Chapman and Therapy.

Raven Press, New York,

and Vol.

J.D. 12.

198’). pp.

197 Wilier, J.C., Comparative study of perceived pain and nociceptive flexion reflex in man, Pain, 3 (1977) 69-80. Wilier, J.C., Anticipation of pain-produced stress: electrophysiological study in man, Physiol. Behav., 25 (1980) 49-51. Wilier, J.C., Studies on pain. Effects of morphine on a spinal nociceptive flexion reflex and related pain sensation in man, Brain Res., 331 (1985) 105-114.

Wilier, J.C., Boureau, F. and Albe-Fessard, D., Supraspinal influences on nociceptive flexion reflex and pain sensation in man, Brain Res., 179 (1979) 61-68. Willer, J.C., Roby, A. and Le Bars, D., Psychophysical and electrophysiological approaches to the pain-relieving effects of heterotopic nociceptive stimuli, Brain, 107 (1984) 1095-l 112.