International Journal of Psychophysiology 71 (2009) 264–268

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International Journal of Psychophysiology

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Altered resting psychophysiology and startle response in Croatian combat veteranswith PTSD

Tanja Jovanovic a,⁎, Seth D. Norrholm a, Andrea Jambrošić Sakoman b,Slavica Esterajher b, Dragica Kozarić-Kovačić b

a Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, GA, USAb Referral Centre for the Stress Related Disorders of the Ministry of Health of the Republic of Croatia, Regional Center for Psychotrauma, Department of Psychiatry,Dubrava University Hospital, Croatia

⁎ Corresponding author. Emory University School of MBehavioral Sciences, 49 JesseHill JrDr, Suite 311,Atlanta,GA3fax: +1 404 778 1488.

E-mail address: tjovano@emory.edu (T. Jovanovic).

0167-8760/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ijpsycho.2008.10.007

a b s t r a c t

a r t i c l e i n f o

Article history:

Posttraumatic stress disorde Received 1 August 2008Received in revised form 22 October 2008Accepted 23 October 2008Available online 5 November 2008

Keywords:PTSDPsychophysiologyHeart-rateSkin conductanceStartle reflex

r (PTSD) is a prolonged reaction to an extremely traumatic experience. One of thecore symptoms of PTSD is hyper-arousal which can be the result of an elevated activation of the autonomicnervous system. Including psychophysiological assessment methods in PTSD research can point to theneurobiological bases of the disorder. The studies of psychophysiology of PTSD to date have mostly measuredreactivity. The aim of the current study was to compare resting state psychophysiology and startle reflexes inPTSD patients and controls in a sample of Croatian combat veterans. We measured heart-rate, respiratorysinus arrhythmia, skin conductance, and eyeblink muscle contraction during an acclimation period andduring the presentation of startle stimuli in 45 male PTSD patients and 33 male healthy controls.We found that PTSD patient had elevated baseline heart-rate and decreased respiratory sinus arrhythmiacompared to the controls. Furthermore, PTSD patients had impaired habituation to the startle probe, butthere was no group difference in initial startle magnitude. There was also no group difference in skinconductance level or skin conductance response. Startle habituation and baseline heart-rate appear to offerthe most reliable psychophysiological indices of PTSD. This finding replicates trends in the literature in a newpopulation of PTSD patients.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Posttraumatic stress disorder (PTSD) is a prolonged reaction to anextremely traumatic experience. It is frequently difficult to make aPTSD diagnosis with absolute certainty. Due to the complicateddifferential diagnosis of PTSD and the fact that the diagnosis is basedlargely on self-report of symptoms, PTSD is not difficult to malinger. If‘secondary gain’ is involved, the problem is exaggerated, which isespecially present in forensic evaluations. PTSD can be isolated orcomorbid with other psychiatric disorders (Kozaric-Kovacic andBorovecki, 2005a), which can also be malingered (Kozarić-Kovačićet al., 2003; Kozaric-Kovacic and Borovecki, 2005b).

Based on the DSM-IV criterion of hyper-arousal in PTSD (APA,1994), several investigators have examined the utility of psychophy-siological recording as an aid to accurately diagnosing the disorder(see Orr et al., 2004 for recent review). Most of these studies haveemployed multiple psychophysiological methodologies to record

edicine, Dept of Psychiatry &0303,USA.Tel.: +14047781485;

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cardiovascular, electrodermal, and electromyographic activity. Thefirst two systems are under the control of the autonomic nervoussystem, whereas the last is under the control of the central nervoussystem (Cacciopo et al., 2004). The cardiovascular measurementsinclude: (1) electrocardiograms (ECG) as a measure of both sympa-thetic and parasympathetic nervous system activity and (2) respira-tory sinus arrhythmia (RSA) as an index of hear-rate variability orparasympathetic nervous system activity (Porges, 2007). Electroder-mal activity (EDA) measures changes in sweat gland activity and thesechanges aremeasured from the skin on the fingers. Electromyographic(EMG) assessments include measuring activity from the control facialexpression and eyeblink.

Early studies of the utility of physiology in assessing PTSD usedcombat-related stimuli to evoke arousal in Vietnam veterans withPTSD. Studies successfully discriminated veterans with or withoutPTSD on the basis of heart-rate responses to combat sounds(Pallmeyer et al., 1986). In studies using scripts, in which everyparticipant describes an actual traumatic event from their combatexperience (Pitman et al., 1987), PTSD patients show a greaterphysiological response compared to traumatized persons withoutPTSD. A large study of 1461 Vietnam veterans demonstrated thatphysiological data offer a useful and objective, although notindependent, assessment of the disorder (Keane et al., 1998).

265T. Jovanovic et al. / International Journal of Psychophysiology 71 (2009) 264–268

Although exaggerated startle response was one of the earliestsymptoms related to combat stress, the psychophysiological evaluationof startle in PTSD patients has not yielded consistent results; in fact, thisfinding is themost equivocal of all (reviewed in Orr et al., 2004). Studiesof Gulf War veterans with PTSD found both self-reported (Southwicket al.,1995) and physiologically (Morgan et al., 1996) exaggerated startlecompared to non-PTSD veterans. On the other hand, Vietnam veteranswith PTSD did not show increased startle (Grillon et al., 1996), unlesstheywere subjected to a threatening context (Grillon et al.,1998). Grillon(Grillon and Baas, 2003) concluded that increased baseline startle maybe related to the recencyof combat exposure andmaydecline after a fewyears. Several studies have examined physiological activity duringstartle and have found increased heart-rate (HR) and skin conductance(SC) responses, and slower skin conductance habituation in PTSDpatients compared to controls (Metzger et al., 1999). The abovedescribed script-driven imagery method has been used with manydifferent PTSD populations: World War II veterans (Orr et al., 1993),Korean War veterans (Orr et al., 1993), Vietnam War veterans (Pitmanet al., 1990), Israeli War veterans (Shalev et al., 1992), and Vietnam Warcombat nurses (Carson et al., 2000). In all of the above studies, PTSDpatients exhibit a stronger HR and SC response to scripts than non-PTSDtrauma survivors.

In order to better understand the development and time course ofPTSD symptomatology and the psychophysiological expression ofthese symptoms, it is important to examine physiological reactivityacross a broad spectrum of traumatized populations. To date,traumatized populations have been studied from the victims ofterrorist attacks (DiGrande et al., 2008), the Middle Eastern theater ofcombat (Southwick et al., 1995; Shalev et al., 1992), and the SoutheastAsian theater of combat (Pitman et al., 1990). The present study is thefirst to examine psychophysiological responses and startle reflex in aCroatian PTSD population under resting conditions. The analysis ofpsychophysiological reactivity across a wide range of traumatizedpopulations may lead to the identification of hallmark symptomsassociated with specific trauma types and the tailoring of moreeffective treatment strategies.

2. Methods and materials

2.1. Participants

The study included 78 male participants: 45 PTSD patients treatedat the University Hospital Dubrava, Croatia, and 33 healthy controls.PTSD patientswere recruited from inpatient and outpatient services ofthe hospital. Age and gender-matched healthy volunteers wererecruited from the general population. All participants were informedof the research study and signed an informed consent form approvedby the Committee for Ethical Conduct of Research, University HospitalDubrava. The average age of the PTSD patients was 40.4±5.0 years,and the age of the controls was 38.1±8.9 years. The majority of theparticipants (89% of 78) completed high school. All PTSD patientswerewar veterans from the Serbo–Croatian war (1991–1995) and the PTSDwas due to combat-related traumatic events. Average combatexposure was 3 years.

Inclusion criteria: Confirmed diagnosis of chronic PTSD by theClinician-Administered PTSD Scale (CAPS; Blake et al., 1990), Croatianversion. Exclusion criteria: Current substance abuse or dependence;suicidal ideation; head injury or neurological disorder; psychotic andbipolar symptoms; hearing or visual impairment; liver or kidneydisease and cardio-vascular disease.

2.2. Psychophysiological assessment

The psychophysiological data was collected using Biopac MP150 forWindows (Biopac Systems, Inc., Aero Camino, CA). We recordedelectromyographic (EMG), electrodermal activity (EDA), and electro-

cardiogram (ECG) activity, and respiration. All psychophysiological datawere saved to the hard drive of a Windows XP computer. All data weresampled at 1000 Hz, digitized at 16 bit A/D resolution, and amplifiedusing the respective modules of the Biopac system. The acquired datawere filtered, rectified, and smoothed in MindWare software (Mind-Ware Technologies, Inc, Gahanna, OH) and exported for statisticalanalyses. TheEMGsignalwas amplifiedbya gain of a 1000, rectified, andfiltered with low- and high-frequency cutoffs at 28 and 500 Hz,respectively. A 60 Hz notch filter was also applied. The EDA signal wasamplified by2 μS/V and smoothed with a rolling filter of 100 data pointsper block. The ECG signal was amplified by a gain of a 1000, filteredwitha Hamming windowing function, and with a 60 Hz notch filter.

The following dependent variables were generated from theacquired data. For EMG, we measured the magnitude of the eyeblinkmuscle contraction during the startle response. For EDA, we analyzedtonic skin conductance level (SCL) and skin conductance response(SCR) to the startle probe. The dependent variables for ECG includedtonic heart-rate (HR in beats per minute) and heart-rate change inresponse to the startle probe (in interbeat-intervals, IBIs). Respiratorysinus arrhythmia (RSA) quantified by spectral analysis of the time-sampled interbeat interval series, according to the methods recom-mended by the SPR Committee on Heart Rate Variability (Berntsonet al., 1997). Because respiratory parameters can impact RSA, we alsorecorded respiration as recommended by the Committee. Respiratorymeasures were taken to evaluate the possibility that observedexperimental effects were potentially secondary to changes isrespiration; however, we did not analyze respiration amplitude per se.

EMG activity was recorded from Ag/AgCl electrodes (Biopac EL504pre-gelled electrodes for EMG) placed over the orbicularis oculi muscle.EDAwas assessed using two finger electrodes (Biopac EL507 electrodespre-gelledwith isotonic paste for EDA) on the index andmiddlefinger ofthe non-dominant hand. Heart-rate was recorded from electrodes(Biopac EL503 pre-gelled electrodes for ECG) placed on the chest, one1 cmbelow the right clavicle and the other below the rib cage on the leftside. Respiration rate was measured via an elastic chest band placedacross the sternum. All electrodes were Ag/AgCl disposable pre-gelledelectrodes, but each was specific to the data being acquired (BiopacSystems, Inc). This studywas a part of a larger baseline assessment studyfor future studies; while more stimuli were presented to the subjects,here we report on the pre- and post-startle phase of the study.Importantly, noneof the stimuli used in this studywere aversive and theparticipants were explicitly told that there would be no painful oruncomfortable events during the study. The data reported here werecollected from the start of the study visit: no interviews or otherpsychophysiological assessments preceded the study.

2.3. Startle procedure

The acoustic startle response (eyeblink component) was measuredvia EMG of the right orbicularis oculi muscle. Two 5 mm Ag/AgClelectrodes filled with electrolyte gel were positioned approximately1 cm under the pupil and 1 cm below the lateral canthus. Theimpedances for all subjects were less than 6 kΩ. Subjects were seatedand asked to look at a blank computer monitor approximately 1 m infront of them. All acoustic stimuli were delivered binaurally throughheadphones (Maico, TDH-39-P).

Once the electrodes were attached, the participants were seated ina chair with a computer monitor in front of them. The participantswere specifically told that no aversive or threatening stimuli would begiven; theywere asked only to relax and look at themonitor in front ofthem. The session began with a three-minute acclimation phaseconsisting of 70-dB [A] SPL broadband noise, which continued as thebackground noise throughout the session. During these 3 min theparticipants was instructed to relax and sit quietly. Since this was abaseline period during which the subjects acclimated to the researchenvironment and electrode placement, no startle probes were

Fig. 2. Mean+Standard Error tonic skin conductance by group and trial. Solid line = PTSD,dashed line = Control. ACL = acclimation phase, trials 1–7 startle trials.

266 T. Jovanovic et al. / International Journal of Psychophysiology 71 (2009) 264–268

delivered. However, during this time, resting levels of EDA and ECGwere acquired and sampled at five time points, with inter-trialintervals of 30 s. The acclimation phase was followed immediately bythe startle phase. The startle phase consisted of seven startle probetrials, with randomized inter-trial intervals of 9–22 s. The startle probe(noise burst) was a 108-dB [A] SPL, 40-ms burst of broadband noisewith instantaneous rise time.

2.4. Statistical analyses

The dependent variables were the following physiological mea-sures: Startle, Tonic Skin Conductance (SC), SC change, Tonic Heart-rate (HR), respiratory sinus arrhythmia (RSA), and HR reactivity.Startle magnitude was measured from the EMG of the orbicularis oculimuscle; we used the peak amplitude recorded between 20 and200 ms after the startle probe offset. Tonic SC was averaged over 6 sduring the 5 acclimation phase trials and for 6 s after the offset of thestartle probes. SC change was defined as the average increase (from a1 s pre-startle baseline) from 3 to 6 s after the startle probe offset.Tonic HR and RSA measures were averaged over 10 s during theacclimation trials and after each startle probe trial. HR reactivity wascalculated by averaging the IBI change (from the 1 s pre-startlebaseline) during the first 3 s after the startle probe offset.

In order to examine the effects of the startle probe on the abovephysiological variables we used a 2×7 mixed analysis of variance(ANOVA) with GROUP (PTSD vs. CONTROL) as the between-groupsfactor, and TRIAL (7 STARTLE TRIALS) as the within subject factor.Interaction effects were followed-up by one-way ANOVAs. Because wewere interested in differential habituation, we also tested the poly-nomial contrasts over the 7 startle trials separately for the two groups. Inorder to avoid sphericitywith the7 trials,weused theHuynh–Feldt termof the repeated-measures ANOVA. For the tonic measures (Tonic SC andTonic HR) we compared the last acclimation trial to the first startle trialusing a 2×2mixed ANOVAwith GROUP (PTSD vs. CONTROL) and PHASE(ACL vs. STARTLE). Alpha was set at 0.05. Effect sizes of the individualeffects are reported using partial Eta square (η2). All analyses wereconducted using SPSS 13.0 for Windows (SPSS, Inc.).

3. Results

3.1. Startle magnitude

We analyzed the peak startle amplitude using a 2×7 mixed ANOVAwith GROUP (PTSD vs. CONTROL) as the between-groups factor, andTRIAL (7 STARTLE TRIALS) as the within subject factor. We found asignificant main effect of TRIAL (F(6,76)=5.24, pb0.001, η2=0.06);however, there was no GROUP difference in startle magnitude and nointeraction of GROUP and TRIAL (see Fig. 1). Given that we were

Fig. 1. Mean+Standard Error startle response by group and startle trial number. Solidline = PTSD, dashed line = Control.

interested in differential habituation we performed polynomial con-trasts for each group separately. We found a significant linear term forthe CONTROL group (F(1,32)=9.17, pb0.01, η2=0.22), but no quadratictrend in the CONTROL group (F(1,32)=0.32, p=0.57, η2=0.01). The PTSDpatients demonstrated both a significant linear term (F(1,44)=6.32,pb0.05, η2=0.12) and a significant quadratic term with a greater effectsize (F(1,44)=7.64, pb0.01, η2=0.15). This quadratic curve suggests thatthe PTSD patients had impaired habituation in that the startlemagnitude to the later trials increased relative to the middle trials (seeFig. 1). In order to further explore this possibility, we categorized thesubjects as startle “Habituators” and “Non-Habituators”. “Habituators”were defined by having the lowest startle magnitude on the 7th trial; ifthe startle magnitude on the 7th startle trial was higher than any of theother trials, the subject was categorized as a “Non-Habituator”. Similarpsychophysiological response curves have been observed in PTSDpatients who do not show habituation (Rothbaum, B.O., personalcommunication). A Chi-square analysis of GROUP by HABITUATORshowed that 89%of the PTSDpatients (40 of the 45) did not demonstratenormal habituation compared to 66% of the CONTROLS (22 of 33), (χ2(1,N=78)=5.77, p=0.01).

3.2. Electrodermal activity

Tonic SC levels during the startle trials were analyzed using a 2×7mixed ANOVA with GROUP (PTSD vs. CONTROL) as the between-groups factor, and TRIAL (7 STARTLE TRIALS) as the within subjectfactor. Again, we found a significant effect of TRIAL (F(6,76)=16.50,pb0.001, η2=0.18), but no effect of GROUP or interaction of GROUPand TRIAL (see Fig. 2). The polynomial contrasts for both groupsrevealed significant linear, quadratic, and cubic terms, with the effectssizes for quadratic terms indicating the best fit (CONTROLS, (F(1,32)=20.92, pb0.001, η2=0.39), PTSD, (F(1,44)=14.38, pb0.001, η2=0.25).As seen in Fig. 2, the quadratic term describes the initial increase fromtrial 1 to trial 2 which reaches an asymptote at trial 4.

We also analyzed the tonic SC levels during the acclimation phaseand startle phase using a 2×2 mixed ANOVA with GROUP (PTSD vs.CONTROL) as the between-groups factor, and PHASE (ACL vs. STARTLE)as the within subject factor. This comparison is depicted by the insertgraph in Fig. 2. We again found a significant effect of PHASE (F(1,76)=70.16, pb0.001, η2=0.48), but no effect of GROUP or interaction ofGROUP and PHASE. Tonic SC was higher during the STARTLE phasethan the ACL phase for the CONTROLS (F(1,32)=24.56, pb0.001,η2=0.43) and PTSD, (F(1,44)=49.98, pb0.001, η2=0.53) groups.

SC change in response to the startle probes was analyzed with a2×7 mixed ANOVAwith GROUP (PTSD vs. CONTROL) as the between-groups factor, and TRIAL (7 STARTLE TRIALS) as the within subjectfactor. Fig. 3 shows the SC change data. As with the previous analyses,we found a significant effect of TRIAL (F(6,76)=20.68, pb0.001,η2=0.21), but no effect of GROUP or interaction of GROUP and TRIAL.

Fig. 3. Mean+Standard Error skin conductance change by group and startle trialnumber. Solid line = PTSD, dashed line = Control.

267T. Jovanovic et al. / International Journal of Psychophysiology 71 (2009) 264–268

Again, the polynomial contrasts for both groups revealed significantlinear, quadratic, and cubic terms. However, in this case the lineartrend had the greatest effect size (CONTROLS, (F(1,32)=30.88,pb0.001, η2=0.49), PTSD, (F(1,44)=32.87, pb0.001, η2=0.42), indicat-ing SC response habituation over trials.

3.3. Electrocardiogram activity

Tonic HR during the startle trials was analyzed using a 2×7 mixedANOVA with GROUP (PTSD vs. CONTROL) as the between-groupsfactor, and TRIAL (7 STARTLE TRIALS) as thewithin subject factor. Fig. 4shows the tonic HR data for GROUP and TRIAL. As opposed to theearlier analyses, we found a significant main effect of GROUP (F(1,76)=10.81, p=0.001, η2=0.12), but no effect of TRIAL and no interaction ofGROUP by TRIAL. The PTSD patients' HRwas on average 10 BPMhigherthan the HR of the controls. The analysis of GROUP by PHASE using a2×2 ANOVA revealed a significant effect of GROUP (F(1,76)=11.20,p=0.001, η2=0.13), as well as PHASE (F(1,76)=3.86, p=0.05, η2=0.05),but no interaction effect of the two variables. These data are depictedin the insert graph in Fig. 4. Follow-up comparisons of the PHASEwithin each group indicated that the PTSD patients tended to havelower HR during startle (F(1,44)=3.36, p=0.07, η2=0.07); there was noeffect of PHASE in the CONTROL group.

Analysis of RSA during the startle trials was analyzed using the 2×7mixed ANOVA with GROUP (PTSD vs. CONTROL) as the between-groups factor, and TRIAL (7 STARTLE TRIALS) as the within subjectfactor. The results mirror those of the HR data: there was a significantGROUP difference (F(1,76)=11.23, p=0.001, η2=0.13) with PTSDpatients having lower RSA compared to the CONTROL group (seeFig. 5). As with the HR there was no effect of TRIAL or TRIAL by

Fig. 4. Mean+Standard Error tonic heart-rate by group and phase. Solid line = PTSD,dashed line = Control. ACL = acclimation phase, trials 1–7 startle trials.

GROUP interaction. Analyses of GROUP by PHASE showed a significantmain effect of PHASE (F(1,76)=5.06, pb0.05, η2=0.06), as well asGROUP (F(1,76)=18.58, pb0.001, η2=0.20), but no interaction effect(see insert for Fig. 5). Follow-up comparisons showed that only thePTSD patients had a significant increase in RSA from the ACL phase tothe STARTLE phase (F(1,76)=5.08, pb0.05, η2=0.10).

The analysis of HR reactivity to the startle probes was analyzedusing the 2×7 GROUP by TRIAL ANOVA. This analysis did not revealany significant main effects or interaction effects. The polynomialcontrasts were also not significant.

4. Discussion

The present study is the first to report physiological assessments ina Croatian sample of combat-related PTSD. This study assessed restinglevels of EMG, EDA, and ECG activity as well as during startleresponses to loud noises. We did not find evidence of exaggeratedbaseline startle response in PTSD subjects compared to controls. Asmentioned above, the data in the literature are mixed in this regard(Orr et al., 2004); it appears that exaggerated startle might be limitedto a shorter period post trauma (Grillon and Baas, 2003) than is thecase for the Croatian veterans, whowere in combat about 10 years ago.It is also possible that the exaggerated startle response is onlyobserved in stressful contexts (Grillon et al., 1998). However, we foundthat PTSD patients did not habituate to the startle probe in the sameway that controls did. PTSD patients showed an initial decrease instartle magnitude to the startle probes but then increased respondinglater in the session. A significantly higher proportion of PTSD subjectsdid not show linear habituation curves compared to controls. Startlehabituation deficits have been reported in Israeli patients whodeveloped PTSD after trauma — the startle deficit was seen at anassessment 4 months after trauma indicating that it developed as partof the other PTSD symptoms (Shalev et al., 2000). However, severalstudies that examined startle responses in PTSD found normal levelsof startle habituation (Pitman et al., 1987, 1990; Orr et al., 1993). Inpart, the differences between the present study and previous workfrom the literature are due to methodological differences. Pitman andcolleagues typically use a startle probe of longer duration (500 ms)than that which we used (40 ms) and give more startle trials (15).Shalev and colleagues found that PTSD patients required more than 6trials to habituate while the controls required less than 4 on average.Therefore, by 15 trials, all subjects may show habituation. While thismay indicate that startle habituation deficits are too subtle to beclinically useful, if the difference is robust it may provide insight intoother aspects of PTSD symptomatology.

The degree towhich an individual habituates to the acoustic startleprobe may represent a trait characteristic as has been observed in

Fig. 5.Mean+Standard Error respiratory sinus arrhythmia (RSA) by group and trials. Thevalue plotted is the natural log of ms2. Solid line = PTSD subjects, dashed line = controls.ACL = acclimation phase, trials 1–7 startle trials.

268 T. Jovanovic et al. / International Journal of Psychophysiology 71 (2009) 264–268

previous work on conditioned fear extinction (Norrholm et al., 2006).In the Norrholm study, psychiatrically healthy individuals werecharacterized as “extinguishers” or “non-extinguishers” based onthe rate at which their fear-potentiated startle diminished during anextended extinction session. It is our hypothesis that, similar to fearextinction, an individual's rate of habituation to the startle probe isinfluenced by both intrinsic and extrinsic factors. It was anticipatedthat we would observe “non-habituators” in both the control andPTSD groups. The decreased number of “habituators” in the PTSDgroup compared to the control groupmay reflect “non-habituators” bytrait as well as “non-habituators” as a result of trauma exposure andthe development of PTSD symptoms. The current study cannotdistinguish whether impaired habituation is a pre-existing factor ora consequence of trauma exposure or PTSD.

We analyzed both tonic skin conductance level and skin con-ductance response to the startle probes. There were no groupdifferences in either measure of EDA; however, tonic SC increasedduring the startle trials in both groups, suggesting that general arousalmay have been sensitized in both groups. On the other hand, the skinconductance response to the startle probes habituated with eachstartle probe in all subjects. The discrepancy between EMG and SChabituation may reflect differences in how the startle reflex ismodulated compared to the autonomic nervous system control ofEDA, in that SC may show more rapid and sustained habituation tostimuli. As a marker of anxiety rather than arousal, startle responsemay be a more sensitive index of deficient habituation.

We found that heart-rate was elevated by approximately 10 beatsper minute in the PTSD patients compared to controls. However,heart-rate did not appear to be affected by startle stimuli since therewas no effect of phase. Neither group of subjects showed heart-ratehabituation over the trials. Elevated basal heart-rate has beenobserved in numerous studies with PTSD patients (Buckley andKaloupek, 2001; Hopper et al., 2006). This elevation appears to berelated to the chronicity of the disorder indicating greater cardiovas-cular risk for PTSD patients (Buckley and Kaloupek, 2001). Respiratorysinus arrhythmia, as an index of parasympathetic activity, was lowerin PTSD subjects. Furthermore, RSA increased during the startle phasein PTSD patients. While it is unclear why RSA increased in the morearousing context, these data suggest alterations in parasympatheticactivity in addition to the sympathetic system hyper-arousal;alterations that contribute to cardiovascular risk (Hopper et al.,2006; Porges, 2007).

A limitation of the study was the lack of a comparison group withequivalent levels of trauma exposure but without PTSD. Thus thegroup differences that we observed could be due to trauma itself,rather than to the disorder. Future studies should examine healthycontrol samples as well as trauma exposed individuals in order toparse out the differential contribution of trauma and PTSD.

In summary, the present study found deficits in startle habituationin Croatian PTSD patients, and replicated earlier findings of elevatedbasal heart-rate in PTSD subjects. This sample is unique in theliterature in that the patients are young enough to be largelymedicallyhealthy, and yet have been severely traumatized 10 years ago anddeveloped chronic PTSD. Showing consistent psychophysiologicalalterations associated with PTSD across diverse populations lendsmuch support for the stability of the association of PTSD symptomswith psychophysiological biomarkers.

Acknowledgements

This research was supported by the Croatian Ministry of Science,Education and Sport project: Integrative diagnostical model for thestress-related disorders (PI, D. Kozaric-Kovacic), and the National

Institutes of Mental Health Kirschstein National Research ServiceAward Individual Fellowship 1F32 MH070129-01A2 (PI, T. Jovanovic).

References

American Psychiatric Association, 1994. Diagnostic and Statistical Manual of MentalDisorders, 4th ed. APA, Washington (DC).

Berntson, G.G., Bigger, J.T., Eckberg, D.L., Grossman, P., Kaufmann, P.G., Malik, M.,Nagaraja, H.N., Porges, S.W., Saul, J.P., Stone, P.H., van der Molen, M.W., 1997. Heartrate variability: origins, methods, and interpretive caveats. Psychophysiology 34,623–648.

Blake, D.D., Weather, F.W., Nagy, L.M., Kaloupek, D.G., Klauminker, G., Charney, D.S.,Keane, T.M., 1990. A clinical rating scale for assessing current and lifetime PTSD. TheCAPS-1. Behav. Ther. 13, 187–188.

Buckley, T.C., Kaloupek, D.G., 2001. A meta-analytic examination of basal cardiovascularactivity in posttraumatic stress disorder. Psychosom. Med. 63, 585–594.

Cacciopo, J.T., Tassinary, L.G., Bernston, G.G., 2004. Handbook of Psychophysiology, 2nded. Cambridge University Press, Cambridge (MA).

Carson, M.A., Paulus, L.A., Lasko, N.B., Metzger, L.J., Wolfe, J., Orr, S.P., Pitman, R.K., 2000.Psychophysiologic assessment of posttraumatic stress disorder of Vietnam nurseveterans who witnessed injury or death. J. Consult. Clin. Psychol. 68, 890–897.

DiGrande, L., Perrin, M.A., Thorpe, L.E., Thalji, L., Murphy, J., Wu, D., Farfel, M., Brackbill,R.M., 2008. Posttraumatic stress symptoms, PTSD, and risk factors among lowerManhattan residents 2–3 years after the September 11, 2001 terrorist attacks.J. Trauma. Stress 21 (3), 264–273.

Grillon, C., Baas, J., 2003. A review of the modulation of the startle reflex by affectivestates and its application in psychiatry. Clin. Neurophysiol. 114, 1557–1579.

Grillon, C., Morgan 3rd, C.A., Southwick, S.M., Davis, M., Charney, D.S., 1996. Baselinestartle amplitude and prepulse inhibition in Vietnam veterans with posttraumaticstress disorder. Psychiatry Res. 64, 169–178.

Grillon, C., Morgan 3rd, C.A., Davis, M., Southwick, S.M., 1998. Effects of experimentalcontext and explicit threat cues on acoustic startle in Vietnam veterans withposttraumatic stress disorder. Biol. Psychiatry 44, 1027–1036.

Hopper, J.W., Spinazzola, J., Simpson, J.B., van der Kolk, B.A., 2006. Preliminary evidenceof parasympathetic influence on basal heart rate in posttraumatic stress disorder.J. Psychosom. Res. 60, 83–90.

Keane, T.M., Kolb, L.C., Kaloupek, D.G., Orr, S.P., Blanchard, E.B., Thomas, R.G., Hsieh, F.Y.,Lavori, P.W., 1998. Utility of psychophysiological measurement in the diagnosis ofposttraumatic stress disorder: results from a Department of Veterans Affairscooperative study. J. Consult. Clin. Psychol. 66, 914–923.

Kozaric-Kovacic, D., Borovecki, A., 2005a. Prevalence of psychotic comorbidity incombat-related post-traumatic stress disorder. Mil. Med. 170 (3), 223–226.

Kozaric-Kovacic, D., Borovecki, A., 2005b. Malingering PTSD. In: Corales, T.A. (Ed.), Focuson Posttraumatic Stress Disorder Research. Nova Science Publishers.

Kozarić-Kovačić, D., Borovečki, A., Udovičić, S., Kocijan-Hercigonja, D., 2003. SimuliraniPTSP. Društvena istraživanja 12, 541–561.

Metzger, L.J., Orr, S.P., Berry, N.J., Ahem, C.E., Lasko, N.B., Pitman, R.K., 1999. Physiologicreactivity to startling tones in women with posttraumatic stress disorder.J. Abnorm. Psychology 108, 347–352.

Morgan 3rd, C.A., Grillon, C., Southwick, S.M., Davis, M., Charney, D.S., 1996. Exaggeratedacoustic startle reflex in Gulf War veterans with posttraumatic stress disorder. Am.J. Psychiatry 153, 64–68.

Norrholm, S.D., Jovanovic, T., Southwick, S.M., Vervliet, B., Myers, K.M., Davis, M.,Rothbaum, B.O., Duncan, E.J., 2006. Conditioned fear extinction and reinstatementin a human fear-potentiated startle paradigm. Learning and Memory 13, 681–685.

Orr, S.P., Pitman, R.K., Lasko, N.B., Hertz, L.R., 1993. Psychophysiological assessment ofposttraumatic stress disorder imagery inWorldWar II and Korean combat veterans.J. Abnorm. Psychology 102, 152–159.

Orr, S.P., Metzger, L.J., Miller, M.W., Kaloupek, D.G., 2004. Psychophysiologicalassessment of PTSD, In: Wilson, J.P., Keane, T.M. (Eds.), Assessing PsychologicalTrauma and PTSD, 2nd ed. Guilford Press, New York (NY).

Pallmeyer, P., Blanchard, E.B., Kolb, L.C., 1986. The psychophysiology of combat-inducedpost-traumatic stress disorder in Vietnam veterans. Behav. Res. Ther. 24, 645–652.

Pitman, R.K., Orr, S.P., Forgue, D.F., de Jong, J.B., Claiborn, J.M., 1987. Psychophysiologicassessment of posttraumatic stress disorder imagery in Vietnam combat veterans.Arch. Gen. Psychiatry 44, 970–975.

Pitman, R.K., Orr, S.P., Forgue, D.F., Altman, B., de Jong, J.B., Hertz, L.R., 1990.Psychophysiologic assessment to combat imagery of Vietnam veterans withposttraumatic stress disorder. J. Abnorm. Psychology 99, 49–54.

Porges, S.W., 2007. The polyvagal perspective. Biol. Psychol. 74, 116–143.Shalev, A.Y., Orr, S.P., Pitman, R.K., 1992. Psychophysiological assessment to traumatic

imagery in Israeli civilian patients with posttraumatic stress disorder. Am.J. Psychiatry 49, 870–875.

Shalev, A.Y., Peri, T., Brandes, D., Freedman, S., Orr, S.P., Pitman, R.K., 2000. Auditorystartle response in trauma survivors with posttraumatic stress disorder: aprospective study. Am. J Psychiatry 157, 255–261.

Southwick, S.M., Morgan 3rd, C.A., Darnell, A., Bremner, J.D., Nicolaou, A.L., Nagy, L.M.,Charney, D.S., 1995. Trauma-related symptoms in veterans of Operation DesertStorm: a 2-year follow-up. Am. J. Psychiatry 152, 1150–1155.