This study was supported in part by grants R0I-AA13892 (RS), K02-DA17232 (RS) and M01-RR00125 (Yale GCRC).
Reprint requests: Helen Fox, Department of Psychiatry, Yale University, School of Medicine, Substance Abuse Center, Connecticut Mental Health Center, 34 Park Street, New Haven, CT 06519; Fax:1-203-974-7076; E-mail: firstname.lastname@example.org
Background: Research has shown that exposure to stress/negative affect and to alcohol cues can each increase alcohol craving and relapse susceptibility in alcohol-dependent individuals. However, whether the emotional and physiological states associated with stress-induced and alcohol cue-induced craving are comparable has not been well studied. Therefore, this study examined the craving, emotional, and physiological responses to stress and to alcohol cues in treatment-engaged, 4-week abstinent, alcohol-dependent individuals using analogous stress and alcohol cue imagery methods.
Method: Twenty treatment-seeking, alcohol-dependent participants (18 males/2 females) were exposed to a brief 5-minute guided imagery procedure that involved imagining a recent personal stressful situation, a personal alcohol cue-related situation, and a neutral-relaxing situation, 1 imagery per session presented in random order. Alcohol craving, anxiety and emotion rating scales, cardiovascular measures, and salivary cortisol were compared across the 3 conditions.
Results: Exposure to stress and to alcohol cues each produced significant increases in alcohol craving, anxiety, and negative emotions and decreases in positive emotions. Stress-induced alcohol craving was significantly correlated with increases in sadness, anger, and anxiety ratings, but alcohol cue-induced craving was associated with decreases in positive affect (joy and neutral relaxed state) and increases in anxiety and fear ratings. Furthermore, stress increased systolic and diastolic blood pressure responses, but significant increases in salivary cortisol were only observed in the alcohol cue condition.
Conclusions: Although both stress and alcohol cues produce increases in anxiety associated with alcohol craving, each produced a dissociable psychobiological state involving subjective emotional, cardiovascular, and cortisol responses. These data could have significant implications for understanding the specific psychobiology associated with stress or alcohol cue exposure and their potential effects on alcohol relapse susceptibility.
Increasing attention in preclinical research has shown that brain stress and reward systems are altered with chronic alcohol abuse and that these changes may contribute to increased alcohol seeking or “craving” and vulnerability to relapse (Breese et al., 2005; Katner and Weiss, 1999; Koob, 2004; Weiss and Porrino, 2002). Animal studies using the reinstatement model have shown that both stress exposure, cue exposure and combined stress and cue exposure increase alcohol seeking in alcohol-experienced and dependent laboratory animals, with each set of cues involving specific neural circuits (Le et al., 2002, 2005; Liu and Weiss, 2002; 2003). This research has also underscored the need to differentiate between alcohol-experienced drinkers and those who are dependent on alcohol, with the alcohol-abstinent and nonabstinent state being important to study in understanding the circuitry and psychobiological state that increases motivation for drinking.
Similarly, in relation to alcohol cue-induced craving during abstinence, research has also shown that the degree to which cue exposure elicits state anxiety and irritability may mediate cue-induced craving in dependent drinkers (Chiang et al., 2002; McCusker and Brown, 1991). Other studies have shown that increased negative mood (Cooney et al., 1997; Litt et al., 1990; Rubonis et al., 1994) and state anxiety (Szegedi et al., 2000) is associated with enhanced cue-induced craving in alcoholic individuals, nonalcoholic individuals, and social drinkers. These data indicate that anxiety and negative affect can be a component of cue-induced craving under some conditions, which, in dependent individuals, may be reflected by a “shift” in hedonic set point with an increase in negative affect and emotionality (Koob and Le Moal, 1997, 2001). However, to our knowledge, there has been no systematic comparison of the subjective emotional and physiological state associated with stress and cue-induced craving, during early abstinence, a period of high vulnerability to relapse.
On the basis of the previous preclinical and clinical research, we hypothesize that in abstinent alcoholic individuals, stress and alcohol cues will produce increases in alcohol craving and negative affect with comparable levels of psychobiological activation when compared with a neutral/relaxed state. We tested this hypothesis in inpatient, treatment-engaged alcoholic individuals with 4 weeks of documented abstinence. Clarification of the specific psychobiological and affective components underlying stress and cue-related alcohol craving could lead to a better understanding of how alcohol dependence may affect the pathophysiology of alcohol craving and hedonic regulation. This, in turn, may have important implications for the treatment of alcoholism.
Twenty recently abstinent alcohol-dependent participants [18 male (M)/2 female (F)] were recruited from those applying for inpatient treatment via advertisements placed in local newspapers. The Structured Clinical Interview for DSM-IV, Axis I Disorders (SCID I; First et al., 1997), was administered to confirm criteria for alcohol dependence. Exclusion criteria included DSM-IV dependence for any drug, excluding nicotine. Participants using or requiring prescribed medications or otherwise not in good health were also ineligible. All participants underwent stringent medical assessments that included electrocardiography and laboratory tests of renal, hepatic, pancreatic, hematopoietic, and thyroid function. All participants gave written consent and the Human Investigation Committee of the Yale University School of Medicine approved the study.
A within-subjects design was used with condition 3 (stress, alcohol cue, and neutral) and timepoint 6 (repeated assessments over time in each laboratory session) as the repeated measures factors. The 3 conditions were presented on 3 consecutive testing days with only 1 stimulus presentation per day. Condition order was assigned randomly and counterbalanced. All participants had to be abstinent for 28 days before laboratory sessions, to allow for normalization of alcohol withdrawal-related physiological changes. Research staff and participants were blind to imagery condition and the content of the scripts assigned to each laboratory session.
Subjects were admitted to the Clinical Neuroscience Research Unit (CNRU) at the CMHC for study participation. The CNRU is a locked inpatient treatment research facility with no access to alcohol or drugs and very limited access to visitors. As subjects were treatment seeking, they participated in 2 to 4 weeks of standard group alcohol and drug counseling treatment for alcohol addiction during their inpatient stay.
During the first week of inpatient stay, subjects were administered structured baseline assessments measuring psychiatric and substance use history. In the second week, scripts for the guided imagery induction were developed as described in previous studies (Sinha et al., 2003). Briefly, the stress imagery script was based on subjects' description of a recent personal stressful event that was experienced as “most stressful.”“Most stressful” was determined by having the subjects rate their perceived stress on a 10-point Likert scale where 1=“not at all stressful” and 10=“the most stress they felt recently in their life.” Only situations rated as 8 or above were accepted as appropriate for script development. The alcohol cue script was developed by having subjects identify a recent situation that included alcohol-related stimuli and resulted in subsequent alcohol use (e.g., being at a bar and watching others drink). A neutral script was developed from the subjects` description of a personal nondrug-related neutral-relaxing situation. Participants related the details of each scenario to a clinical interviewer, who subsequently devised a personalized script of the event using the standardized techniques reported in Sinha et al. (2003). All scripts were then recorded onto an audiotape by the interviewer for use in the laboratory sessions.
On each testing day, subjects abstained from breakfast and were brought into the testing room at 7:45 am. The laboratory sessions were conducted early in the morning to eliminate any effects of dietary variation among the participants. All subjects were allowed an morning smoke break at 7:30 am to ameliorate any nicotine craving. A blood pressure cuff was placed on the subject's preferred arm to monitor blood pressure and a pulse sensor was placed on the subject's forefinger to obtain a measure of pulse. Self-reports of craving and anxiety were completed after set up. This was followed by a 1-hour adaptation period during which the subjects were instructed to practice relaxation. At 9:00 am, subjects were provided headphones and given the following instructions for the imagery procedure: “Close your eyes and imagine the situation being described, ‘as if’it were happening right now. Let your body and mind get completely involved in the situation, doing what you would do in the real situation.” The length of each script was approximately 5 minutes. Heart rate and blood pressure were continuously monitored during the 5-minute baseline period, imagery, and for the 5 minutes immediately following recovery.
Alcohol craving, mood ratings, heart rate, blood pressure, and salivary measurements were made straight after set up, at 2 baseline timepoints: one 20 minutes before imagery presentation (−20 timepoint) and one 5 minutes before imagery presentation (−5 timepoint), and again immediately following imagery (0 timepoint) and periodically every 15 minutes after the imagery period (+15, +30, +45, +60, and +75 timepoints). After the final assessments at 10.35 am, the blood pressure cuff and pulse sensor were removed and breakfast was served.
Subjective Measures: Alcohol Craving. Participants were requested to rate the intensity of their desire to have an alcoholic drink at the current time. The desire for using alcohol was assessed using a 10-point visual analog scale (VAS) in which 0=“not at all” and 10=“extremely high.”
Anxiety. Participants were requested to rate how tense, anxious, and/or jittery they felt at that moment using a similar 10-point VAS anchored as above.
Subjective Measures: Differential Emotion Scale (DES). An abbreviated 30-item version of the DES (Izard, 1972) was administered to assess specific positive and negative emotional states at baseline and immediately following imagery and recovery. The scale requires participants to rate the extent to which various emotional words were able to describe their feelings at the present time on a 5-point Likert scale. Items were attributed to 5 subscales, comprising fear, anger, joy, sadness, and neutral/relaxed state. Scores ranged from 5 to 25 on each subscale.
Cardiovascular Measures. An SD-700 Monitor (IBS Corporation, Waltham, MA) was used to assess blood pressure. A pulse sensor was attached to the subject's finger and connected to the SD-700 Monitor to provide a continuous measure of pulse.
Salivary Cortisol Assessment. Saliva samples were collected as a marker of hypothalamic–pituitary–adrenal (HPA) axis response (Fox et al., 2006) using a cotton swab that participants were instructed to place between their tongue and cheek for approximately 2 to 3 minutes until the swab was completely saturated. Participants were required to focus their gaze on a segment of lemon being squeezed 2 feet away from them to stimulate saliva flow. The saliva swab was then collected in plastic tubes, which were placed directly on ice and stored at −20°C. Saliva samples were assayed in duplicate to obtain levels of free cortisol following standard radioimmunoassay kits with no modifications (Coat-A-Count Cortisol Kit, Diagnostic Products Corporation, Los Angeles, CA) at the Yale General Clinical Research Center's Core Laboratories. The intra-assay coefficients of variation ranged from 3.0 to 5.1%.
Linear mixed effect (LME; Laird and Ware, 1982) models were implemented to analyze the data, both at baseline (−5 timepoint) and imagery response, using SAS version 8 (SAS Institute, Cary, NC). Baseline responses were assessed using condition (stress, alcohol cue and neutral) as the fixed effect factor and subjects as the random effect factor. For response analyses, the within-group factors of condition (stress, alcohol cue, and neutral) and timepoint (varying levels including baseline) represented the fixed effects. Subjects represented the random effect. Linear mixed effects are particularly well suited when the design calls for repeated measurements within the same individual that can lead to positive correlations between measurements. Such models are also useful if there are missing data, as it prevents the exclusion of subjects with missing data points (Littell et al., 1996).
A series of Spearman's rho correlational coefficients were also used to assess the associations between stress and cue-induced alcohol craving, the emotional subscales of the DES, and physiological responses. An averaged response across timepoints was computed for each measure to assess correlations.
Participants comprised 18 M and 2 F. Fifteen were Caucasian, 4 African Americans, and 1 Hispanic. The mean age for the total sample was 38.2±7.4 years, and the mean number of years in education was 13.1±1.6. Seventeen participants smoked regularly, smoking a mean of 15.1±7.8 cigarettes per day. On average, participants had consumed alcohol for 18.1±7.7 years, having consumed their first alcoholic drink at 14.9±3.7 years old and consuming alcohol on a regular basis at 19.4±4.5 years old. All participants had consumed alcohol on an average of 57.9±29.3 days in the 90 days before entering inpatient treatment, and the mean number of drinks consumed per occasion was 15.7±7.2. This amounted to a mean of 926.9±711.7 drinks consumed per person, in the 90 days before treatment.
In addition, 5 (25%) of the participants also met DSM-IV criteria for lifetime anxiety disorder and 2 (10%) met criteria for lifetime mood disorders.
As no main effect of condition was observed for any of the dependent measures, baseline was included in the LME models.
Response to Stress and Drug Cue Imagery
Subjective Measures. A main effect of condition was observed for both self-reported alcohol craving [F(2, 38)=25.0, p<0.0001] and anxiety [F(2, 38)=18.8, p<0.0001]. Higher alcohol craving was reported in the stress (p<0.0001) and alcohol cue (p<0.0001) conditions compared with the neutral condition, and higher craving was also reported in the alcohol cue compared with stress condition (p=0.02; see Fig. 1A). Significantly higher anxiety was also reported in the stress and alcohol cue conditions compared with the neutral (p<0.0001 in both cases; see Fig. 1B).
Findings from the DES indicated that a main effect of condition was observed for all negative emotions (see Fig. 1C–1E): anger [F(2, 38)=20.1, p<0.0001], fear [F(2, 38)=5.3, p<0.009], and sadness [F(2, 38)=18.1, p<0.0001]. In all cases, reported negative emotion was significantly higher in the stress condition (p<0.0001 for anger; p=0.003 for fear; p<0.0001 for sadness) compared with the neutral condition. Significantly higher fear (p<0.03) and a trend for sadness (p<0.06) were also reported in the alcohol cue condition compared with the neutral condition. Significantly higher anger (p<0.0001), fear (p<0.03), and sadness (p=0.0003) were also reported in the stress compared with the alcohol cue condition.
In relation to positive emotion, a main effect of condition was also observed for joy [F(2, 38)=9.7, p<0.0004] where significant decreases were observed in the stress condition (p=0.0003) compared with the neutral condition, and in the stress condition compared with the alcohol cue condition (p=0.001; see Fig. 2A). A main effect of condition was also observed for scores on the neutral/relaxed scale [F(2, 38)=6.7, p=0.003] where participants reported significantly lower scores in the stress (p=0.0008) and alcohol cue (p=0.03) conditions, compared with the neutral conditions (see Fig. 2B).
Significant Condition × Timepoint Interaction
Significant condition × timepoint interactions were also observed for all self-report measures (p<0.0001, in all cases). Extended analyses indicated that higher alcohol craving was reported in the stress and cue conditions compared with the neutral condition at the imagery timepoint (0; p<0.0001 in both cases) and the initial recovery timepoint (+15; p=0.03 in both cases). Higher alcohol craving was also reported in the cue compared with the neutral condition at both the second (+30; p=0.03) and third (+45; p=0.03) recovery timepoints.
Significantly higher anxiety was reported in both the stress and cue conditions compared with the neutral condition at the Imagery timepoint (0; p<0.0001 in both cases). Increased anxiety was also reported at the initial recovery timepoint (0) in the stress compared with the neutral condition (p=0.04) and in the cue compared with neutral condition at the second recovery timepoint (+30; p=0.04).
In the case of alcohol craving and anxiety, the significant condition × timepoint interactions show that participants took significantly longer for their scores to return to baseline levels. In the case of all other subjective measures, differences in condition were observed at the Imagery timepoint only.
A main effect of condition was observed for both SBP [F(2, 38)=3.9, p<0.03] and DBP [F(2, 38)=5.7, p=0.007]. In both cases, significantly higher blood pressure was seen in the stress compared with the neutral condition (p=0.01 for SBP and p=0.004 for DBP). Participants also showed significantly increased DBP in the stress compared with alcohol cue condition (p=0.009; see Fig. 3A and 3B). No differences between conditions were observed for heart rate.
Salivary Cortisol Measures
A main effect of condition was observed [F(2, 38)=11.4, p<0.0001], indicating that participants showed increased levels of cortisol in the alcohol cue condition compared with both the neutral condition (p<0.0001) and the stress condition (p=0.001; see Fig. 3C).
Significant correlations were obtained between stress-induced alcohol craving and anxiety (r=0.65; p<0.002), anger (r=0.45; p=0.04), fear (r=0.44, p=0.05), and sadness (r=0.75, p=0.0001) when averaged across each timepoint. No significant associations were observed between stress-induced alcohol craving and the positive subscales joy and relaxed state, heart rate, blood pressure, and cortisol responses.
Significant correlations were also obtained between cue-induced alcohol craving and anxiety (r=0.53; p<0.02), fear (r=0.67, p=0.001), joy (r=−0.59, p=0.006), and relaxed state (r=−0.52; p<0.02) when averaged across each timepoint. No significant correlations were found between alcohol cue-induced craving and responses of anger, sadness, heart rate, blood pressure, or cortisol responses.
Findings from the current study indicate that both stressful and alcohol cue-related imagery significantly increase alcohol craving in recently abstinent alcohol-dependent individuals compared with a neutral/relaxing imagery condition. In both the stress and cue conditions, these consistent increases in craving also occurred alongside significant increases in negative affect and significant decreases in positive affect. This enhanced stress-induced and alcohol cue-induced craving state was also accompanied by physiological arousal where a simultaneous increase in blood pressure was seen in the stress condition and a simultaneous increase in salivary cortisol was observed in the alcohol cue condition.
Exposure to both personal stress and alcohol cue imagery significantly increased emotions of anxiety, anger, fear, and sadness and significantly decreased feelings of joy and relaxation in the group. Importantly, these changes in emotion were accompanied by increases in stress and cue-induced alcohol craving. Stress and cue-induced increase in alcohol craving in recently abstinent abusers corroborates prior research that has identified both as being important to alcohol relapse in addicts (Breese et al., 2005; Cooney et al., 1997; Sinha 2001; Weiss and Porrino 2002) as well as being an impetus for increased alcohol consumption in social drinkers (de Wit et al., 2003). A prior study by Sinha et al. (2000) using the same methodology, also found comparable increases in stress and cue-induced fear, sadness, anger, and anxiety in a group of recently abstinent cocaine abusers. This is the first study to report similar findings in abstinent alcoholic individuals across 2 different cue environments presented using an analogous paradigm.
Importantly, our correlational analyses also showed that although both stress and cue-induced alcohol craving states increased anxiety and fear, other qualitative differences were observed in relation to emotional distress. While stress-induced craving was significantly associated with an increase in negative emotions, with the highest correlations for sadness, followed by anxiety, anger, and fear, alcohol cue-induced craving was predominantly associated with an anxiety/fear state and a decrease in positive emotion (joy and relaxed state). This supports the fact that craving is a multifaceted phenomenon with considerable variance in the underlying psychobiology of the craving state associated with stress versus that related to alcohol cues (Feldman et al., 1998, 2000; Melendez et al., 2002). The possibility that varying components of emotional distress may underlie the stress and cue-related craving state is therefore concordant with brain imaging studies that have identified neural correlates specific to emotional states such as sadness, anger, and fear (Murphy et al., 2003; Phan et al., 2004) as well as studies that have also shown emotion-specific blood pressure responses in major categories of emotions (Montoya et al., 2005; Sinha et al., 1992). In view of this, our findings may suggest that the neural and physiological correlates of emotional distress underlying stress-induced and cue-induced craving may also be different.
In physiological measures, the current findings showed an interesting dissociation between response to stress and alcohol-cue imagery exposure in relation to both blood pressure and salivary cortisol. While a significant increase in SBP and DBP was observed following exposure to stress imagery, no similar effects of alcohol cue-related increases in blood pressure were seen. Conversely, increased cortisol levels were observed in the alcohol-cue condition only.
Transient withdrawal hypertension has been well documented as a common feature of alcohol abstinence (Ceccanti et al., 2006; King et al., 1994) and alcohol consumption has been associated with elevated blood pressure and hypertension in several subsets of drinkers including light/social drinkers (Nakanishi et al., 2002), heavy drinkers (Gillman et al., 1995; Marmot et al., 1994), and alcohol-dependent individuals (King et al., 1994). These transient blood pressure dysregulations have been associated with exaggerated cardiovascular stress responses (Bernardy et al., 2003; King et al., 1996). Stress-induced increases in blood pressure in the current study are consistent with these findings and indicate that a stress-related hyperarousal response may also represent an important component of the craving state in alcoholic subjects during early abstinence. Consistent with the current findings, recent studies have also found that although robust increases in cue-induced subjective craving have been observed in alcoholic patients, concomitant changes in physiological markers such as blood pressure, skin conductance, and temperature have not always been shown to be specific to alcohol cues (Reid et al., 2006; Thomas et al., 2005).
Findings from the salivary cortisol data indicated that an increased response was only observed in the alcohol cue condition. Interestingly, no significant difference was observed between the stress and neutral imagery condition. In relation to this latter point, it is relatively well established that abstinent alcoholic individuals experience blunted HPA axis response to stress and anxiety (Lovallo et al., 2000). Previous research has shown HPA-axis hyporeactivity in abstinent alcoholic individuals in response to a range of stress-related pharmacological challenges (Adinoff et al., 2005) as well as a variety of stressors including cold pressor and mental arithmetic (Errico et al., 1993), combined mental arithmetic and isometric handgrip (Bernardy et al., 1996), and public speaking (Lovallo et al., 2000). Our findings indicating a lack of stress-induced HPA response is consistent with these findings and different from the response that we previously documented with abstinent cocaine-abusing individuals (Sinha et al., 2000, 2003).
While prior evidence has shown that cue-induced cocaine craving may be characterized by HPA activation (Berger et al., 1996; Sinha et al., 2000, 2003), findings from the current study indicate that increased cue-related cortisol levels may also be an important component of the cue-induced craving state in abstinent alcohol abusing individuals. This increase in alcohol cue-induced cortisol may reflect a more conditioned, appetitive cue response and, as such, is consistent with preclinical studies that have assessed the mediating role of glucocorticoids on mesolimbic dopamine and the rewarding properties of drugs (Marinelli et al., 1998; Marinelli and Piazza, 2002). In humans, a recent study showed that increased cortisol levels were associated with amphetamine-induced dopamine release in regions of the striatum and putamen as well as increases in positive drug effects (Oswald et al., 2005). Similarly, in cigarette smokers, dose-related increases in HPA hormones have also been reported alongside rapid increases in positive subjective ratings (Mendelson et al., 2005). In support of this, cortisol release has not only been associated with dysphoria but also elation and excitation (Brown et al., 1993) as well as emotional arousal and conflictual situations in healthy females (Gerra et al., 1996).
It may be the case, then, that HPA activation in the cue condition is the result of a combined appetitive desire for the drug as well as a distress state possibly related to deprivation and “wanting” of drug. It is also important to note that a wealth of previous studies have shown that high expectation of alcohol consumption increases both autonomic arousal and the desire to drink in alcohol-dependent individuals (Kaplan et al., 1983, 1984; Laberg, 1986; Turkkan et al., 1989). Findings from the current study suggest that the arousal of stress system markers may also underlie the cue-induced craving state in the context of very low alcohol consumption expectancies. In the current study, this was due to the fact that participants were staying in a locked inpatient unit with no access to alcohol.
The variations in the emotional components associated with stress versus cue-induced alcohol craving may be an appropriate reflection of the stress and cue-related physiological dissociations observed in this study. While increases in anger and sadness may be a probable component of stress-related alcohol craving, decreases in joy and relaxed state may be considered more likely responses to an increase in alcohol cue-induced anticipatory cortisol levels. These interpretations must, however, remain tentative pending further research due to the fact that very few studies have actually assessed the specific role of mood, emotionality, and subjective experience in mediating stress and cue-related cortisol release in normal healthy volunteers (Reuter, 2002). Moreover, prior studies have emphasized that the magnitude of stress-induced cortisol response may be related to a multitude of environmental variables including the degree of emotion experienced (Nejtek, 2002), the persistence of an event, prior frequency of an event, habituation (van Eck et al., 1996) as well as individual extent of subjective and/or physiological response (Szegedi et al., 2000).
The limitations of the present study relate to the absence of a healthy control group, which may be required to assess more accurately the potential changes in stress and alcohol cue-induced negative affect and pathophysiology associated with the craving state, as well as ascertain the extent to which an actual “shift” in reward set point may have occurred. Despite these caveats, the findings clearly show that an increase in subjective negative affect and a decrease in positive affect are associated with both stress and alcohol cue-related imagery compared with neutral imagery in recently abstinent alcoholic individuals. The findings also show that stress and alcohol cues each elicit alcohol craving with a dissociable emotional and physiological state in abstinent alcoholic individuals, which is different from that observed in abstinent cocaine–abusing individuals (Sinha et al., 2000). Clarifying the emotional and physiological components underlying stress and alcohol cue-related craving may also shed light on some of the mechanisms associated with relapse and clinical outcome. For example, in cocaine-abusing individuals, stress-induced HPA arousal has been significantly associated with drug-use factors relating to relapse (Sinha et al., 2006); however, blunted HPA responses have been associated with alcohol relapse (Adinoff et al., 2005; Junghanns et al., 2005). Moreover, while the negative affect component of the craving state may provide an important focus for the reduction of both stress and cue-related relapse to alcohol, highlighting dissociable stress and cue-related emotional distress states may assist in the development of better-tailored pharmacological and clinical interventions to target both reward and distress components of the craving state.
We also wish to thank the staff at the Substance Abuse Treatment Unit, Clinical Neuroscience Research Unit, and the General Clinical Research Center at Yale University School of Medicine and those at the Yale General Clinical Research Center Laboratory for their assistance in completing this study.