Tracking the Life Course. The Emotional Brain Across the Life Course

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Wisconsin Longitudinal Study: Tracking the Life Course Principal Investigator: Hauser, Robert M.

The Emotional Brain Across the Life Course Project 8 Leader: Davidson, Richard J.

a. Specific Aims

This project will use magnetic resonance imag-

ing (MRI) to obtain high resolution informationabout the morphometry of particular brain regionsimplicated in emotion and emotion regulation and

their levels of functional activation in a highly

stratified biological subsample of 500 high schoolgraduates in the Wisconsin Longitudinal Survey(WLS). The WLS has followed the lives of sam-

ple members from their senior year (1957) to the present with very little attrition, and it has sup-

plemented adolescent socioeconomic and psycho-logical measures with rich longitudinal data oneducation, careers, economic status, family, social

activities, and health.Respondents will undergo functional and struc-

tural MR imaging, along with brain electrical ac-tivity measures. The circuitry that will be fea-

tured will include the amygdala, hippocampusand different territories of the prefrontal cortex

(PFC). Each of these structures has been impli-cated in different aspects of emotion and emotionregulation and is part of the central circuitry that

is likely crucial for understanding how cumula-tive psychosocial burden can have deleterious ef-

fects upon health. For example, the hippocampus plays a crucial role in the regulation of the hypo-

thalamic-pituitary-adrenal axis. The hippocampus is a site that contains a very high density of

glucocorticoid receptors and in animal studies, ithas been found that chronically high levels ofglucocorticoids will produce hippocampal cell

death (see McEwen, 1998 for review). In hu-mans, MRI studies have revealed hippocampal at-

rophy in patients with specific psychiatric disor-ders that involve chronic stress—both depression

and post-traumatic stress disorder. For the for-mer, it has been reported that the cumulativenumber of days depressed is inversely associated

with hippocampal volume (see Sapolosky, 2000,for review). The hippocampus plays an important

role in context-dependent emotional responding(see Davidson, Jackson & Kalin, 2000). An im-

portant consequence of hippocampal dysfunctionis the display of “normal” emotion in inappropri-

ate contexts. The prototypic example of this is in post-traumatic stress disorder where high levelsof fear and anxiety that might be appropriate for

the original traumatic context are displayed re-

peatedly in safe environments. The failure tomodulate emotion in a context-appropriate fash-

ion is likely a consequence of hippocampal dys-function (Davidson et al., 2000). It should benoted that in primates, in contrast to rodents, there

appears to be relatively few glucocorticoid recep-

tors (GR) in the hippocampus (Sanchez et al.,2000) and thus, whatever impact chronic expo-sure to high levels of cortisol might have in the

hippocampus, such effects may not operatethrough GRs. Moreover, Sanchez et al. (2000)

have reported relatively dense GR distributions inseveral neocortical areas including temporal, pre-frontal and anterior cingulate cortices. The vol-

ume and shape of these regions will be extractedin this project through voxel-wise deformation-

based morphometry.In addition to the hippocampus, the amygdala

and prefrontal cortices are other key structures inthe circuitry of emotion regulation and also play

an important role in regulating peripheral biologythat may be consequential for health (see David-son & Irwin, 1999; Davidson, Putnam & Larson,

2000). The amygdala plays an important role inthe detection of cues of threat as well as in the

coordination of the behavioral, autonomic andhormonal responses that accompany responding

to aversive stimuli. The dorsolateral prefrontaland orbitofrontal cortices play crucial roles in dif-

ferent aspects of emotion regulation. These areasof the brain enable us to maintain emotion in theabsence of immediate cues for its elicitation (e.g.,

maintaining positive affect while pursuing distantgoals) and also facilitate the rapid recovery of

negative affect following exposure to a stressfulevent.

For each of the brain regions identified above, both structural and functional abnormalities have been observed. The proposed work will include

both structural and functional assessments of cor-tical and subcortical territories of the brain.

In light of the fact that both age and gender have been found to be critically important variables in

the determination of hippocampal volume,Pruessner et al., (2001) have recently noted that

such findings “underscore the need to include so-ciodemographic variables in functional and ana-tomical MRI designs” (p. 194). This project in-





deed will be one of the first, and certainly, the

most systematic effort of this sort ever undertakenand it will provide a comprehensive assessment of

the association of life-course trajectories, includ-ing gender and socioeconomic status, on the mor-

phometric measures we propose to obtain. With

the functional data we will acquire, the nature of

the dataset will be unparalleled and will enable usfor the first time to examine relations that go fromsocial and demographic factors across the life

course, e.g., cumulative exposure to adversity oradvantage, to brain structure and function.Finally,

we will also obtain physical exam and laboratorymeasures of health status to examine in relation tothe psychosocial and imaging measures.

Aim 1: To assess with structural MRI the volume

and shape of the hippocampus, amygdala and sev-eral territories of the prefrontal cortex in a WLS

subsample. We predict that the volume of theamygdala (De Bellis et al., 2000) will be posi-

tively correlated with anxiety symptoms and otherindices of negative affect. We predict that thevolume of the hippocampus (Sapolsky, 2000) will

be reduced in individuals with a greater cumula-tive exposure to adversity. Moreover, hippocam-

pal volume in particular will be inversely corre-lated with cortisol.

Aim 2: To assess with functional MRI and a spe-

cific emotion regulation task designed to probethe circuitry described above the functional statusof the amygdala, hippocampus and prefrontal cor-

tex. We predict that greater cumulative exposureto adversity will be associated with accentuated

activation of the amygdala following the offset ofa negative stimulus and with inability to voluntar-

ily suppress amygdala activation through con-trolled efforts to attenuate negative emotion. Therequirement to regulate negative emotion will

also be associated with less activation of the cer-tain regions of the prefrontal cortex in subjects

with greater exposure to adversity. In addition,on a task requiring emotional memory, we predict

less activation of the hippocampus in more vul-nerable subjects exposed to adversity.

Aim 3: To examine relations between electrophysiological measures of prefrontal activation

asymmetry and morphometric and functional

brain imaging measures as well as measures ofdispositional affect, well being, exposure to ad-

versity and cortisol.

Aim 4: To obtain data on physical health using

both physical exam and laboratory indices. These

measures will be used to examine relations among psychosocial, neuroimaging and health variables.

b. Background and significance

The results of a number of studies usingdiverse methodologies assessing normal andclinical populations are consistent with the hy-

pothesis that specific anterior cortical regions inthe left hemisphere are relatively more activated

during the experience or expression of certain positive emotions while other cortical regions of

the right hemisphere are relatively more activatedduring the experience or expression of certain

negative emotions (see Davidson & Tomarken,1989; Davidson, 1995; Davidson & Irwin, 1999,for reviews). Following from the analysis by

Schnierla (1959) of the importance of approachand withdrawal over the course of phylogeny and

the speculations by Kinsbourne (1978; see alsoKinsbourne & Bemporad, 1984), we have inter-

preted the findings on asymmetry and affect to re-flect differences in anterior systems mediating

approach and withdrawal (e.g., Davidson et al.,1990; Davidson & Tomarken, 1989; Davidson,1998) with certain regions of the left prefrontal

cortex playing a role in an approach system andother regions of the right anterior cortical zone

playing a role in a withdrawal system. These cor-tical regions are undoubtedly part of larger sys-

tems that collectively constitute approach andwithdrawal systems. I have recently reviewed(Davidson, 1994; Davidson & Sutton, 1995;

Davidson, 1998; Davidson & Irwin, 1999) rele-vant animal and human data and proposed on the

basis of these reviews that the approach systemfacilitates appetitive behavior and generates cer-

tain types of positive affect that are approach-related. This form of positive affect arises in the

context of moving toward a desired goal (seeCarver & Scheier, 1990; Lazarus, 1991; Stein &Trabasso, 1992). The representation of the appeti-





tive goal state is hypothesized to be implemented

in dorsolateral prefrontal cortex (PFC), partic u-larly on the left side. The medial and orbital zones

of the PFC appear to play an important role inmaintaining representations of behavioral-reinforcement contingencies in working memory

(Thorpe et al., 1983). In addition, output from the

medial PFC to the nucleus accumbens, partic u-larly the caudomedial shell region has been impli-cated in the expression of goals in action plans

and in the anticipation of reward (Schultz et al.,1995; see Davidson & Sutton, 1995; Davidson,

1998, for reviews). We have recently found thatsubjects with greater left prefrontal activation alsoshow less activation in the amygdala (Abercrom-

bie et al., 1996). This finding is consistent withanimal data (Morgan et al., 1993). Thus, the pat-

tern of activation associated with positive affectand approach includes both cortical activation and

subcortical inhibition consistent with models thathave emphasized the inhibitory role of prefrontal

cortex in emotion regulation (e.g., Tucker, 1981;Liotti & Tucker, 1995). Note that strong recipro-cal connections exist between the amygdala and

medial prefrontal cortex (Amaral et al., 1992) that provide the requisite anatomical substrate for this

inhibitory relation.The withdrawal system facilitates the with-

drawal of an individual from sources of aversivestimulation and generates certain forms of nega-

tive affect that are withdrawal-related, such asfear and disgust. The right prefrontal cortex acti-vates during such withdrawal-related emotional

states and may be associated with the heightenedvigilance, particularly to threat-related cues, that

is apparent during such emotions (e.g., MacLeodet al., 1986). It appears that the amygdala is criti-

cally involved in this system (LeDoux, 1987;1992). In addition, the anterior temporal cortexalso appears to be activated during withdrawal-

related negative emotion as does the anterior cin-gulate and insular cortex (e.g., Rauch et al. ,1995)

and the hypothalamus (Smith et al., 1990). We published one of the first studies to demonstrate

in humans activation of the amygdala using fMRIin response to aversive pictures (Irwin et al.,

1996). In addition, we performed an FDG-PETstudy using an extended picture presentation

paradigm that we developed and validated (Sutton

et al., 1997) in which we demonstrated reliable

changes in regional glucose metabolism duringobjectively verified (with startle) appetitive and

aversive affect. Appetitive emotion was associ-ated with activation in the left inferior and medial

prefrontal cortex, left nucleus accumbens and left

superior lateral prefrontal cortex and premotor re-

gion. Aversive emotion was associated with acti-vation in the right lateral prefrontal cortex and theright amygdala (the latter was demonstrated with

MR-PET coregistration) (Sutton et al., 1997).Over the past 10 years, my laboratory has been

engaged in a program of research on individualdifferences in electrophysiological measures oftonic asymmetric anterior activation in normal

adults, patients with affective disorders, childrenwho differ in their temperamental style and in

rhesus monkeys (with Kalin). We have estab-lished that individual differences in EEG meas-

ures of prefrontal activation asymmetry are reli-able (Tomarken et al., 1992a). They are both sta-

ble over time and show excellent internal consis-tency reliability. We have now demonstrated thisrepeatedly in adults, children and rhesus monkeys

(see Davidson, 1995, 1998 for reviews). We havealso established the validity of these individual

differences by showing that they predict disposi-tional mood (Tomarken et al., 1992b), reactivity

to experimental emotion elicitors (Wheeler et al.,1993), temperament in children (Davidson, 1992),

temperament in rhesus monkeys (Davidson et al.,1993), immune function (Kang et al., 1991;Davidson et al., 1999) and affective disorders

(Henriques & Davidson, 1990; 1991). We havealso used FDG-PET to examine individual differ-

ences in patterns of regional glucose metabolismin a manner similar to that we have developed for

EEG (Schaefer et al., 2000). Using MRI-coregistration procedures to extract metabolic ratein the amygdala and other discrete subcortical

structures, we have demonstrated that such meas-ures of metabolic rate are stable over time and

that depressed patients with higher levels ofamygdala metabolism report more intense dispo-

sitional negative affect (Abercrombie et al.,1998). We have also begun to mechanistically

characterize the time course of emotional reactiv-ity and examine relations between measures ofthe kinetics of affective responding and individual





differences in both electrophysiological measures

of prefrontal asymmetry and hemodynamic meas-ures of regional prefrontal activation (see David-

son, Putnam & Larson, 2000).Over the past six years we have performed a

number of pilot studies directly relevant to the

major aims of this Project. Findings from these

studies will be briefly reviewed in the next sec-tion. Several general themes will be highlightedhere. We have begun a major collaboration with

the Hauser’s, Carol Ryff and Burt Singer using asmall sample of respondents from the Wisconsin

Longitudinal Study (WLS). For the first time inthe history of this study, which began as a strictlysocio-demographic study, we brought subjects to

campus for laboratory evaluation. We studiedthem in the psychophysiology laboratory in addi-

tion to conducting a physical examination and ob-taining measures that reflect allostatic load and

immunocompetence. The latter measure con-sisted of antibody titers to influenza vaccine.

Among the psychophysiological measures we ob-tained were brain electrical activity and cardio-vascular measures. The electrophysiological and

cardiovascular data were obtained under both baseline and challenge conditions. These meas-

ures were obtained from subjects who were clas-sified as resilient (lives of adversity accompanied

by reports of high levels of psychological well- being) and several comparison groups. The sam-

ple size for the biological data ranged froma p proximately 70 to 100 depending upon themeasure. Our initial analyses focused on

examining specific questions that were basedupon a priori data and/or theory. One of our

major hypotheses concerned relations betweenindividual differences in asymmetric prefrontal

activation and immune function. In earlier workwe found that subjects with greater relative left

prefrontal activation had higher baseline levels of

natural killer cell (NK) function (Kang et al.,1991). In more recent studies (Davidson et al.,

1999) we replicated this earlier finding with alarger sample of subjects and demonstrated that

the relation is present throughout the continuumof prefrontal activation asymmetry, not just in a

comparison of extreme groups. We alsoestablished that subjects with greater relative left-sided prefrontal activation at baseline show a

smaller decrease in NK function in response to

function in response to both a naturally occurring

and an experimentally-induced stressor. Thesedata were based exclusively upon relatively crude

electrophysiological measures and used an in vi-tro measure of immune function. In the WLS pi-lot study, we found consistent relations between

baseline left-sided prefrontal activation and

higher antibody titers in response to influenzavaccine. This finding was present for most of the

prefrontal electrode sites but was not present in

the posterior scalp region, underscoring the speci-ficity of the effect. We also found that subjects

with greater left-sided prefrontal activation at baseline consistently reported fewer symptoms ofdispositional negative affect, depression, anxiety

and perceived stress.Another issue that we have pursued in the WLS

data is the question of emotion regulation (seeDavidson, Putnam & Larson, 2000; Davidson,

Jackson & Kalin, 2000; Jackson et al., 2000a).We have distinguished between automatic and

voluntary forms of emotion regulation thoughthere is little evidence on the relations betweenthese forms of regulation. For example, we do

not know if individuals who are good at voluntaryregulation of negative affect are also adept at

automatic regulation of negative emotion (seeDavidson, Jackson & Kalin, 2000, for discus-

sion). In the WLS pilot study, we presented unpleasant, pleasant and neutral pictures during

which we exposed subjects to startle probes thatoccurred either during picture presentation or 1.5seconds following picture presentation. The latter

probe time was used to capture automatic regula-tion. Subjects who recover quickly from negative

challenges should show decreased startle magni-tude during this post picture period while those

less facile in recovering from negative challengeshould show greater startle magnitude at thistime. In these analyses, we use the probe during

the picture presentation as the first predictor in aregression equation to equate for differences in

reactivity to the emotional stimulus itself. Wethen examined, in a second step in the regression

model, the amount of variance accounted for inthe post-picture startle response by electrophysio-

logical measures of asymmetric prefrontal activa-tion. In this analysis, we found that the prefrontalasymmetry indices accounted for a large percent-





age of the variance (65%) in the magnitude of the

post-negative picture startle response after thevariance accounted for by the startle magnitude

during the picture was removed. The direction ofthe effect was that subjects with greater left-sided

prefrontal activation showed a diminished startle

magnitude during the post-picture period suggest-

ing that these individuals were better able to regu-late their negative affect and return to baselinemore quickly. A specific example from these

data will be presented below in the PreliminaryStudies section.

In another task we administered to the WLS re-spondents, we had them write about the most ex-treme positive and most extreme negative experi-

ences in their lives. In the three-minute period af-ter a five-minute writing epoch, subjects were in-

structed to sit quietly and think about the materialthey had just written. We collected both brain

electrical activity measures and startle measuresduring this period. Those subjects who showed

the most extreme negative affect as indexed bythe highest magnitude startle following the nega-tive compared with the positive writing period

had the smallest rise in antibody titers followingvaccine (r=.42).

What we were not able to examine in the WLSdata was the detailed neurocircuitry of emotion

and affective style since we did not collect neuro-imaging data and relied solely on measures of

brain electrical activity to make inferences aboutregional brain function. These measures of brainelectrical activity are only sensitive to cortical

function and thus, activity in subcortical regionscritically important for emotion was not assessed.

Moreover, we did not obtain any morphometricdata in the WLS data collection to date. Recent

findings have suggested that changes in the vol-ume of the hippocampus, amygdala and selectneocortical areas may be associated with chronic

exposure to stress (Sapolsky, 2000), and the WLSdata will provide ample evidence of such expo-

sure across the life course. Moreover, the oppor-tunity to relate such measures to both fMRI

measures of regional brain activation in responseto emotional challenges and also to measures of

cortisol that have been hypothesized to play acausal role in the production of tissue atrophy isunprecedented. In short, we will collect a large

corpus of imaging data as well as objective in-

formation on health status on subjects for whomextensive history of sociodemographic informa-

tion is available.

c. Preliminary Studies

In this section, I first review data from my labora-tory using electrophysiological measures of acti-vation that provided the early evidence on the dif-

ferential role of the left and right prefrontal cortexin various aspects of emotion and affective style.

I then turn to neuroimaging data.

A. Electrophysiological measures of anterior

asymmetry as a trait-like index: Psychometricevidence: A critical initial question that required

an answer was the extent to which electrophysio-logical measures of activation asymmetries in an-

terior scalp regions were stable over time and ex-hibited other psychometric characteristics desir-

able for a trait-like index. Investigators who use physiological measures as dependent variablesrarely examine the psychometric characteristics of

these measures, yet if they are to be used in indi-vidual differences and psychopathology research,

it is imperative to examine them in this way. Ac-cordingly, we (Tomarken, Davidson, Wheeler &

Kinney, 1992) performed the first psychometricevaluation of this kind for electrophysiological

measures of frontal and anterior temporal alpha power asymmetry measures.

We focused on alpha power as a dependent

measure for several reasons. First, some electro- physiologists have argued that in the waking

adult, power in the alpha band (8-13 Hz) is in-versely related to activation (e.g., Shagass, 1972;

Lindsely & Wicke, 1974). Second, we have sys-tematically examined power in other bands inmany published articles (e.g., Davidson et al.,

1990a, b; Davidson et al., 1995). We have repeat-edly found that individual differences in alpha

power asymmetry are more consistently related totheoretically-predicted psychological measures

than asymmetry measures derived from other bands. Third, in experiments where we have ma-

nipulated either cognitive or affective task vari-ables to induce a change in asymmetry, we haverepeatedly found that the most consistent task-





dependent changes are found in the alpha band,

with decreased alpha power observed in the hemi-sphere hypothesized to be most activated by the

task and/or increased alpha power in the oppositehemisphere (see Davidson et al., 1990 for a de-tailed consideration of this issue). It should be

noted that we still examine power across the en-

tire spectrum to ascertain whether asymmetries inother frequency bands also account for variancein mood or other emotion-related variables. We

have specifically been interested in evaluating power in the gamma band (centered around 40

Hz) to evaluate claims that power in this band is adirect measure of activation (Spydell & Sheer,1982; Miltner et al., 1999), in contrast to alpha

power which is indirectly related to activation.Although other studies had examined the stabil-

ity of spectral power per se (e.g., Fein et al., 1984;Gasser et al., 1985), it was critical to examine

asymmetry of power since a determinant of abso-lute power is skull thickness (e.g., Leissner et al.,

1970), which will be largely responsible for hightest-retest reliability of power. However, meas-ures of asymmetry are not particularly influenced

by skull thickness, since substantial asymmetriesin skull thickness are not present (see discussion

in Tomarken et al., 1992b). 90 right-handed subjects were tested on two occasions separated by

approximately three weeks. EEG was recordedfrom left and right mid-frontal and anterior tem-

poral scalp sites referenced to both Cz and computer-derived ears [for the derivation of an earsreference, we now always use a computer-derived

average, rather than physical linking since slightimpedance differences between the ears can cause

substantial variations in measured asymmetry.This problem does not arise if each ear is sepa-

rately recorded to a common reference, since thehigh input impedance of the amplifiers effectivelyeliminates the impact of any variation within the

normal range of electrode impedance]. At each ofthe two sessions, 8 one-minute trials of resting

brain activity were acquired. These one minutetrials were divided into 4 eyes-open and 4 eyes-

closed trials that were presented in counterbal-anced order. A fast Fourier transform was per-

formed on 2 second chunks of artifact-free EEGand extracted with overlapping Hamming win-dows (see Tomarken et al., 1992b in Appendix

for additional details). Power in the delta (1-4

Hz), theta (4-7 Hz), alpha (8-13 Hz), beta 1 (13-20 Hz) and beta 2 (20-30 Hz) bands was extracted

and converted to power density (µV²/Hz). Powervalues were log-transformed to normalize theirdistribution. For the purpose of analyses assessing

the stability of EEG asymmetry, weighted means

across each of the eight baselines within a sessionwere computed. Asymmetry values were obtained

by subtracting the power density in the left hemi-

sphere electrode from the power density in thehomologous right hemisphere lead. We pooled

over eyes-open and eyes-closed measures becausethe correlation of asymmetry between eyes-openand eyes-closed trials was uniformly high and the

aggregated measure led to greater stability compared with separate eyes-open or eyes-closed

measures for most bands. In the article presentingthese data (Tomarken et al., 1992a), we also pre-

sent the results separately by eyes condition forarchival purposes.

The findings of most importance from this studywere that measures of frontal (F3/F4) and anteriortemporal (T3/T4) alpha power asymmetry were

stable over time. The intraclass correlation for thefrontal sites was .66 and for the anterior temporal

sites it was .72. We also computed coefficient alpha, a measure of internal consistency reliability

within each session across the 8 baseline trials, aswell as across the 2 sessions (16 trials). The inter-

nal consistency reliability for the midfrontal re-gion (across the 16 baseline trials) was .90 and forthe anterior temporal region it was .94. These

findings were the first to show that EEG measuresof anterior asymmetry were psychometrically re-

liable. We focused on alpha power because of awealth of data that indicate an inverse relation be-

tween alpha power and activation in the awake, behaving adult (see Pivik et al., 1993 for a generalreview; Davidson, Chapman et al., 1990 for an

empirical demonstration; Michel et al., 1999 foranother empirical demonstration and extension to

magnetoencephalography; and Oakes et al., 2001,from my lab where we developed a method to ex-

amine on a voxel-wise basis the relation betweenhemodynamic imaging methods and the intensity

of brain electrical sources located in a coregis-tered brain volume).





Since this first study, we have had the opportu-

nity to examine the test-retest stability of EEGmeasures of activation asymmetry from multiple

scalp sites in a much larger sample of subjects.We pooled the data across several cohorts testedover the past three years where the interval be-

tween assessments was six weeks. Subjects were

tested at the same time of day on each testing oc-casion. We tested a total of 175 subjects (N=88females), all of whom were right-handed. The

procedure for the assessment of baseline EEGwas identical to that described for the initial study

above. EEG was recorded from 29 scalp sites(FP1/2, AF3/4, F3/4, F7/8, FC3/4, FT7/8, T3/4,T5/6, C3/4, CP3/4. CP5/6, P3/4, PO3/4, FZ, PZ

and Cz) and re-derived off-line to an average ref-erence and an derived-ears reference. For the av-

erage ears reference, the mean ICC for alpha power (8-13 Hz) asymmetry scores across site

was .53. For the average reference, the mean ICCfor asymmetry scores across site was .66. The

correlations for mid-frontal asymmetry (F3/4) are.55 for the AA reference and .74 for the averagereference; for anterior temporal asymmetry the

same correlations were .54 and .70 (all p’s<.0001). These values are generally consistent

with the effects we previously reported using asmaller sample size, only females and a shorter

interval between test occasions. We had the opportunity to examine longer-term stability by

computing the ICC’s based upon the means ofAssessment 1 and 2 (held 6 weeks apart) and thesession during which the startle task was pre-

sented (see below), which took place an averageof 273 days following the second assessment

(N=55 for these analyses). The mean ICC forasymmetry scores across region for the average

ears reference was .58. The ICC for the mid-frontal asymmetry score was .62 and the ICC forthe anterior temporal asymmetry score was .61.

These data indicate that when aggregation can be performed, good test-retest stability over a rela-

tively long period is observed. Comparisons ofstability estimates separately for males (N=87)

and females (N=88) revealed no gender differ-ences. There were also no significant gender dif-

ferences in measures of asymmetry from any ofthe anterior scalp regions, though males did have

slightly greater relative left-sided activation (e.g.,

for F8-F7, M for males=.034; M for females=.01).Tomarken (who was a post-doc in my lab at the

time the first set of studies in my lab on this topicwere completed and who is now at Vanderbilt)showed that if multiple assessments are obtained

in each year (i.e., 2 or 3 assessments), the test-

retest reliability over a one year period is veryhigh (>.80; Tomarken et al., 1994).

Another important parameter of reliability is in-

ternal consistency reliability that is measured bycoefficient alpha. By extracting measures of each

of the 8 one-minute baselines at each session wecan compute coefficient alpha, which reflects theextent to which each individual baseline trial is

representative of the aggregate index across trials.The mean coefficient alpha across site based upon

8 trials was .90 for the ears reference (range=.86to .94) and .88 for the average reference

(range=.77 to .94). These data indicate that theseelectrophysiological measures of asymmetry have

excellent internal consistency reliability.We also examined the test-retest stability of

residualized alpha power measures using intra-

class correlations. As we have explained in detailelsewhere (Wheeler et al., 1993; Pivik et al.,

1993; Davidson, Jackson & Larson, 2000), if onewishes to examine power at a single individual

site (rather than compute an asymmetry score) itis necessary to residualize alpha power by whole

head power since a major contributor to overalldifferences in alpha power across individuals isskull thickness. Residualizing in this way effec-

tively eliminates skull thickness contributions.The sample size was again the 175 subject data

set described above. We found that the intraclasscorrelations for this measure were excellent, rang-

ing from .70 to .89, with a mean across site andreference of .83.

B. Relation of EEG asymmetry to dispositionalmood and reactivity to emotion elicitors: Having

established that measures of anterior brain electri-cal asymmetries were stable over time and exhib-

ited excellent internal consistency reliability, wewere now in a position to examine how such

measures predicted features of emotional reactiv-ity and how they were associated with affectiveand anxiety disorders. One of the first questions





we asked was whether subjects selected on the

basis of stable and extreme electrophysiologicalasymmetry differed in ratings of dispositional

mood (Tomarken, Davidson, Wheeler & Doss,1992a). We administered the Positive and Nega-tive Affect Scale (Watson et al., 1988), a factor-

analytically-derived and relatively pure measure

of dispositional positive and negative affect. Sub- jects simply rate the degree to which a series ofadjectives characterizes how they generally feel.

Examples of the positive adjectives include inter-ested, strong, enthusiastic, proud and alert; nega-

tive adjectives include distressed, upset, nervous, jittery and afraid. We examined whether thosesubjects in the top and bottom 25% of the asym-

metry score distribution on both assessment occa-sions differed in their ratings of dispositional

positive and negative affect. We found that subjects in the left frontal group (i.e., those with less

alpha on the left side and more alpha power onthe right side) reported significantly more positive

and less negative affect than their right frontally-activated counterparts. A similar pattern was ob-served for subjects classified on the basis of sta-

ble and extreme anterior temporal activationasymmetry. When we examined the data correla-

tionally using the entire range of the distributionon asymmetry scores, we generally found that

electrophysiological measures were significant predictors of PANAS measures for those subjects

who exhibited stable asymmetry across time. Thefindings from this study indicated that classifyingsubjects exclusively on the basis of electrophysio-

logical measures of anterior asymmetry, we could predict their self-reported dispositional affect.

Our findings with the PANAS have been inde- pendently replicated by Jacobs and Snyder

(1996).More recently, we (Sutton & Davidson, 1997)

have administered several additional self-report

instruments that we predicted should be associ-ated with anterior activation asymmetry. In one

study (N=57), we administered the scales thatwere designed to assess individual differences in

Gray’s (1994) Behavioral Activation (BAS) andBehavioral Inhibition (BIS) systems (Carver &

White, 1994). Examples of items from the BASscale include: “When I’m doing well at some-thing, I love to keep at it” and “When I want

something, I usually go all out to get it.” Items

from the BIS scale include: “Criticism or scold-ing hurts me quite a bit” and “I worry about mak-

ing mistakes.” Items are answered on a 1 to 4scale that ranges from “very true for me” to “veryfalse for me.” The BAS scale consists of three

separate sub-scales--Reward Sensitivity, Drive

and Fun-Seeking. Carver and White (1994) havedemonstrated good reliability for these sub-scales.Using the derived-ears reference and our standard

alpha power band (8-13 Hz), we found that subjects with greater relative left-sided prefrontal ac-

tivation in both mid-frontal (F3/4) and lateralfrontal (F7/8) regions had higher scores on theoverall BAS (for F3/4, r=.28, p=.07; for F7/8,

r=.35, p<.01) as well as on the Drive subscale in particular (for F3/4, r=.28, p=.03; for F7/8, r=.38,

p<.005). Subjects with greater relative right-sided prefrontal activation reported more behavioral in-

hibition (for F3/4, r=-.41, p<.002; for F7/8, r=-.47, p<.0005). We also computed a within subject

difference score that reflected the relative strengthof the BAS over the BIS by standardizing thescores on these scales and then taking a difference

score within subjects. Subjects with greater rela-tive left-sided prefrontal activation had higher

BAS relative to BIS scores (for F3/4, r=.43, p<.001; for F7/8, r=.53, p<.0001). Relations be-

tween activation asymmetry and scores on thisindex are specific to anterior scalp regions. Meas-

ures derived on the basis of the average referencedata showed the same pattern of effects, thoughwere less robust. Analyses based upon residual-

ized power measures at separate left and righthemisphere scalp sites indicates that the effects

described above are indeed due to a relative dif-ference in activation between the hemispheres

since the correlations with asymmetry scores areconsistently much higher than the correlationswith individual hemisphere residualized power

values. In most cases, the latter indices by them-selves were not significantly related to the BAS

and BIS measures, while the asymmetry score didsignificantly predict these scores. Thus, contrary

to what might have been expected, BAS scoreswere not related primarily to activation differ-

ences in the left prefrontal region and BIS scoreswere not related primarily to activation differ-ences in the right prefrontal region. Scores on





these scales were best predicted by the asymmetry

metric, implying that the balance of activation inthese systems is the most important determinent

of at least self-reports of these behavioral tenden-cies.

We next wished to examine whether such meas-

ures of asymmetry might predict reactivity to

standardized emotion elicitors. In three studies(reported in two articles: Tomarken, Davidson &Henriques, 1990; Wheeler, Davidson &

Tomarken, 1993) we obtained support for the hypothesis that baseline measures of prefrontal

asymmetry predict reactivity to emotion elicitors.In particular, those subjects with greater baselineright-sided prefrontal activation reported more in-

tense negative affect in response to the negativeemotional film clips while those with greater

baseline left-sided prefrontal activation reportedmore intense positive affect in response to posi-

tive film clips (see Wheeler et al., 1993 in Ap- pendix). An aggregate (across two sessions)

measure of prefrontal asymmetry correlated .45with an index of positive affect and -.49 withnegative affect. We also computed a measure of

generalized affective reactivity that representedthe sum of positive affect in response to the posi-

tive film clips and negative affect in response tothe negative film clips. Frontal asymmetry was

unrelated to this measure of generalized affectivereactivity (r=-.01). In this study, we also obtained

baseline mood ratings at the time the EEG as-sessment was obtained. The baseline measures offrontal asymmetry were unrelated to subjects cur-

rent mood, but did predict their reactivity to theemotional film clips, even when baseline mood

was statistically partialled in a hierarchical regres-sion. In the published article that presents these

data, we examine relations between reactivitymeasures and residualized power values at indi-vidual left and right hemisphere sites, as noted

above.Collectively, the studies reviewed above on

baseline asymmetries, mood, and emotional reac-tivity in normals indicate that baseline measures

of anterior asymmetry predict self-reports of dispositional mood but are unrelated to the current

mood or emotion that a subject reports at the timeof EEG assessment (perhaps because of the low

base rates and consequently low variability of re-

portable emotion while sitting and resting). The

electrophysiological measures do predict reactiv-ity to emotion elicitors. In particular, those sub-

jects with greater left-sided frontal activation(both absolute and relative; this issue was specifi-cally examined in the Wheeler et al. study) report

more intense positive affect to positive elicitors

while subjects with greater right-sided anterioractivation report more intense negative affect inresponse to negative elicitors. These findings

support the diathesis/stress conception of individ-ual differences in prefrontal asymmetry that I

have advanced (see in particular, Davidson, 1993and Davidson, 1998 where this position is explic-itly articulated). On this view, individual differ-

ences in anterior activation act as diatheses thatalter an individual’s vulnerability to positive and

negative elicitors, provided the requisite elic itor is presented.

C. Anterior activation asymmetry and immune

function: A growing corpus of evidence suggeststhat certain parameters of immune function areresponsive to psychological events that elicit

emotion. In particular, it is by now a common ob-servation that stress, such as final exams, be-

reavement and divorce, can cause a decrease inthe cellular immune response (see review by Kie-

colt-Glaser & Glaser, 1991). These are all eventsthat have a strong affective component. A notable

fact about these studies is the pronounced indi-vidual variability in both baseline measures aswell as in the magnitude of change of the immune

measures. In light of the observation that individ-ual differences in anterior asymmetry predict im-

portant features of affective responsivity, we wereinterested in examining the extent to which base-

line measures of anterior asymmetry might ac-count for some of the variability across individu-als in immune function.

A second body of literature also points toward possible relations between asymmetric brain func-

tion and immunity. The pioneering work ofGeschwind argued for an association between

hemispheric specialization as reflected in handed-ness and autoimmune disorders (Geschwind &

Galaburda, 1985). Handedness, and hemisphericspecialization more generally, capture only asmall component of the variance across individu-





als in hemispheric function. A number of investi-

gators (e.g., Levy, 1983) have highlighted thedistinction between hemispheric specialization

and hemispheric activation and have noted thatvariations in the latter are superimposed upon theformer. More direct evidence for the existence of

a connection between cerebral asymmetry and

immune function has been provided by animalstudies in which the effects of unilateral corticallesions on immune function were evaluated (Ba-

rneoud et al., 1987; Neveu, 1988; Neveu et al.,1986; Renoux et al., 1983). Renoux et al. (1983)

and Neveu et al. (1986) showed that ablation ofthe left fronto-parietal cortex of mice, whichwould result in a pattern of relative right-sided ac-

tivation, decreased immune responses, whereascomparable lesions of the right cortex either had

no effect or increased immune responses.By comparing subjects with extreme and stable

patterns of left and right-sided prefrontal activa-tion, we could examine whether naturally occur-

ring (in contrast to lesion-induced) asymmetriesaccounted for variance across individuals in base-line measures of immune function (see Kang,

Davidson et al., 1991 in Appendix). We selected20 subjects from a cohort that was tested on two

occasions on EEG measures. 10 subjects showedextreme and stable left-frontal activation and 10

showed extreme and stable right-frontal activa-tion. These subjects were brought to the labora-

tory where blood samples were taken and severalself-report measures were administered. The ex-

perimenters and lab techs were all blind to group

status. We examined natural killer (NK) cell ac-tivity, lymphocyte proliferation to mitogen stimu-

lation (concanavalin A (Con A), phytohemmag-glutinin (PHA) and pokeweed (PWM)), with each

mitogen presented at three different concentra-tions. In addition, the helper/suppressor T-cell ra-tio was determined and plasma cortisol was also

obtained. Our results indicated that the right fron-tal subjects had significantly lower NK activity

compared with their left-frontal counterparts. Thisdifference was apparent at the two lower effec-

tor:target cell ratios. No group differences inlymphocyte proliferation or in T-cell subsets were

found. In addition, no difference in plasma corti-sol was found, nor was cortisol correlated withany of the immune measures. Self-report meas-

ures of trait anxiety and depression did not differ-

entiate between groups nor did these measurescorrelate with immune function. This was the first

study in normal humans to demonstrate a relation between a parameter of immune function and in-dividual differences in asymmetric hemispheric

activation.

More recently, we attempted to conceptuallyreplicate our finding of NK differences betweensubjects who differ on electrophysiological meas-

ures of frontal asymmetry. Rather than select ex-treme groups, we wished to determine whether

individual differences in prefrontal asymmetrywere associated with immune function in a groupof unselected subjects. In this study, we (David-

son et al., 1999) assessed baseline measures of brain electrical activity on two occasions sepa-

rated by 6 weeks in our standard paradigm in 24subjects. In a third session, we brought subjects

back to the laboratory for a blood sample, fromwhich measures of NK activity were obtained.

We found that the aggregate measure of frontalasymmetry from the initial two sessions was sig-nificantly correlated with NK activity at the two

effector:target cell ratios that were associated withfrontal asymmetry in our first study (for 11:1,

r=.46, p=.02; for 33:1, r=.51, p=.01). This indi-cates that subjects with lower asymmetry scores

(more relative right-sided frontal activation) hadlower levels of NK activity, thus replicating our

initial finding on an unselected group.In this more recent study, in addition to examin-

ing relations between asymmetry and baseline

NK activity, we were also interested in whetherour asymmetry measures would predict change in

NK activity in response to negative elicitors. We predicted that subjects with more right-sided ante-

rior activation would show a larger decrease in NK activity to the negative event. We studied thisquestion in two ways. The first strategy involved

the use of academic stress as a naturally occurringnegative event. We obtained blood samples from

subjects at a point in the semester during whichno exams were being taken and at a second point

24 hours prior to the subject’s most important fi-nal exam (based upon their own report). We

found a large and significant decrease in NK ac-tivity during the final exam period compared tothe earlier time point (p<.02 for both 33:1 and





100:1 ratios), replicating Kiecolt-Glaser et al.

(1984; Glaser et al., 1986). Most critical to ourhypothesis was whether the baseline measures of

prefrontal activation asymmetry predicted thechange in NK activity from the first to the secondassessment. To answer this question we focused

on the 100:1 effector:target cell ratio since the

greatest variability was observed at this ratio. Wecomputed a hierarchical regression where thevariable to be predicted was NK activity at the fi-

nal exam period. The first step in the model was NK activity at the pretest (i.e., the mid-semester

blood draw at a time of little academic stress).The second step in the model was the frontal EEGasymmetry variable. We found that the pretest

NK measure accounted for a non-significant 6%of the variance in the final exam NK measure

(F(1,22)=1.40, p=n.s.). The frontal asymmetryvariable entered as Step 2 in the model accounted

for an additional 21% of the variance beyond thataccounted for by the pretest NK measure

(F(1,22)=6.06, p=.02). The sign of the betaweight indicated that subjects with greater rela-tive right-sided prefrontal activation had a larger

decline in NK function at the final exam periodcompared to the baseline period. Our single

measure of frontal asymmetry accounted for 21%of the unique variance in the decline in NK func-

tion from the baseline to the final exam period.Since many aspects of a student’s life change

during final exam period (e.g., diet, sleep), wealso wished to obtain a more well-controlledmeasure of this relation. In addition, we were in-

terested in assessing possible immune changes inresponse to both negative and positive challenges.

Accordingly, we brought subjects back to thelaboratory later in the year during a period they

judged to be relatively non-stressful and exposedthem to two 30 minute film clips. One was de-signed to elicit sadness. The clip, from the movie

“Beaches”, depicted a mother dying of cardio-myopathy and her interactions with her 10 year-

old child. More than one third of the subjectscried in response to this clip. The happy clip was

a medley of segments from the “Lady and theTramp”, the Olympics and “Parenthood.” Clips

were selected based upon normative ratings from144 subjects. The order of clips was randomizedacross subjects. Blood samples were obtained

prior to film exposure (baseline) and then after

each clip. Overall, there was no significant pre-to- post film change in NK activity. However, we

found that the magnitude of change in response tothe film clips was significantly predicted by ourmeasures of anterior asymmetry. We examined

the data in a manner identical to that described

above for the final exam analyses. Hierarchicalregressions were computed where the variable to

be predicted was the NK activity following the

film clip. Step one in the model was always the NK activity at baseline and Step two was the EEG

asymmetry measure. In response to the happyfilm clip, we found that subjects with greater rela-tive left-sided prefrontal activation (F4-F3) had

significantly higher NK activity at the 100:1 ratioafter removing the variance associated with base-

line NK activity (F(1,19)=4.19, p=.05), thoughthis effect accounted for only 3.5% of the vari-

ance. Prefrontal asymmetry from the F8-F7 leadssignificantly predicted NK activity following the

happy film clip at the 11:1 and 33:1 ratios(F(1,19)=6.07, p=.02; F(1,19)=4.27, p=.05, re-spectively), accounting for 6.2% and 5.2% of the

variance respectively, after removing the varianceaccounted for by baseline NK activity. A mar-

ginal effect was found at the 100:1 ratio(F(1,19)=3.90, p=.06). These findings indicate

that subjects with greater relative left-sided acti-vation show higher levels of NK activity follow-

ing the happy film clip after the variance in the pre-film baseline NK is removed. Similar effectswere found for the anterior temporal region,

though they failed to reach significance. In re-sponse to the negative film clips, we found that

subjects with greater relative right-sided anteriortemporal activation had less NK activity (for the

33:1 ratio) after the negative film clip, followingremoval of the variance accounted for by prefilm

baseline NK activity (F(1,19)=4.38, p=.05). Simi-

lar though marginally significant effects werefound for other effector:target ratios and with the

prefrontal scalp sites. Collectively, these data in-dicate that although main effects for the 30 min.

film clips on NK activity are not present, some ofthe variability in NK response to the film clips is

significantly predicted by individual differencesin anterior activition asymmetry, after removingthe variance accounted for by baseline NK activ-





ity. Subjects with greater relative left-sided ante-

rior activation show a larger increase in NK activ-ity in response to the positive film clip, while

those with greater right-sided anterior temporalactivation show a larger decrease in NK activityin response to the negative film clip.

D. Preliminary data from the Wisconsin Longitu-dinal Study: As noted in the Background andSignificance section above, we have been col-

laborating with Hauser, Ryff and Singer on thelaboratory component of the WLS study for the

past four years. This effort has involved the

testing of approximately 120 respondents fromthe WLS sample (though not all were available

for each measure). These individuals came tocampus for a 1.5 day visit during which they

partic ipated in an extensive protocol in my labo-ratory that included recording of brain electrical

activity, cardiovascular activity and startle. Pre-liminary data from this study were presentedabove in the Background and Significance sec-

tion. What we wish to emphasize here is the co-herent network of associations that we observed

when we examined relations among prefrontal ac-

tivation, startle, immune function and self-report

measures of well-being, distress, depression and perceived stress. We found systematic relations between left prefrontal activation and various

well-being subscales including Self-Acceptance,Purpose in Life, and Positive Relations with oth-

ers. This is illustrated in Figure 1.

In general, higher levels of distress and depres-

sion were associated with lower antibody titerlevels to influenza vaccine, as was increased

right-sided activation and greater startle magni-tude following the negative compared with the

positive writing period. Figure 2 presents data

from this study showing relations between startle

magnitude and immune function. These data in-dicate that subjects with larger magnitude startleresponses following the negative compared with

the positive writing period show lower levels ofantibody rise (r=-.50).

We also presented standardized positive, nega-tive and neutral pictures in this study and examinedthe magnitude of startle to acoustic probes that

were presented both during the stimulus and 1.5 secfollowing the stimulus, the latter of which was used

to assess recovery. We found that subjects withgreater left-sided prefrontal activation showed a

greater diminution of startle magnitude in the inter-

val after the negative picture. Figure 3 below illus-trates this finding. We have not yet collected anyimaging data in this study and so conclusions about

the functional neuroanatomical bases of these ef-fects remains speculative. This is something we

intend to pursue over the next two years with the

fresh WLS sample proposed here.

E. Functional magnetic resonance imaging of thehuman amygdala and prefrontal cortices in re-

sponse to affective stimuli: Valence effects andindividual differences: We have conducted sev

eral studies over the past five years to interrogate

Baseline asymmetry (FC3/4) and

self-acceptance

Well-being: Self-acceptance

9080706050403020

M e a n a s y m m

e t r y ( + = g r e a t e r l e f t a c t i v e )

.6

.4

.2

-.0

-.2

-.4

-.6

r =.352

p <.001

n = 97

Figure 1: Relations between baseline prefrontal asymmetry

and Self-Acceptance. Higher numbers on the ordinate denote

reater relative left-sided refrontal activation

Startle Response during Negative - Positive

Thinking Task and Immune Response

-2

-1.5

-1

-0.5

0

0.5

1

1.5

-4 -2 0 2 4 6 8Log 2 transformed antibody titer fold-rise

r = - .501

p = .0056

n = 29

Figure 2: Higher numbers on the ordinate denote greater relative st r-

tle magnitude during the negative versus positive thinking periods.

Abscissa reflects antibody rise to influenza vaccine





the functioning of the human amygdala with fMRI.As noted above, the amygdala is a key site in thecircuitry underlying emotion and both functional

and structural differences have been associatedwith psychopathology and stress. We published

one of the first studies to document the capacity offMRI to detect signal in the amygdala in response

to negative emotional stimuli (Irwin et al., 1996).

For this study, we used unpleasant, pleasant andneutral pictures and found that the amygdala wassignificantly activated in response to negative ver-sus neutral stimuli. We did not find this pattern in

response to positive versus neutral stimuli.More recently, we sought to examine relations

between individual differences in the magnitude ofamygdala activation and dispositional negative and

positive affect. In this study (Irwin et al., 2001) 14subjects were presented with unpleasant and neu-tral pictures in a block design. We quantified the

magnitude of MR signal change for each subject in

the amygdala in response to the unpleasant versusneutral pictures. We describe our imaging parame-ters and analytic strategy in detail below since our

proposed research will use a similar though notidentical sequence since our proposed work will be

conducted at 3T. P

The image acquisition protocol consisted of 10scans, the details of which are provided only forthose scans relevant to the description below: 1)

an axial 3D spoiled gradient-recalled echo scan[SPGR; echo time (TE)/repetition time (TR) =

8/35 ms, field of view (FOV) = 24 x 24 cm, flip

angle (α) = 30º, number of excitations (NEX) = 1,

matrix 256 x 128, reconstructed to 256 x 256, 124

slices, slice thickness = 0.9 - 1.2 mm, scan time =9’37”] graphically prescribed to cover the entire

brain volume; 2) a coronal 3D SPGR scan

(TE/TR = 10/35 ms, α = 30º, NEX = 1, FOV = 24x 24 cm, matrix = 256 x 128, reconstructed to 256

x 256, 28 slices, slice thickness = 1.0 mm, scantime = 2’27”) covering a 28 mm region beginningat approximately the middle of the pons, posteri-

orly, which provided the image data for localiza-tion of the amygdalae; 3) a coronal T1-weighted

spin-echo scan (TE/TR = 20/500, α = 90º, NEX =1, FOV = 24 x 24 cm, matrix = 256 x 128,

reconstructed to 256 x 256, 23 slices, slicethickness = 7 mm, interslice spacing = 1 mm,

scan time = 2’24”) which provided the slice loca-tions from which functional image data would be

acquired. This scan was manually prescribed suchthat one slice was centered on the amygdalae.This was defined such that the posterior edge of

this slice was positioned 1 mm anterior to the lo-

Frontal Pole EEG Asymmetry and Late Startle Reactivity Following Picture Off set

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

-1.5 -1 -0.5 0 0.5 1 1.5 2 2.5

Late Startle Reactivity (Negative - Neutral)

r = - . 41

p < .0 3

n = 3 2

Figure 3: Higher numbers on the ordinate reflect greater relative left-sided prefrontal activation. Positive numbers on the abscissa

denote greater startle magnitude 1.5 s following the offset of a negative versus neutral picture





was positioned 1 mm anterior to the location

where the hippocampus could first be identified inthe image data acquired in scan 2; 4) a coronal

T2*-weighted gradient-echo echo-planar scan

(TE/TR = 50/3000 ms, α = 90º, NEX = 1, FOV =

24 x 24 cm, matrix = 64 x 64, same interleavedslice parameters as scan 3, 1 image per slice, scan

time = 0:03) based on the Mansfield (1977) andthe blood oxygen level dependent contrast(Ogawa, 1992) methods, was used to acquire

functional image data. The pulse sequence usedfor this scan was customized to use a Shinnar-

LeRoux slice-selective pulse (Pauly, 1991) tominimize slice cross-talk and increase the signal-

to-noise ratio (Mock, 1997). The final scan pro-vided the functional image data using the sameimaging parameters as scan 4 except that 191 im-

ages were acquired from each slice location (scantime = 9’33”).

Image data were acquired on a General Electric(Waukesha, WI) EchoSpeed 1.5 Tesla scanner

equipped with high-speed, whole-body gradients(2.2 g/cm, 100 ms rise time) and a standard clini-

cal whole-head transmit-receive quadrature bird-cage headcoil.

The functional image data were reconstructed

off-line running on a Sun SPARC Ultra 1 (SunMicrosystems, Mountain View, CA) without the

application of any filters to the k-space data (cf.,Lowe, 1997) and with a band-pass filter to correct

for asymmetries in the analog-to-digital signalconversion (King, 1995) All other image datawere reconstructed on-line.

All individual subject timeseries datasetswere adjusted to correct for any possible head

movement (Cox, 1996). To identify paradigm-correlated MR signal increases, the time series

from each voxel were fitted to a hemodynami-cally-delayed box-car reference function which

modeled the alternating stimulus blocks using athree-parameter (amplitude, mean, slope) least-squares method (Lowe, 1999). The hemodynamic

delay was estimated to be 6 sec (i.e., 2 functionalimages) by examining MR signal changes in the

amygdalae. The first 5 images from each trial ac-quired while the subject viewed the work “Begin”

were discarded. Thus, for each trial, for each subject, 184 images (i.e., 191 acquired images - 5

discarded images - 2 images to account for hemo-

dynamic delay) were included in the fitting pro-

cedure. The fitting procedure yielded a statistical parametric map where the voxel values were the

Student’s t statistic.Using Analysis of Functional NeuroI-

mages (AFNI, Version 2.00, Cox, 1996; Cox,

1997) each subject’s anatomical data were trans-

formed into the Talairach and Tournoux (1988)stereotaxic coordinate system. Then, the statisticalmaps were coregistered to the transformed ana-

tomical mage data using nearest-neighbor interpo-lation and resampled to 1 mm isotropic voxels to

create new statistical maps. These new statisticalmaps were combined across subjects using in-house code by summing the square of the (un-

thresholded) voxel values to create a group-wiset² map. The distribution of t² is approximated by

the χ² distribution (Hotelling, 1931; Worsley,1995).

Using a maximal estimation of the searchvolume for the region of the amygdalae (Pruess-

ner, 2000), the group-wise map was thresholdedto visualize contiguous clusters of activation = 10

mm3, corresponding to a corrected false-positive

rate of p = 0.05 per cluster, as estimated using thesimultaneous inference tool within AFNI. Using

in-house code (TRO) written in Interactive DataLanguage (Version 5.2, Research Systems, Inc.,

Boulder, CO), image masks based on the group-wise clusters in the amygdalae were applied to

each subject’s image data to identify the subject-wise Student’s t values for each cluster. Milli-metric coordinates reported below are in reference

to the Talairach and Tournoux stereotaxic coordi-nate system. The term “activation” is used to de-

scribe greater mean MR signal during the viewingof the negative compared to neutral stimuli.

False-color ROIs are shown coregistered to amean anatomical image derived from the 14 sub-

jects. We then examined the correlation betweenthe subject-wise Student t-values and scores onthe PANAS positive and negative affect scales

(see appendix for manuscript).Figure 4 illustrates the activation in the amyg-

dala we detected with fMRI in response to unpleasant versus pleasant pictures (left side of the

image is the right side of the brain) while the fig-ure on the right presents a scatter plot depicting

the strong positive correlation between disposi-





tional negative affect and the magnitude of right-sided amygdala activation elicited in response to

the unpleasant compared with the neutral stimuli

(see Irwin et al., 2001 for details).We also had the opportunity to examine changes

in activation in other brain regions in response tothe unpleasant versus neutral stimuli since we ac-

quired data from the entire brain volume. View-ing unpleasant pictures consistently activated dif-

ferent sectors of the prefrontal cortex. Figure 5 il-lustrates the pattern of prefrontal activation in re-

sponse to the unpleasant (compared with the neu-tral) pictures (right side of the brain is on the left

side of image). The sagittal image at the left indi-cates the slice locations for the coronal slices on

the right.Recently, in a sample of normal and depressed

patients we examined the ex-

tent to which the prefrontalactivation elicited by unpleas-

ant pictures was significantlymore right-sided and whetherthis differed between patients

and controls. Figure 6 illus-

trates the data from this study(Davidson et al., 2001).

We found greater right-sided

prefrontal activation in themiddle and superior prefrontal

gyri and this pattern of activa-tion was present in both nor-mal controls and in patients in

an acute depressive episode.These findings are consistent with our data using

considerably more crude electrophysiologicaltechniques (e.g., Davidson, Ekman et al., 1990).

Unfortunately, our acquisition sequence usingwhole brain imaging with 7 mm slices in this

study resulted in significant susceptibility artifactin the region of the orbital prefrontal cortex(OFC) and we were thus unable to obtain ade-

quate signal from this region to examine. Recentwork using a somewhat different echoplanar

pulse sequence with a higher field strength andspecifically shimming to obtain good signal qual-

ity from the OFC has found significant asymmet-ric activations in response to monetary reward

and loss in the direction predicted on the basis ofour prior data and theory (O’Doherty et al., 2001).

Figure 5: Saggital image at left denotes

the slice locations for the coronal images

displayed to the right. Activations repre-

sent the contrast between viewing nega-

tive versus neutral pictures. Left side of

the image is right side of the brain.

Figure 4: Left: activation of the right amygdala in response to negative versus neutral pictures

(N=14); Right: relation between magnitude of MR signal change in the right amygdala and

dispositional negative affect





One important aspect of the functional imag-ing paradigm proposed in the present application

involves voluntary regulation of emotion using a paradigm that we have deve loped and extensively

validated using psychophysiological measures in-cluding facial EMG and emotion-modulated star-tle (Jackson et al., 2000b). We recently com-

pleted our first fMRI study using a variant of this paradigm that requires subjects to either passively

view emotional pictures or to voluntarily “main-tain” the emotion during a delay period following

the presentation of the picture. We were specifi-cally interested in the MR signal change duringan 8 second delay period that occurred immedi-

ately following a 6 second exposure of an emo-tional (or neutral) picture. Following the 8 sec-

ond delay period during which subjects were re-quested to either maintain the emotion or not, a

signal to “RELAX” was presented that cued subjects to cease whatever regulatory strategy in

which they were engaged. Using event-relatedfMRI we were specifically interested in interro-

gating brain activity during the delay period.Figure 7 presents data from this study (Schaeferet al., 2001) illustrating voxels in the amygdala

that were significantly more active during themaintain versus passive conditions in the delay

period.

G. Hippocampal morphometry and its associationwith depression and anxiety: We recently exam-ined the relation between hippocampal volume

and mood and anxiety in a group of 40 subjects(25 patients with major depressive disorder and

15 healthy controls screened for an absence oflifetime history of psychopathology in themselves

and their first degree relatives).We present here the details of

our procedures for drawingROI’s for the hippocampussince they will serve as a basis

for the work we propose

Figure 6: Left: MR signal change in response to negative versus neutral pictures in the regions of the left and right prefrontal cortex

identified by the cross-hairs in the image on the right in normal controls (N=14) and depressed patients off medication (N=16).

Right: location of maximal activation in the prefrontal cortex across groups.

0.00

0.05

0.10

0.15

Controls Patients

Right

Left

Figure 7: Voxels within theamygdala that are signifi-

cantly more activated duringvoluntary emotion regulation

(“MAINTAIN”) comparedwith passive viewing (Schae-

fer et al., 2001)





in this application. Using the same basic MR

methods, we have also established reliable criteriafor drawing ROIs for the amygdala (see Schaefer

et al., 2000).The MRI image data underwent the following

preprocessing steps: (1) reformatting into a single

3-dimensional volume (ANALYZE [R.Robb,

Mayo Clinic] format); (2) psuedo-histogram re- binning to set the highest 0.1% of values to the99.9 percentile level, enhancing the apparent con-

trast in the brain regions of interest; and (3)smoothing using a 3-dimensional anisotropic an-

nealing algorithm (Perona & Malik 1990; Gerig etal. 1992), which preserves edges and small fea-tures while smoothing large homogeneous areas.

The criterion for smoothing was that similar pixelclusters smaller than 2-4 pixels should be re-

moved, but pixel clusters larger than 4 pix-elsshould remain.

In-house software (SPAMALIZE) was used todefine regions of interest. This software displays

axial, coronal, and sagittal views simultaneously,and allows the user to draw in any of the views toquickly construct a 3-dimensional Volume-of-

Interest (VOI) with pixel-level precision. Vol-umes for the whole brain and the cerebellum were

determined using automated segmentation tech-niques (Oakes et al. 1999) followed by manual

corrections if needed. The hippocampal VOIswere rapidly defined manually with the aid of

software that limited the VOI to grey matter, andwere then refined without the grey-matter limita-tion.

Hippocampus VOIs were traced and edited on both sagittal and coronal slices. Sagittal criteria

follow: On the lateral-most slices, the hippocampus borders were defined superiorly by the fim-

bria, anteriorly by the alveus, posteriorly by theCSF of the lateral ventricle, and inferiorly by thewhite matter of the temporal lobe. On more me-

dial slices, a white matter tract appearing poste-rior to the hippocampus was excluded. For most

subjects, the amygdala could be readily distin-guished from the hippocampus on sagittal slices

by defining the alveus (a white matter tract) as theanterior border of the hippocampus. On the me-

dial-most slices, the head and tail of the hippo-campus are separated by thalamic nuclei. At this

point, the tail was no longer traced sagittally be-

cause of an inability to exclude the gyrus fascio-

laris and the fasciola cinera.Coronal criteria follow: The posterior portion of

the hippocampus was defined as being borderedlaterally by the white matter of the fornix (or theCSF of the lateral ventricle in places where the

fornix was indistinguishable), medially by CSF,

inferiorly by white matter, and superiorly by thesplenium of the corpus callosum (moving anteri-orly, the superior border is defined by the gyrus

fasciolaris and the fasciola cinera, and then by thefimbria). For the most anterior portions of the

hippocampus, the amygdala delineates the supe-rior edge of the hippocampus, while the inferior

border was defined by white matter resulting in

the subiculum being included in the hippocampalvolume.

To account for individual differences in overall brain size, total cerebral volume is used as a re-

gressor in the analyses. Because not all scans in-cluded the entire cerebellum, the cerebellum was

excluded from whole brain measurements.The group of subjects we tested in this study

consisted of relatively young community volun-

teers. The mean ages were 33.2 years for the depressed sample and 37.4 years for the controls.

This is a potentially important factor since mostof the prior data on hippocampal atrophy in de-

pression were derived from older samples (see re-view in our article, Rusch et al., 2001; in appen-

dix). We first wished to determine if our methodsfor drawing hippocampal volumes were reliable.Accordingly, we had two raters draw volumes in-

dependently on 5 subjects and computed the in-traclass correlations between them. Intra-class

correlations indicated reliable tracing of both theleft (IC = .97, p = .007) and right (IC = .80, p =

.10) hippocampi.. Consistent with other recentreports (Pruessner et al. 2000; Mervaala et al.2000), we found that right corrected hippocampal

volumes were be significantly larger than left cor-rected hippocampal volumes.

In our sample, the volumes of the hippocampusdid not differ between the depressed and control

subjects using measures of either absolute volumeor of corrected volume (corrected for whole brain

volume) for either the left, right or total hippo-campus. Half of our depressed sample met crite-ria for melancholic depression and when we ex-





amined this group separately compared with the

other depressed patients and controls, no groupdifferences were found. Power calculations indi-

cated that this was not an artifact of the study be-ing underpowered.

Interestingly, for the both the depressed patients

and the controls, right hippocampal volume and

total volume were correlated with trait anxiety.Figure 8 illustrates this finding. The failure tofind group differences between depressed patients

and controls in this study may, as noted earlier, bea product of the relatively young age at which

these patients were tested. Given findings that

indicate a relation between number of cumulativedays depressed and hippocampal volume in such

patients (Sheline et al., 1996), it may well be thatexamining an older group of patients will yield

more consistent findings. In the proposed re-search, we will be able to take advantage of the

extensive corpus of psychosocial and demo-graphic data on these respondents to examine

which factors are most strongly associated withvolume reductions in the hippocampus.

The discovery of a positive correlation betweentrait anxiety and right and total corrected hippo-campal volumes was novel. Animal studies have

suggested a role for the hippocampus as part of acoping system for stressful and anxiety-rich situa-

tions, with the dentate gyrus (Henke 1990; Bel-zung 1992) and ventral subiculum (Herman et al.

1998) being specifically implicated. Furthermore,it has been hypothesized that hippocampal hyper-

activity may be a potential cause of generalized

anxiety (McNaughton 1997). Again, we intend tofollow-up these data in the current project with a

much larger sample size and a much more complete battery of mood and affect measures in therespondents.

d. Research Design And MethodsThe central goal of the work proposed in this

project is to interrogate brain mechanisms that

might underlie vulnerability andresilience in a moderately large,

representative and highly stratifiedsample of WLS participants. We

propose to test 500 subjects over

the course of this five year project period.

The major hypotheses pursued in

this project are:

1. Brain electrical activity meas-ures of prefrontal activationasymmetry will be associated

with measures of dispositionalnegative affect, anxiety and psy-

chological well-being. Specifi-cally, subjects with greater right

prefrontal activation will report more negative af-fect and anxiety and show higher levels of corti-

sol compared with their more less anxious andmore positive counterparts. Subjects with greaterleft-sided prefrontal activation are predicted to re-

port higher levels of psychological well-being andto show a more resilient profile on both self-

report and other biological measures.2. The volume of the hippocampus will be in-

versely correlated with measures of cumulativestress and depression. However, in light of ournew data indicating that hippocampal volume cor-

relates positively with trait anxiety (Rusch et al.,2001), we will also examine the role of anxiety in

moderating this relation. We also predict thathippocampal volume will be correlated with

measures of cortisol available from these subjects.We will also examine possible gender differences

since recent data indicate a decline in hippocampal volume for men but not for women (Pruessner

Anxiety Correlation (Depressives)

40

50

60

70

80

0.001 0.00125 0.0015 0.00175 0.002 0.00225 0.0025

Right Corrected Volume

A n

x i e t y S c o r e

Figure 8: Relation between trait anxiety and hippocampal volume in depressed

patients (N=25).

r=.75

p=.00





et al., 2001). Gender will be examined in all of

our analyses.3. The volume of the amygdala will also be posi-

tively correlated with anxiety symptoms (De Bel-lis et al., 2000). We have established proceduresfor accurately drawing the amygdala on MR in

my laboratory (see Schaefer et al., 2000) and will

use these in the current work.4. Using fMRI to measure the BOLD signal in

response to a task designed to elicit emotion and

to assess the voluntary regulation of emotion, we predict that greater cumulative exposure to adver-

sity will be associated with larger magnitudeamygdala signal in response to aversive comparedwith neutral stimuli. In addition, we predict

greater activation of the amgydala during a delay period immediately following the presentation of

the emotional stimulus. We also predict that re-spondents with more cumulative adversity will be

less able to voluntarily regulate negative affectand will show a larger amygdala signal in a con-

dition that requires them to voluntarily attenuatetheir negative affect.

5. In the task designed to assess reactivity and

regulation of emotion, a series of novel emotional pictures will be presented. We will examine the

magnitude of hippocampal signal in response tothe presentation of these novel pictures. We pre-

dict that subjects exposed to more cumulative ad-versity will show less hippocampal activation

than their more advantaged counterparts. We also predict that hippocampal activation will be corre-lated with recognition memory and with levels of

basal cortisol.6. We will examine relations between the anatomi-

cal and functional data, specifically for the amyg-dala and hippocampus. These, as well as other

analyses involving the anatomical data will usenew voxel-wise deformation-based morphometricmeasures that have been pioneered by our new

colleague at UW, Moo Chung, who is a Co-Investigator on this proposal. We will conduct

exploratory analyses examining the relation be-tween individual differences in electrophysiologi-

cal measures of prefrontal activation asymmetryand the morphometric and functional MRI data.

Subjects:

The subjects will consist of 500, randomly se-

lected WLS participants. Because the examina-tions will take place over a 4 -year period, at thetime of testing, subjects will be between the ages

of 63 and 67. The random sample will be repre-

sentative of the graduate cohort, but highly strati-fied by gender, adolescent cognitive ability, edu-cational attainment, marital status, and employ-

ment history.1 There are numerous questions that

will be addressed in this project and separate

power calculations were performed for all of themajor classes of variables. For every neural vari-able, the effect size ranges from moderate to

large. For example, in research examining differ-ences in hippocampal volume between depressed

patients and controls, the effect size is large. Inthe Bremner et al. (2000) study, the effect size is

d=.99; in the Sheline et al. (1999) study, the effectsize is d=.80. Sheline et al. (1999) also report a

correlation of r=.60 between hippocampal volumeand total number of days depressed over thecourse of the lifetime. This latter correlation re-

sults in a power of .80 to detect this relation usinga .05 two-tailed alpha level with a sample size of

19. In our studies of relations between electrophysiological measures of prefrontal activation

asymmetry, well-being and emotion-modulatedstartle, the magnitude of most correlations ranges

between r=.3 and r=.5 (see Preliminary Studiesabove). A power of .8 to detect a relation at the.05 (2-tailed) level between our electrophysio-

logical measure of asymmetric prefrontal activa-tion and these other variables would require a

sample size of 84 subjects for a correlation of .3.The rationale for a sample size of 500 is so that

we can maximize our range on the variables of in-terest and could then form smaller subgroups thatare still sufficiently large to provide adequate

power to test our hypotheses.

1 Aside from its much smaller size, the sample used in my

earlier work with Ryff and Singer was stratified by psycho-

logical characteristics and was not fully representative of the

WLS cohort. The new sample will also be 5 to 10 years olderat the time of examination, and I will have the benefit of a

new wave of life-history data.





Procedures:

Subjects will begin their assessment procedures

in the morning by checking into the GeneralClinical Research Center (GCRC). During theirstay at the GCRC, a Registered Nurse (under Dr.

Muller's supervision) will conduct comprehensive

physical examinations (see protocol in Appen-dix). These data will complement and corrobo-rate self-report data. In our pilot studies, we have

found that subjects do not always report findingsthat may be of significance for predicting future

health and daily functioning. Examples includecarotid arterial bruits, abnormal heart sounds (S3,S4), lower extremity edema, anosmia, osteoarthri-

tis of the hands, lower extremity varicosities, andfibromyalgia and other regional pain syndromes

such as bursitis or tendenitis. The physical examreveals vessel changes in the eye, usually due to

hypertension, that may be predictive of future brain, kidney, or heart damage. Vibration loss in

the lower extremities may indicate nerve damagein subclinical diabetes; subclinical diabetes is alsotested by measuring glycosylated hemoglobin

(HbA1c, laboratory test). Forgotten injuries andsurgeries are found by asking about scars. Nota-

tions on teeth alignment, repair, and false teethmay reflect both behavioral and economic factors.

Cardiac findings in the physical exam may be re-flected in the cardiovascular measures of emo-

tional reactivity and allostatic load (cortisol, epi-nephrine). Other physical findings such as in-tegument scars, sun exposure skin lesions, striae,

arcus senilis, nail lesions, skin and mucus mem- brane moisture, heart murmurs, ear crease, sub-

clinical sinusitis, grip strength, peak flow (lungfunction) may be predictive of future health. We

view these examinations as a valuable opportu-nity to identify potential predictive markers of

physical health with possible linkage to social and

demographic factors and other biological assess-ments of allostatic load, immune function, and

cerebral activation asymmetry.Standard laboratory blood tests will be per-

formed in the GCRC and can be used a generalmarkers of health. The complete blood count

may indicate anemia. Abnormalities in electro-lytes (sodium, potassium, bicarbonate) indicateabnormal regulation in the kidney and cardiovas-

cular systems. Elevations in liver enzymes can

indicate occult alcohol abuse (GGT) or intrinsicliver disease (GGT, AST, bilirubin). Mild eleva-

tions in creatinine can indicate early kidney insuf-ficiency. Modestly low serum albumin can be amarker for inflammation or malnutrition, and the

sedimentation rate is a sensitive marker of in-

flammation. Other abnormalities in routine labo-ratory tests such as total protein, calcium, lactatedehydrogenase, and alkaline phosphatase can in-

dicate a myriad of diseases. Glycosylated hemo-globin is an excellent marker for occult diabetes

mellitus. Cholesterol and HDL cholesterol aregood markers for future risk of cardiac disease.Our preliminary analyses of a simple variable

such as total numbers of abnormalities on physi-cal examination or total numbers of abnormalities

on standard laboratory blood tests show correla-tion with social relationship variables. Further

analyses will examine variables ranked as to pos-sible risk for future morbidity and mortality.

Less routine laboratory blood tests such as C-reactive protein and homecysteine have clearlinks to increased risk of coronary artery disease

(Ridker et al. 2000). Dihydroepiandrosterone-sulfate (DHEA-S) and interleukin-6 are markers

of aging and immune-dysregulation, with de-creases in DHEA-S with age and decreased

health, inversely related to increases in inter-leukin-6 (Daynes et al, 1993; Papanicolaou et al,

1998). Three cytokine markers are strongly asso-ciated with links between psychosocial factorsand the immune system: interleukin-6, tumor ne-

crosis factor and interleukin-2 receptor. Tumornecrosis factor is closely linked with interleukin-6

as a proinflammatory cytokine, but interleukin-6has separate links to psychosocial factors such as

depression (Dentino et al, 1999). A third cyto-kine marker, interleukin-2 receptor, has inde-

pendent links to schizophrenia and major depres-

sion (Maes et al, 1995). The use of three cyto-kines in combination with other tests of inflam-

mation such as the sedimentation rate, using mul-tivariate analyses increases our potential to assign

the cytokine to simple underlying occult infla m-mation versus a more subtle and complex rela-

tionships to psychological and physical health.After spending the morning in the GCRC, sub-

jects will have lunch. Following lunch they will





be taken to an MR simulator that consists of the

shell of an MR scanner, along with the bed and amock head coil of the precise diameter used in the

actual scanner. We use this apparatus to accli-mate subjects to the MR environment and to fa-miliarize them with the behavioral tasks that will

be presented in the scanner. In addition, we have

digitized the sounds of the actual scanner and play them to the subjects so that they will knowahead of time exactly what to expect during the

actual scanning session. We have found thesimulator session to be important in reducing sub-

jects’ anxiety about the upcoming scan and to fa-cilitate the acquisition of higher quality data.

At the simulator session, subjects will also com-

plete a number of standardized self-report instru-ments including an assessment of handedness

(Chapman & Chapman, 1987), along with a groupof instruments that assess mood and dispositional

affect that we have found to be related to individ-ual differences in prefrontal activation. These in-

clude the Positive and Negative Affect Scales(Watson et al., 1988); the Behavioral Inhibitionand Activation Scales (Carver & White, 1994);

the SpielbergerState-Trait Anxi-

ety Scale (Spie l- berger et al.,

1983), the MoodAffect Symptom

Questionnaire(MASQ; Watsonet al., 1995) and

the Daily Retro-

spective Question-

naire (Kahneman,2001; see appen-

dix). In addition,all subjects will begiven a SCID in-

terview to assesscurrent and past

history of psychopathology accord-

ing to the DSM-IV(First et al., 1995).

Following thesimulation sessionand the comple-

tion of the self-report and interview measures,

subjects will be taken into the scanner. Thescanning sequence will consist of anatomical

scans following by functional scans. The ana-tomical scans will be obtained with a 3D whole

brain T1-weighted SPGR, 30° flip angle, 1mm

axial slices with 0mm skip. The functional data

will be collected in a time series using T2*-weighted gradient echo EPI sequences on our 3TGE scanner. Data will be acquired in the sagittal

orientation, TE=30 ms, TR=3000 ms, slice thic k-ness=4mm, with a .5mm skip. These are scan-

ning parameters that we are currently using on the3T and they are providing high quality data.

The anatomical acquisition protocol will permitdeformation-based morphometric analyses to be

performed on a voxel-wise basis by Moo Chung

(see Chung, 2001). An example of volumetricmeasurement using Chung’s deformation-based

algorithms is presented in Figure 9 below. Thisfigure illustrates changes over time in a longitudi-

nal study and highlights in red areas that increasein volume, in blue areas that decrease in volume

and in yellow areas that display displacement in

Figure 9: Deformation-based morphometry illustrating longitudinal changes in volume and displace-ment Chun et al., 2001





location over time.

The task will be based upon recent event-relatedstudies with emotional stimuli that have been con-

ducted in my laboratory (e.g., Putnam et al., 2001).A total of five “runs” will be presented. Each runwill consist of 75 different picture presentations.

All presentations will be unique. Pictures are se-

lected from the International Affective Pictures Se-ries (Lang et al., 1998). Each picture will be pre-sented for 3 s, with an ISI of 12 Ss. An equal num-

ber of positive, negative and neutral pictures will be presented. The first two runs will be passive

viewing though subjects will be told that they will be tested for their memory of the pictures after thescan. The final three runs will consist of a regula-

tion task. Immediately after the presentation ofeach picture, subjects will be presented with either

an arrow up, an arrow down or a circle. The circledenotes that they are to maintain the emotion that

they are experiencing; the up arrow denotes thatthey should accentuate the emotion they are experi-

encing and the down arrow indicates that theyshould attenuate the emotion they are experiencing.We have performed extensively initial research

with this task and know that both emotion-modulated startle (Jackson et al., 2000b) and MR

signal change in the amygdala (Schaefer et al.,2000) are modified in response to emotion regula-

tion instructions. At six seconds post picture off-set, the word “RELAX” will appear on the screen

for one second informing the subjects to cease theemotion regulation strategy that they have invoked.To assess the impact of the instructed regulation

strategy on MR signal change, we will specificallyinterrogate the activity in the six-second post-

picture period prior to the instruction to RELAX.Following the scanning session, subjects will be

presented with a recognition memory test to probefor memory of the pictures presented. A subset of150 of the pictures that were presented, with an

equal number selected across the different condi-tions (passive viewing, accentuate, attenuate and

maintain) of each valence, along with an equalnumber of recognition foils will be presented.

Each picture will be presented for 2 seconds witha 2 second ISI and subjects will simply have to

press one of two buttons following each picture toindicate whether it was a new or old picture.

We will pilot this task to insure that activation in

the hippocampus can be detected. If we do notsee reliable signal in the hippocampus in middle

aged adults during this task, we will modify it toinclude a basis set of pictures that the subjectswill have been familiarized on prior to the scan-

ning session (see Constable et al., 2000). These

familiar pictures would then be interspersed withthe novel pictures during the picture presentation

paradigm. This might be required to elicit hippo-

campal activation based upon recent imaging data(Constable et al., 2000).

Finally, following the recognition task, subjectswill be escorted to the electrophysiology testingroom at Keck where baseline EEG will be re-

corded with the geodesic sensor net system ac-cording to our standard protocol (Tomarken et al.,

1992). We will acquire eight 1-minute trials ofEEG, half during eyes open and half during eyes

closed, presented in counterbalanced order.

Data reduction, analyses and hypotheses: Theelectrophysiological data will be analyzed as pre-viously described in a long series of publications

(see Davidson, Jackson & Larson, 2000 for exten-sive description). Measures of prefrontal activa-

tion will be computed from spectral measures ofalpha power from a composite of the prefrontal

scalp sites. In addition, source localization meth-ods will be used to compute the sources of signals

in theta, alpha, beta and gamma bands and relatethem to the key variables of interest (see Pizza-galli et al., 2001, from our lab). The source local-

ization method we have been extensively usingfor spectral EEG data is Low Resolution Electro-

magnetic Tomography (LORETA; Pascual-Marqui, 1999). LORETA computes the three-

dimensional intracerebral distributions of currentdensity for specified EEG frequency bands. Insimulations comparing five source localization

techniques using linear solutions for the EEG in-verse problem, only LORETA reliably localized

sources in three-dimensional space (Pascual-Marqui, 1999). One important difference be-

tween the LORETA algorithm and previously published source localization methods (e.g.,

BESA; Scherg & Von Cramon, 1986) is thatLORETA does not assume a specific number ofsources for solving the inverse problem. The only





assumption implemented is that neighbor neu-

ronal sources are likely to be similarly active (i.e.,have similar orientations and strengths). This as-

sumption is well supported by animal single unitrecordings (e.g., Llinas, 1988). Mathematically,the assumption of simultaneous and synchronous

activation of neighboring neurons is implemented

by computing the “smoothest” of all possible ac-tivity distributions. A recent study (Worrell et al.,2000) provided direct cross modality validation

by showing that LORETA generators of ictal dis-charge were remarkably close to the locations of

MRI-identified epileptic foci.The LORETA version we have implemented in

our laboratory uses a three-shell spherical head

model registered to the Talaraich brain atlas (Ta-laraich & Tournoux, 1988), as well as EEG elec-

trode coordinates derived from cross-registrations between spherical and realistic head geometry

(Towle et al., 1993). Computations will be re-stricted to cortical gray matter and hippocampus

using the digitized Talaraich and probability at-lases of the Brain Imaging Centre, Montreal Neu-rologic Institute. If a voxel’s probability of being

gray matter is higher than 33% and higher thanthe probability of being white matter or cerebro-

spinal fluid, that voxel is labeled as gray matter.The solution space contains 2394 voxels, and the

spatial resolution is 7 mm.The LORETA analyses consist of three steps:

First, for every subject, all available artifact-free2048-ms EEG epochs derived from average refer-ence data are subjected to cross-spectrum analy-

sis. Second, LORETA computes current densityas the linear, weighted sum of the scalp electrical

potentials and then squares this value for eachvoxel to yield power of current density. Finally,

for every subject and every band, the LORETAsolution is normalized to a total power of 1 andlog-transformed. LORETA units are proportional

to square amperes per square meter. A Monte-Carlo procedure is used to estimate the false posi-

tive rate in testing the null-hypothesis of no rela-tion between the chosen experimental variable or

group identification and the LORETA signal (seePizzagalli et al., 2001 for an example of this ap-

proach from our lab).The structural MR data will be analyzed as de-

scribed in the Preliminary Studies section to ob-

tain measures of hippocampal volume (see Rusch

et al., 2001, in appendix). The volume of amyg-dala core nuclei and the non-core amygdala

(Sheline et al., 1999) will be separately obtainedsince the former has been found to be positivelycorrelated with hippocampal volume (r=.68) and

to be associated with number of days depressed

voer the course of a lifetime in patients with re-current major depression (Sheline et al., 1999).The core nuclei of the amygdala will be defined

by the white matter tracts surrounding them. In-cluded will be the basal nucleus, accessory basal

nucleus and the lateral nucleus (medial portion).Excluded are the periamygdaloid areas, the me-dial nucleus and the central nucleus. The non-

core amydala is defined by measuring the totalamygdala and subtracting the core amygdala.

The anterior boundary of the amygdala will bevisualized in coronal section and is the first slice

in which the temporal stalk connects to the whitematter of the insula. Dorsally, visualized in cor-

onal section, the border is defined in anterior sec-tions by the endorhinal sulcus between the basalforebrain and temporal lobe, and posteriorly in

sagittal sections by a horizontal to the posteroinfe-rior edge of the temporal stem with the temporal

horn of the lateral ventricle. Ventrally, visualizedin sagittal section, the amygdala is bounded by a

horizontal line connecting to the ventral/anterioredge of the hippocampus. Posteriorly, observed

in sagittal section, the amygdala is bounded by its border with the hippocampus. Medially, seen incoronal section, the amygdala is bounded by the

subarachnoid space. Laterally, seen in coronalsection, the amygdala is bounded by white matter.

Corrected volumes, corrected for overall differ-ences in cerebral volume will be calculated. De-

formation-based morphometric measures will beutilized to perform voxel-wise searches for mor-

phometric relations with select variables and

group definitions (Chung et al., 2001).Event-related functional MRI data will be ana-

lyzed as we have described in recent reports (Put-nam et al., 2001). Briefly the following steps will

be performed on the data: volume registration,linear detrending and then non-linear fitting using

a gamma-variate function to model the hemody-namic response. The primary dependent measurefrom this data reduction process will be percent





signal change though other indices such as area

under the curve will also be computed and exam-ined on an exploratory basis.

The data from the physical exam will be quanti-tated by summing the number of abnormalitiesdetected. The specific laboratory tests on the

blood samples will be examined by first determin-

ing the pattern of intercorrelation among themeasures and reducing the variable set by aggre-gating across measures. These aggregate meas-

ures will then be used in regression models wherekey psychosocial and socioeconomic variables are

entered as predictors. In addition, they will beused in analyses that examine relations betweenthe brain imaging variables and peripheral biol-

ogy and physical health.Using the four primary corpora of data (physical

health and peripheral biomarkers; EEG; structuralMR measures; functional MR measures), rela-

tions with aggregate summary variables from theWLS data set will be examined to test the follow-

ing major hypotheses:1. We predict that we will replicate previous find-

ings and observe that subjects with increased

left-sided prefrontal activation on the electrophysiological measures will report greater levels

of well-being, particularly on the subscales ofSelf-acceptance, Positive Relations with Others,

Purpose in Life and Environmental Mastery.We also predict that individuals with greater

left-sided prefrontal activation will have fewer physical health problems as revealed in the physical exam and a healthier profile of periph-

eral biomarkers, particularly IL-6 (i.e., lowerlevels).

2. A major addition to the new WLS data collec-tion will be the inclusion of Kahneman’s Daily

Retrospective Questionnaire that is derived fromexperience-sampling studies and is more closelyassociated with momentary affect measures

compared with memory-based retrospectivemeasures. For each of our key measures of

brain function, we will systemically compare thetraditional memory based measures with Kah-

neman’s new measure and predict that relationswill be systemically more robust with the new

Kahneman measure.3. Examine relations between stressful life events,

coping styles and functional and structural

measures. We predict that increased exposure to

adversity and stressors will be associated withsmaller hippocampal volumes. While we did

find smaller hippocampal volumes in our recentstudy with depressed patients (Rusch et al.,2001), this study was performed with relatively

young individuals (average age=35 years). Cu-

mulative exposure to adversity over a consid-erably longer period of time is predicted to haveconsequences for the hippocampus. Exposure to

adversity is also predicted to be associated withsmaller amygdala core nuclei since it was this

measure of amygdala anatomy that was moststrongly correlated with hippocampal volume inSheline et al.’s (1999) recent data. We also pre-

dict thatanxiety will be positively associatedwith noncore amygdala volume, so trait levels of

anxiety will be included as a predictor in all ofthese analyses. In the analyses of stressful life

events, we will take advantage of the detailedand differentiated information available in the

WLS corpus of data to examine for possible dif-ferences in types of stressors, including subjec-tive economic strains. The moderating effects

of coping strategies and styles will be includedin these analyses using regression-based analytic

approaches. In light of the recent data from theMontreal group, we predict hippocampal vol-

ume reductions with age in men, but not inwomen (Pruessner et al., 2001). These hypothe-

ses will be examined with both region-of-interest analyses as well as voxel-wise whole

brain searches using deformation morphometry

methods (Chung et al., 2001) that will allow usto examine measures of shape in addition to

volume.4. Examine relations between the cognitive meas-

ures and the structural and functional measuresof the brain. We predict that hippocampal vol-ume will be associated with variations in verbal

memory. Individual differences in verbal flu-ency and in working memory are predicted to be

associated with structural and functional varia-tions in prefrontal cortex. These hypotheses will

again be examined using both voxel-wise whole brain searches as well as region-of-interest

(ROI) analyses using a probabilistic atlas(Chung et al., 2001).





5. On the fMRI measures, we predict that in-

creased exposure to adversity and greater expo-sure to stressors will be associated with be asso-

ciated with greater magnitude activation of theamygdala in response to unpleasant pictures andfollowing the presentation of the unpleasant pic-

tures. In our previous fMRI studies with emo-

tional pictures, we have not found many groupdifferences in response to the positive pictures

but we will test for relations between our key

subject variables (well-being, anxiety and dispo-sitional negative and positive affect) and whole-

brain reactivity to positive pictures. In the regu-lation task, we predict that instructions to at-tenuate negative affect will be associated with

less facility at modulating amygdala reactivity tonegative pictures in the post-picture period in

subjects with increased exposure to adversity.This is conceptually analogous to the findings

using emotion-modulated startle that were pre-sented in the Preliminary Studies section. All

fMRI analyses will be performed on data cor-rected for overall amount of gray matter, waswell as the uncorrected data.

6. Examine relations between the peripheral bio-markers and the structural and functional meas-

ures of the brain. We predict that decreasedhippocampal volume, increased amygdala acti-

vation both during the following negative pic-ture presentation and possibly decreased volume

in certain regions of the PFC (detected withdeformation-based morphometric methods) will

be associated with a profile of biomarkers in the

less healthy direction. These include higher body mass index, greater waist-hip ratio, lower

levels of DHEA, higher levels of total choles-terol and HDL cholesterol. and higher levels of

IL-6.





e. Human Subjects

1. The subjects for this study will consist of 500

respondents from the WLS sample. Half the subjects will be female. Subjects will range in agefrom 63 to 67 years.

All subjects will be carefully screened for possible presence of metal implants and/or cardiac pacemakers and will be excluded if any such im-

plants are suspected (because of MR imaging).

2. The sources of the research material will bedata collected in the laboratory strictly for re-search purposes. The principal types of data will

be blood samples, brain electrical activity meas-ures, and MR measures of brain structure and

functional brain activity.

3. The sample will be recruited from the ongoingWLS study. Prospective subjects will be given a

short statement describing the research and pro-vided with an oral description of the project. Anyquestions a prospective subject might have about

the nature of the research will be answered by astaff member associated with the project. Follow-

ing this, subjects will give signed consent to theexperimental procedure.

4. The risks associated with all procedures are

very minimal. There is essentially no risk to theelectrophysiological recording procedures otherthan possible slight irritation at the site of elec-

trode application. The risk associated with theMR procedure is minimal. Subjects with metal

implants or cardiac pacemakers will be excludedfrom participation. Individuals with claustropho-

bia may experience some anxiety in the small bore of the magnet. There may be slight discom-fort in having to lie still during the scanning pro-

cedure. The emotional pictures used in the MRstudy may be slightly disturbing, though they are

culled from readily available media to which citi-zens in this age range are exposed daily.

5. Every effort will be made to provide informa-

tion to subjects to reduce their anxiety about theMR procedures and the electrophysiological re-cordings. Confidentiality will be maintained

throughout the project period. Names will not be

kept on data records and standardized codes will be used at each site. Subjects will be identified

only by a code number.

If any adverse effects occur during the imaging

protocols, appropriate medical intervention is pro-

vided.

6. The risks to the subjects in this research are

minimal. The research proposed offers consider-able promise in furthering our understanding of

the biological bases of vulnerability and resilienceand their relation to health and disease. The po-tential benefits of this research far outweigh the

risks involved.





e.1. Gender and Minority Inclusion

Table 1. Gender and Minority Inclusion

NOTE: This table includes all of the original mem-

bers of the graduate sample (N=10,317), and theselected siblings who responded to the 1977 and/or

1994 surveys (N=5,812), and their living spouses(N=10,148).

Grads &

sibs

American

Indian orAlaskan

Native

Asian or

PacificIslander

Black, not of

HispanicOrigin

Hispanic

White, not of

HispanicOrigin

Other or

Unknown Total

Female 17

1 20 7 8,254 - 8,299

Male 12 2 12 11 7,793 - 7,830

Total 29 3 32 18 16,047 - 16,129

Spouses

Wives 8

1 8 7 4,903 - 4,926Husb 11 1 13 4 5,193 - 5,222

Total 18 2 20 11 10,096 - 10,148

Full-sample

Female 25

2 28 14 13,157 - 13,225

Male 23 3 25 15 12,986 - 13,052

TOTAL 47 5 52 29 26,143 - 26,277

Among Americans aged 60 to 64 in March 2000,66.7% are non-Hispanic white women and men

who completed at least 12 years of schooling (U.S.Bureau of the Census 2000: Table 1a) and thus re-

semble the WLS cohort. The WLS is unusuallyvaluable in its representation of women as well asmen. The WLS cohort, mainly born in 1939, pre-

cedes by about a decade the bulk of the baby boomgeneration that continues to tax social institutions

and resources at each stage of life. For this reason,the study can provide early indications of trends

and problems that will become important as the

larger group passes through its sixties. In addition,the WLS is the first of the large, longitudinal stud-

ies of American adolescents, and it thus providesthe first large-scale opportunity to study the life

course from late adolescence through the mid-60sin the context of a full record of ability, aspiration,

and achievement.2 The WLS overlaps the youngest

cohorts that entered the HRS, and this providesopportunities to check the scope of our findings

(and those of the HRS). Unlike the WLS, the HRSis nationally representative, but it does not cover

the lives of respondents from adolescence forwardto midlife.

The WLS data also have obvious limitations.

Some strata of American society are not repre-sented. Everyone in the primary sample graduated

from high school. (Sewell and Hauser 1975:207-15) estimated that about 75% of Wisconsin youth

graduated from high schools in the late 1950s;

2 There have, of course, been important and influ-ential longer-term studies of the life-course in theU.S. These reflect careful and insightful work, butthey are based on small, local, or highly selectedsamples (Holahan and Sears 1995; Elder 1974;Clausen 1993).





about 7% of siblings in the WLS did not graduate.

There are only a handful of African American,Hispanic, or Asian persons in the WLS. Given the

minuscule share of minorities in Wisconsin whenthe WLS began, there is no way to remedy thisomission. About 19% of the WLS sample is of

farm origin; this is consistent with national esti-

mates in cohorts of the late 1930s. In 1964, in1975, and again in 1992, 70% of the sample livedin Wisconsin, but 30% lived elsewhere in the U.S.

or abroad. Fifty-seven percent of the graduateshave always lived in Wisconsin, and 17% have

lived outside Wisconsin at every contact since1957. The WLS graduates are homogeneous in age,

but their siblings are not, and their ages cover a

broad range, mainly within 8 to 10 years of the ageof graduates.

In summary, the WLS samples consist ofwomen (5323) and men (4994) who graduated

from Wisconsin high schools in 1957 and a randomsample of approximately 5800 of their sisters and

brothers. Because of differences in longevity andwillingness to respond, there are yet more womenthan men currently active in the study. While there

are no restrictions on the ethnic compositions of thesamples, because of the population composition

and schooling outcomes in Wisconsin in the late1950s, the sample contains a very small share of

racial or ethnic minorities, and there is now no wayin which this problem in sample coverage can be

corrected.

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