nn-bc27950b-khalsa 1951 iso natnsci suppdatafinal3 ... · and was following them appropriately. ......

The pathways of interoceptive awareness

Khalsa, SS,* Rudrauf, D,* Feinstein, JS, and Tranel, D.

Supplementary Information

* These authors contributed equally to this work.

Nature Neuroscience: doi:10.1038/nn.2411

Participants

The participants were 11 healthy males and patient 1951 (aka ‘Roger’), a rare

neurological patient drawn from our patient registry (see Supplementary Table 1 for

complete demographics). Roger acquired focal bilateral brain damage following a severe

episode of herpes simplex encephalitis in 1980. Briefly, his brain injury involves

extensive bilateral damage to the insular, anterior cingulate and orbitofrontal cortices,

basal forebrain, hippocampus, amygdala and temporal poles (Fig. 1; for detailed

descriptions of Roger’s profile see1,2). All healthy comparison participants were screened

for the presence of any neurological, psychiatric, cardiac or respiratory disease. None of

the participants were smokers, and all participants demonstrated a normal 12 lead

electrocardiogram (EKG), as assessed by a board certified cardiologist or neurologist..

Tasks

Using a previously developed protocol3, participants rated the experience of

heartbeat sensations following bolus intravenous infusions of isoproterenol (a beta

adrenergic agonist similar to adrenaline) and saline. This protocol was adopted after

preliminary testing with Roger on another heartbeat detection task4,5 (modeled after6)

revealed chance heartbeat detection performance within the normal range (Roger’s

average detection rate = 53%; 14 age–matched male healthy comparison’s average

detection rate = 61%, SD 12%). After each bolus, participants turned a dial to track their

ongoing, moment–to–moment experience of the overall intensity of heartbeat sensations.

The dial could range from 0 (“normal, i.e., no change in intensity”) to 10 (“most ever”).

The dial was always set to zero at the beginning of each infusion, and participants were


specifically instructed to keep the dial at zero if they did not notice any increase in the

intensity of heartbeat sensations above baseline levels. Drug administration was

randomized and double blinded with the assistance of a skilled nurse. Participants were

informed ahead of time that they would be receiving both isoproterenol and saline

infusions at some point during the challenge, and were told what the isoproterenol

sensations might feel like (e.g., “you may notice your heart beating harder and faster,

and/or may feel an increase in your breathing sensations”). Each infusion period lasted

approximately 2 minutes. At the onset of each infusion period participants were verbally

notified (e.g., “infusion starting”). All infusions were delivered a minimum of 3.5

minutes apart.

Since Roger has an anterograde memory impairment, his task instructions were

repeated prior to each bolus. During extensive testing sessions in our laboratory, Roger

has demonstrated an intact ability to follow experimental instructions over prolonged

periods of time (on the order of several minutes and longer). The moment–to–moment

nature of the dial rating protocol employed in the current study was designed to alleviate

potential memory confounds associated with retrospective ratings7,8. Moreover, verbal

reports during all infusion periods indicate that Roger understood the task instructions

and was following them appropriately.

All participants received two challenges of isoproterenol and saline infusions.

During the first challenge, participants rated their experience of heartbeat sensations

immediately following bolus infusions of isoproterenol and normal saline. During the

second challenge, participants again rated their experience of heartbeat sensations

following application of a topical lidocaine anesthetic to the body surface where the


heartbeat sensation was felt maximally during the previous challenge (Fig. 3c), and

participants once again rated their experience of heartbeat sensations.

Infusion protocol

The first challenge consisted of 14 infusions: 7 isoproterenol (0.1, 0.25, 0.5, 0.75,

1.0, 2.0 and 4.0 micrograms (mcg)) and 7 saline. These doses were used to establish the

chronotropic dose 25 (CD25), which is the dose necessary to increase the participant’s

heart rate by 25 beats per minute above baseline, a commonly reported measure of beta

adrenergic receptor sensitivity9–13. The CD25 was calculated by extrapolation from the

slope of a linear regression at each individual’s isoproterenol induced heart rate

response9,12–14.

The second challenge consisted of four infusions: 2 isoproterenol (2.0 and 4.0

mcg) and 2 saline. We only selected the two highest doses for the second challenge, as

we have previously found that all participants receiving the 2.0 mcg dose experience

report changes in heartbeat sensations3.

Each infusion (isoproterenol and saline) consisted of two 3 milliliter (ml) bolus

infusions delivered sequentially through an intravenous catheter. During isoproterenol

infusions, a 3 ml bolus containing the specified dose was delivered, immediately

followed by a 3 ml bolus of saline to flush the line. During saline infusions, a 3 ml bolus

of saline was delivered, immediately followed by an additional 3 ml bolus of saline.

Both bolus volumes were administered in entirety within a 15 second period by a nurse.

This method of delivery minimized the participant’s ability to use external cues to


distinguish between the different infusion types, and ensured rapid and standardized

systemic introduction of isoproterenol.

Anesthetic protocol

Sixty grams of 4% lidocaine topical anesthetic cream (L.M.X.4TM, Ferndale

Laboratories, Inc.) were applied to the body region where each participant reported

maximally feeling their heartbeat sensations during the first infusion challenge (Fig. 3c).

Anesthetic administration was single blinded. To ameliorate possible placebo effects, all

participants were informed that the topical cream would either (a) increase skin

sensitivity to heartbeat sensations, (b) decrease skin sensitivity to heartbeat sensations, or

(c) would not change skin sensitivity to heartbeat sensations at all. In order to achieve an

anesthetic effect the topical cream was left in place for a minimum of 30 minutes prior to

the start of the second infusion challenge. To maintain adequate anesthesia, the anesthetic

was left in place throughout the second infusion challenge.

Anesthetic body surface area coverage was equivalent across all participants

(Roger: 1.3%, healthy comparison participants: mean 1.7%, SD 0.7%)15. After the

conclusion of the second infusion set, quantitative pinprick testing was conducted to

determine whether an anesthetic effect had been obtained. Throughout the testing each

participant was instructed to keep their eyes closed. A series of 18 stimulations were then

applied to the participant’s body, 50% in the anesthetized area and 50% within a 10 cm

radius of adjacent (non–anesthetized) skin. Participants were asked to spontaneously

report whether they had experienced a sharp or dull sensation. A report of “dull” in the

anesthetized area was considered correct, whereas a report of “sharp” in the same area


was incorrect. The opposite criteria were applied to the adjacent non–anesthetized skin.

Any participant achieving > 13 out of 18 trials correct or > 72% correct (p < .05 per

binomial test) was considered to have demonstrated a satisfactory anesthetic effect. The

quantitative sensory testing results showed that all participants demonstrated a

satisfactory anesthetic effect according to this criterion (Roger: 83% correct, healthy

comparison participants: mean 83% correct, SD 11%).

Procedure

The study involved one visit, which always started between 7 and 8am in the

General Clinical Research Center at the University of Iowa. After completing the

consent process a nurse measured each participant’s height and weight. The nurse then

placed a 22 gauge intravenous catheter into the participant’s non dominant dorsal hand

vein, and administered a 12 lead EKG. A physician evaluated the EKG, and the

experiment proceeded only if the EKG was considered normal (all participants displayed

normal EKGs). The participant was led to a quiet room, seated in a comfortable chair,

and was attached to leads for measuring heart rate (lead II EKG). At this point the

participant’s non dominant hand was placed outstretched on a pillow at chest level. A

curtain was positioned with the participant on one side and the nurse and the

experimenter on the other side, to prevent the participant from viewing the preparation

and administration of each infusion. The nurse then measured the participant’s blood

pressure and began the infusion protocol. Participants were instructed not to recline in

the chair during each infusion period, in order to prevent them from using the back of the


chair as an external source to help them detect heartbeat sensations. The entire procedure

lasted approximately 5 hours.

Psychophysiological measures

Physiological data including heart rate were recorded continuously during all

infusions with an MP100 acquisition unit (Biopac Systems, Inc). Dial ratings were

collected with a custom built dial that consisted of a rotating potentiometer with a

continuous range of 0.000 to 5.000 Volts. The average heart rate response during each

infusion was calculated across a 120–second interval immediately following the onset of

each infusion. The average heart rate response was obtained by subtracting the average

heart rate during the 30–second post–infusion window (i.e., before isoproterenol–induced

heart rate changes had occurred) from the average heart rate during the subsequent 90–

second window (i.e., when the isoproterenol induced heart rate changes were most likely

to occur). These time windows were carefully chosen to coincide with the typical delays

observed in the onset of isoproterenol–induced heart rate changes (mainly due to the slow

rate of venous drainage to the heart)9,11-14. All artifacts affecting the instantaneous heart

rate waveform (e.g., movement related, or due to premature ventricular contractions)

were manually identified and removed.

Cross correlations for each participant were calculated in Matlab (Mathworks,

Inc.) from mean centered dial ratings and instantaneous heart rate changes occurring over

the 120–second interval following the onset of each infusion. Dial ratings and

instantaneous heart rate changes for each dose were mean centered by subtracting the

120–second mean for each infusion interval from each time point within that interval.


Supplementary Results

See Supplementary Fig. 1a,b for full results of heart rate response and

interoceptive awareness testing of all seven doses (0.1, 0.25, 0.5, 0.75, 1.0, 2.0 and 4.0

mcg) during the first challenge (i.e., without lidocaine anesthetic). All participants

correctly detected increases in heartbeat sensations at the two highest doses (2.0 and 4.0

mcg), as indicated by a positive dial rating during the infusion period (Supplementary

Fig. 1c). Roger correctly detected increases in heartbeat sensations at the four highest

doses (0.75, 1.0, 2.0 and 4.0 mcg), as further indicated by his verbal responses

(Supplementary Table 2). Cross correlation analysis of the time course of subjective

and objective interoceptive changes revealed comparable zero order and maximum cross

correlations between Roger and the comparison participants (Supplementary Fig. 1d).

On average, Roger tended to generate ratings that were somewhat delayed with respect to

the heart rate changes, with dose–dependent ratings that were similar in amplitude to the

comparison participants.

See Supplementary Fig. 2a,b for full results of heart rate response and

interoceptive awareness testing of the two highest doses (2.0 and 4.0 mcg), following

topical anesthetic application. Under this condition, Roger no longer detected any

increase in heartbeat sensations, as indicated by the absence of dial ratings

(Supplementary Fig. 2b,d,e), and as further indicated by his verbal responses

(Supplementary Table 3). On the contrary, healthy comparison participants’ ratings

were unaffected by anesthetic application, although it remains possible that there could

have been subtle reductions in interoceptive awareness that were not picked up by our


measurement. For example, anesthetic seemed to abolish sensation in one healthy

comparison subject at the 2.0 mcg dose (91% of healthy participants reported a change in

sensation), but this was not the case for the 4.0 mcg dose (100% of participants reported a

change in sensation) (Supplementary Fig. 2d).

Supplementary Table 1. Participant demographic data. Means +/– SD. Chronotropic dose 25 (CD25) is the isoproterenol dose necessary to increase the participant’s heart rate by 25 beats per minute above baseline.

Roger Healthy comparison Age (yrs) 55 54 +/– 9.6

Sex Male 11 Males Body Mass Index 29.4 25.8 +/– 5.1

CD25 (mcg) 8.1 7.8 +/– 3.0 Supplementary Table 2. Verbal reports obtained during the non–anesthetized condition, 4.0 mcg dose. This particular healthy comparison participant used a numerical scale when verbalizing his sensation that went from 0 (“normal, i.e., no change in intensity”) to 10 (“most ever”). HR change refers to increase above baseline heart rate at the start of the infusion.

Roger Comparison participant

HR (bpm) HR change Verbal rating HR (bpm) HR change Verbal rating 90

13

“Still pretty low”

80

8

“3 maybe”

103

26

“It’s increasing

some”

86

14

“4…a sense of fullness…from

going up a couple flights

of stairs”

91

14

“Not beating real hard or

fast”

78

6

“2…maybe 1”

87

10

“Now it’s closer to normal”

72

0

0


Supplementary Table 3. Verbal reports obtained during the anesthetized condition, 4.0 mcg dose. This particular healthy comparison participant used a numerical scale when verbalizing his sensation that went from 0 (“normal, i.e., no change in intensity”) to 10 (“most ever”). HR change refers to increase above baseline heart rate at the start of the infusion.

Roger Comparison participant

HR (bpm) HR change Verbal rating HR (bpm) HR change Verbal rating

94

10

“I haven’t noticed hardly

any…any increase in heartbeat

85

2

“1”

107

23

“Not great heartbeat”

97

14

“2. Within my chest, It’s

slightly faster, the beat has

more power to it, akin to a

quick walk or maybe a full

flight of stairs”

109

25

“Pretty quiet, calm”

95

12

[Where in your body are you

feeling it?] “In the chest under the gel area.”

[Are you feeling it in your skin?] “No. Not in the skin, all within

the chest cavity. It’s like a

pulsing within”

105

21

[Would you describe it as

normal?] “Yes”

80

–3

“0”

99

15

“I think it’s pretty normal”


Supplementary Figure 1. Objective and subjective interoceptive changes during isoproterenol infusions. (a) Mean heart rate response to isoproterenol and saline. (b) Mean time course of heart rate response and subjective dial rating. Bolus infusions occurred at time zero. (c) Percent of participants correctly detecting increases in heartbeat intensity. A participant was considered to have correctly detected an increased heartbeat intensity if they turned the dial above zero at any point during the latter 90 seconds of the infusion period, when isoproterenol induced heart rate changes are most likely to occur. (d) Mean zero lag and maximum cross correlations between heart rate response and subjective interoceptive rating as a function of absolute lag time. Error bars = SE.


Supplementary Figure 2. Objective and subjective interoceptive changes during isoproterenol infusions following topical anesthetic application. (a) Mean heart rate response to isoproterenol and saline. (b) Mean time course of heart rate response and subjective dial rating. (c) Overlaps showing area of topical anesthetic application, corresponding to the region of maximal heartbeat sensation. (d) Percent of participants correctly detecting increases in heartbeat intensity. (e) Mean zero lag and maximum cross correlations between heart rate response and subjective interoceptive rating as a function of absolute lag time. All comparison data depict mean values, except (c). Error bars = SE. References 1. Tranel, D., Damasio, H., Damasio, A.R. in Handbook of Neuropsychology (eds.

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isoproterenol infusions provide a reliable method for assessing interoceptive awareness. Int J Psychophys 72, 34–45 (2009)

4. Khalsa, S., Rudrauf, D. & Tranel, D. Interoceptive awareness declines with age. Psychophysiology (in press).

5. Khalsa, S. S. et al. Interoceptive awareness in experienced meditators. Psychophysiology 45, 671–77 (2008).

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9. Cleaveland, C. R., Rangno, R. E. & Shand, D. G. A standardized isoproterenol sensitivity test. The effects of sinus arrhythmia, atropine, and propranolol. Arch Intern Med 130, 47–52 (1972).

10. Martinsson, A., Lindvall, K., Melcher, A. & Hjemdahl, P. Beta-adrenergic receptor responsiveness to isoprenaline in humans: concentration-effect, as compared with dose-effect evaluation and influence of autonomic reflexes. Br J Clin Pharmacol 28, 83–94 (1989).

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