Brain Serotonin Transporter Distribution in Subjects With Impulsive Aggressivity: A Positron Emission Study With [¹¹C]McN 5652

The study was approved by the institutional review boards of the New York State Psychiatric Institute, Columbia Presbyterian Medical Center, Mount Sinai Hospital, and the Bronx Veterans Affairs Medical Center. Written informed consent was obtained from each subject after explanation of the study procedures. All subjects were free of significant medical problems, had no current or past neurological disorder, had no history of loss of consciousness, were not pregnant or nursing, and had not taken any psychoactive medications in the 3 weeks preceding the PET scan (6 weeks for fluoxetine). Ten patients (five men and five women; mean age=35 years [SD=9, range=18–51]) meeting criteria for intermittent explosive disorder–revised (impulsive aggression) (24) were recruited through advertisements in local newspapers and referrals from outpatient psychiatrists at the Bronx Veterans Affairs Medical Center and Mount Sinai School of Medicine. Patients were considered eligible for the impulsive aggression group if they met research diagnostic criteria for intermittent explosive disorder–revised (25) and the DSM-IV borderline personality disorder “impulsiveness” criterion or the borderline personality disorder “self-damaging” criterion according to a “Module for Intermittent Explosive Disorder-Revised” (prepared by Coccaro et al., personal communication). The Structured Interview for DSM-IV Personality Disorders (26) was used for personality disorder diagnoses. Patients with a history of schizophrenia or other psychotic disorder according to the Structured Clinical Interview for DSM-IV Axis I Disorders (27) were excluded. All subjects with a history of alcohol/drug dependence or substance abuse that had been active in the preceding 6 months were also excluded from the study. Ten healthy comparison subjects (five men and five women; mean age=34 years [SD=8, range=24–49]) with no current or past DSM-IV axis I psychiatric disorder were recruited through advertisements in local newspapers.

The absence of pregnancy and medical and neurological abnormalities was confirmed by a review of the patient’s history and systems, physical examination, routine blood tests including pregnancy test, urine toxicology, and electrocardiogram recordings.

The standard (+)-McN 5652 was a gift from R.W. Johnson Pharmaceutical Research Institute. The precursor for the production of [¹¹C] (+)-McN 5652, (+)-McN butyryl thioester tartrate, was prepared from (+)-McN 5652 by a modified literature procedure (28) as described previously (29). Radiochemical and chemical purity of [¹¹C](+)-McN 5652 in saline was >95%.

PET imaging was performed with the ECAT EXACT HR+ (Siemens/CTI, Knoxville, Tenn.). Sixty-three slices covered an axial field of view of 15.5 cm, axial sampling of 3.46 mm, three-dimensional mode in plane and axial resolution of 4.4 and 4.1 mm, respectively, full width at half maximum at the center of the field of view. An arterial catheter was inserted in the radial artery after completion of the Allen test and infiltration of the skin with 1% lidocaine. A venous catheter was inserted in a forearm vein on the opposite side. Head movement minimization was achieved with a polyurethane head immobilization system (Soule Medical, Tampa, Fla.) (30). A 10-minute transmission scan was obtained before radiotracer injection. [¹¹C]McN 5652 was injected intravenously over 45 seconds. Emission data were collected in the three-dimensional mode for 120 minutes as 21 successive frames of increasing duration (three for 20 seconds, three for 1 minute, three for 2 minutes, two for 5 minutes, 10 for 10 minutes).

Following radiotracer injection, arterial samples were collected every 10 seconds with an automated sampling system for the first 2 minutes, and manually thereafter at longer intervals. A total of 32 samples were obtained per scan. Seven samples (collected at 2, 16, 30, 50, 70, 90, and 120 minutes) were further processed by high-performance liquid chromatography to measure the fraction of plasma activity representing unmetabolized parent compound (18).

A biexponential function was fitted to the seven measured unmetabolized fractions, which was then used to interpolate values between the measurements. The smallest exponential of the unmetabolized fraction curve, λ_par, was constrained to the difference between λ_cer, the terminal rate of washout of cerebellar activity, and λ_tot, the smallest elimination rate constant of the total plasma activity (31).

The input function was calculated as the product of total counts and interpolated unmetabolized fraction at each time point. The measured input function values (Ca(t) [mCi/ml]) were fitted to a sum of three exponentials from the time of peak plasma activity, and the fitted values were used as the input to the kinetic analysis. The initial distribution volume (V_bol [liters]) was calculated as the ratio of injected dose to peak plasma parent concentration. The clearance of the parent compound (C_L [liters/hour]) was calculated as the ratio of the injected dose to the area under the curve of the input function (32).

The high retention (>90%) of free [¹¹C]McN 5652 on the filter precludes the free fraction measurement of [¹¹C]McN 5652; therefore, plasma f₁ was not determined (18).

MRIs, three-dimensional spoiled gradient-recall acquisition in the steady state, were acquired on a GE 1.5-T Signa Advantage system, as previously described (33). MRI segmentation was performed within MEDx (Sensor Systems, Inc., Sterling, Va.), with original subroutines implemented in MATLAB (The Math Works, Inc., Natick, Mass.). Steps for MRI segmentation included correction for field inhomogeneities, fitting of the intensity distribution to a sum of three Gaussian functions, voxel classification, and post filtering (34).

Images were reconstructed to a 128×128 matrix (pixel size of 2.5×2.5 mm²). Reconstruction was performed with attenuation correction that used the transmission data and a Shepp 0.5 filter (cutoff 0.5 cycles/projection ray). Reconstructed image files were then processed with the image analysis software MEDx (Sensor Systems, Inc., Sterling, Va.). If indicated following visual inspection, frames were realigned to a frame of reference using a least-squares algorithm for within-modality coregistration (automated image registration) (35). The results of the frame-to-frame realignment were checked again visually. Following frame to frame registration, the 21 frames were summed to one dataset, which was coregistered to the MRI dataset using automated image registration (35). The spatial transformation derived from the summed PET registration procedure was then applied to each individual frame. Thus, each PET frame was resampled in the coronal plane to a voxel volume of 1.5×0.9×0.9 mm³.

Regions of interest (N=10) and region of reference (cerebellum) boundaries were drawn on the MRI according to criteria derived from brain atlases (36, 37) and published reports (38–41). Analysis was restricted to regions of interest where serotonin transporter density is high enough to provide a reliable signal. Regions of interest included the midbrain (encompassing serotonin transporter dense structures such as the raphe nuclei, substantia nigra, locus ceruleus, ventral tegmental area, and superior and inferior colliculi), thalamus, dorsal caudate, dorsal putamen, ventral striatum, amygdala, entorhinal cortex, hippocampus, parahippocampal gyrus, and the anterior cingulate cortex. A segmentation-based method was used for the neocortical regions, and a direct identification method was used for the subcortical regions (33). For bilateral regions, right and left values were averaged. The contribution of plasma total activity to the regional activity was calculated assuming a 5% blood volume in the regions of interest (42), and tissue activities were calculated as the total regional activities minus the plasma contribution.

Derivation of [¹¹C]McN 5652 regional tissue distribution volumes was performed with kinetic modeling using the arterial input function and a one-tissue compartment model. This model has been demonstrated to provide reliable estimates of total distribution volume for [¹¹C]McN 5652 (16, 18, 19). Total distribution volume (V_T [ml/g]), which is equal to the ratio of tissue to plasma parent activity at equilibrium, was derived as the K₁/k₂ ratio, where K₁ (ml/g/min) and k₂ (1/min) are the unidirectional fractional rate constants for the transfer of the tracer in and out of the brain, respectively (43, 44). Kinetic parameters were derived by nonlinear regression using a Levenberg-Marquart least-squares minimization procedure (45) implemented in MATLAB (The Math Works, Inc., South Natick, Mass.) as previously described (43). Given the unequal sampling over time (increasing frame acquisition time from the beginning to the end of the study), the least-squares minimization procedure was weighted by the frame acquisition time.

Derivation of serotonin transporter parameters was based upon the following assumptions: 1) given the negligible density of serotonin transporter in the cerebellum (46–48), cerebellum total distribution volume was assumed to be representative of equilibrium nonspecific binding; 2) the nonspecific binding did not vary significantly between regions.

Two measures of serotonin transporter availability were calculated. The binding potential (ml/g) was derived as the difference in total distribution volume between the region of interest and the cerebellum, the reference region. The relationship between binding potential and serotonin transporter receptor parameters is given by (49)

where B_max is the regional concentration of serotonin transporter (nmol/liter), and K_D is the in vivo affinity of the tracer for serotonin transporter (nmol/liter).

The specific-to-nonspecific equilibrium partition coefficient (V₃″ [unitless]) was derived as the ratio of binding potential to total distribution volume in the cerebellum. The relationship between V₃″ and serotonin transporter receptor parameters is given by (49)

where f₂ is the free fraction of the nonspecific distribution volume in the brain (f₂=f₁/ cerebellum V_T).

Between-group comparisons were assessed with unpaired two-tailed t tests with a probability of 0.05 set as the level of significance. The a priori hypothesis of this study related to serotonin transporter level in the anterior cingulate cortex, and therefore no correction for multiple comparisons was employed in the examination of this brain region. The other brain regions were also analyzed for between-group differences to determine the specificity of the difference seen in the anterior cingulate cortex. In order to correct for multiple comparisons, these analyses employed a univariate repeated-measures analysis of variance (ANOVA) with brain regions as the within-subject factor and diagnosis as the between-subject factor.

Demographic data for study participants are shown in Table 1. No significant group differences were observed on any demographic factor. One individual in the impulsive aggression group reported taking fluoxetine for 5 months several years before the study; all other participants were medication naive at the time of the scan. A significant degree of psychiatric comorbidity was present in the impulsive aggression group. This was particularly evident with regard to axis II diagnoses. All subjects had at least one personality disorder, the most common being borderline personality disorder (N=7), followed by paranoid (N=4), narcissistic (N=3), obsessive-compulsive (N=4), schizotypal (N=2), antisocial (N=2), histrionic (N=2), dependent (N=1), and avoidant (N=1) personality disorders. At the time of scanning, no subject had an active axis I mood disorder. However, several subjects were retrospectively diagnosed with comorbid mood illness. These included major depressive disorder (N=5), dysthymia (N=1), and bipolar II disorder (N=1). Anxiety disorders were also prevalent in this group, such as generalized anxiety disorder (N=1), adjustment disorder (N=1), social phobia (N=1), obsessive-compulsive disorder (N=1), and body dysmorphic disorder (N=1). In addition, there were two subjects with a history of alcohol dependence and two with a history of alcohol abuse; all had been in remission for greater than 6 months.

Scan parameters, including the injected dose, injected mass, and specific activity, did not differ between groups (Table 1). The plasma clearances of [¹¹C]McN 5652 were similar between the groups. No significant group difference was observed in [¹¹C]McN 5652 nonspecific distribution volume, measured as the cerebellar distribution volume.

Table 2 lists the average size for each region of interest in mm³. There was no difference in anterior cingulate cortex volume between the groups (t=0.18, df=18, p=0.86). For the other regions, a repeated-measures ANOVA showed a significant effect of region (F=121.4, df=1, 21, p<0.0001), no effect of group (F=0.04, df=1, 18, p=0.85), and no group-by-region interaction (F=0.23, df=1, 21, p=0.99).

After injection, the accumulation of [¹¹C]McN 5652 activity was consistent with the known distribution of serotonin transporter in the brain (Figure 1). The kinetic analysis converged in all regions for all subjects. Regional values of the unidirectional fractional rate constant of tracer delivery as well as the regional total distribution volume values are shown in Figure 2. There was no difference between groups in the tracer delivery values for the anterior cingulate cortex (t=–0.52, df=18, p=0.14). Examining all regions, a significant region effect was observed (repeated-measures ANOVA: F=153.0, df=1, 21, p<0.0001), with no difference between groups (repeated-measures ANOVA: F=0.06, df=1, 18, p=0.81) and no group-by-region interaction (repeated-measures ANOVA: F=0.635, df=1, 21, p=0.80). Similarly, for total distribution volume, no group differences were present in the anterior cingulate cortex (t=0.53, df=18, p=0.60). For the other regions, a region effect was observed (repeated-measures ANOVA: F=152.1, df=1, 21, p<0.0001), with no effect of group (repeated-measures ANOVA: F=0.09, df=1, 18, p=0.77) and no group-by-region interaction (repeated-measures ANOVA: F=0.60, df=1, 21, p=0.83).

As shown in Table 3, a significant difference in binding potential between healthy subjects and subjects with impulsive aggression was revealed in the anterior cingulate cortex. For all other regions, no group differences were detected (repeated-measures ANOVA: F=1.24, df=1, 18, p=0.28). A significant regional effect was seen (F=137.1, df=1, 21, p<0.0001), with no group-by-region interaction (F=0.48, df=1, 21, p=0.90).

As shown in Table 4, a significant difference in V₃″ between healthy subjects and subjects with impulsive aggression was revealed in the anterior cingulate cortex. Examination of all regions in a repeated-measure design resulted in no significant difference between the groups (F=2.83, df=1, 18, p=0.11), with a significant regional difference (F=163.6, df=1, 21, p<0.0001) and no region-by-group interaction (F=1.0, df=1, 21, p=0.43). The effect size of the reduction in [¹¹C]McN 5652 V₃″ in all regions examined is presented in Figure 2.

Given the finding of reduced serotonin transporter parameters in the anterior cingulate cortex of patients relative to healthy comparison subjects, we performed a post hoc analysis to determine if this finding was specific to the left or right hemisphere. Table 5 shows the results of this analysis. No difference was seen in total distribution volume in either the right or left anterior cingulate cortex in patients relative to healthy subjects. However, for the outcome measures binding potential and V₃″, patients with impulsive aggression had significantly lower values in the left anterior cingulate cortex than did the healthy subjects, whereas no significant difference was seen in the right anterior cingulate cortex. This finding was not due to differences between the right and left anterior cingulate cortex in healthy subjects, since total distribution volume, binding potential, and V₃″ values were not different in the left relative to the right anterior cingulate cortex. For patients with impulsive aggression, no differences in total distribution volume were seen when comparing the left anterior cingulate cortex with the right anterior cingulate cortex. However, for binding potential and V₃″, the values in the left anterior cingulate cortex tended to be lower than the right anterior cingulate cortex (paired t test, p=0.06). No significant differences were observed for any other region in this analysis.