Schizophrenia is associated with elevated amphetamine-induced synaptic dopamine concentrations: Evidence from a novel positron emission tomography method

In Vivo Microdialysis/PET Study. Subjects.

Four adult rhesus monkeys (Macaca mulatta) were used in this study (weight, 7.4–11.2 kg). They were housed individually on a 12-hr light/dark cycle. Food and water were available ad libitum. All procedures were carried out with strict adherence to the NIH Guide for the Care and Use of Laboratory Animals and had been approved by the National Institute of Mental Health Animal Care and Use Committee.

Microdialysis Probes and Probe Placement.

The microdialysis probes were constructed as described earlier (20, 21). Briefly, we used two fused silica barrels, the inner barrel extending 3–5 mm beyond the distal end of the outer barrel. A short piece (4–6 mm) of dialysis membrane tubing (AN 69 polyacrylonitrile membrane, 240 mm i.d., 300 mm o.d., 40,000 M_r cutoff; Hospal Dasco, Bologna, Italy) was placed over the extended portion of the inner barrel and glued to the inside surface of the outer barrel using cyanoacrylate instant adhesive. All probes were tested in vitro for dopamine recovery. The probes were placed in a beaker containing 10^–7 M dopamine (Sigma) in artificial cerebrospinal fluid buffered with 1.0 mM phosphate (pH 7.4) and supplemented with 0.15 mM ascorbate at 37°C. The probes were perfused at 1 μl/min flow rate for 2 hr before three 25-min collections were made for chromatographic analysis of recovered dopamine utilizing electrochemical detection. The mean recovery from three collections of each probe was considered as the criteria for selecting a probe for in vivo dialysis, with recovery values >30% used for in vivo microdialysis (21, 22). Stereotactic coordinates for the head of the caudate nucleus were determined for individual animals using MRI (23). Specially constructed guide holders were fixed to the skull under sterile surgical conditions to position and secure the probes into the targeted brain regions (24). The animals were allowed to recover for 2–3 weeks prior to any in vivo microdialysis experiments.

In Vivo Microdialysis.

On the morning of each study, monkeys were initially sedated with ketamine (ketamine hydrochloride, 10 mg/kg, i.m.), intubated, and anesthetized with gas isofluorane (1–3%). Animals were kept under light gaseous anesthesia throughout the procedure. Each animal was wrapped in a heating blanket (37°C) and vital signs (heart rate, temperature, and respiration) were monitored throughout the experimental session. Two probes were placed into the head of the caudate nucleus. Artificial cerebrospinal fluid (CSF; 147 mM Na⁺, 3 mM K⁺, 1.3 mM Ca²⁺, 1.0 mM Mg²⁺, 155 mM Cl⁻, buffered with 1.0 mM phosphate, pH 7.3–7.4) supplemented with 0.15 mM ascorbate was continuously perfused through the probes at 1.5 μl/min flow rate using a Harvard microinfusion pump (Harvard Apparatus). Dialysate samples (15 μl) were collected every 10 min in amber-colored glass vials and immediately frozen on dry ice. They were assayed for dopamine using microbore HPLC coupled to electrochemical detection (21). Analytical reagent-grade chemicals were used for preparation of artificial CSF (Mallinckrodt).

PET Procedure.

Animal studies were conducted on a Scanditronix (Uppsala) model PC 2048–15B scanner. Animals were positioned in a head holder developed for nonhuman primate PET studies, and slices were obtained in the coronal plane. Following a transmission scan, [¹¹C]raclopride (2–6 mCi; 1 Ci = 37 GBq) was administered in a bolus/constant infusion over 90 min (25). The bolus dose was equivalent to 40% of the total dose administered and produced nearly constant radioactivity levels by 25–30 min postinjection. Forty minutes after the raclopride bolus, the first dose of amphetamine, 0.2 mg/kg, was administered i.v. Beginning with the raclopride bolus, 31 PET scans (15 slices each; in-plane resolution, 7 mm; slice width, 6 mm) were obtained over 90 min, which concluded the first raclopride study. There was then an ≈90-min rest period until the second raclopride study commenced. The second study involved repeating the procedure from the first study except that 0.4 mg/kg of amphetamine was administered 40 min after the second raclopride bolus.

Dopamine Data Analysis.

Dopamine response was determined by percent change from baseline. Baseline for both 0.2 and 0.4 mg/kg amphetamine studies was determined from a mean of four consecutive samples collected immediately preceding the 0.2 mg/kg amphetamine administration. Postamphetamine dopamine effects were determined using a mean of four consecutive samples following the 0.2 and 0.4 mg/kg amphetamine administrations.

PET Data Processing and Analysis.

Image processing was performed with mirage software developed by the National Institutes of Health PET Center. The images corresponding to 0–5 min of raclopride infusion were added together to form a single “sum” image. Volumes of interest (VOIs) were drawn in the cerebellum and on the left and right striatum (consisting of caudate and putamen combined). After visual inspection, these VOIs were then overlaid onto their corresponding position in each of the 31 individual scans, and samples (mean pixel values) were generated for each VOI. Left and right striatal VOIs were averaged to a single striatal value. These data from five consecutive scans 25–40 min after injection and immediately before amphetamine injection (baseline) and five consecutive scans 65–90 min post-raclopride bolus administration (postamphetamine) were averaged. The specific binding was computed as striatum/cerebellum−1 (26).

Clinical Study. Subject Characteristics.

All subjects gave informed written consent to an institutional review board-approved protocol. Eleven patients with schizophrenia disorder diagnosed according to Diagnostic and Statistical Manual of Mental Disorders, 4th Ed. (DSM-IV) criteria participated in the study. Diagnoses were determined by a diagnostic conference utilizing data from a structured diagnostic interview (SCID) (27), clinical interview by a research psychiatrist, past psychiatric and medical records, and informant interviews. Patients were excluded if they had a history of illegal drug dependance and/or significant drug abuse, severe head trauma resulting in loss of consciousness, and any medical condition that made amphetamine administration contraindicated. The schizophrenic patients were ill for (mean ± SEM) 6.6 ± 1.8 years prior to study participation. Six of the 11 patients had either no or minimal previous antipsychotic drug exposure and were termed the neuroleptic-naive subgroup (four patients had no antipsychotic drug exposure and two had less than 1 week of low-dose neuroleptic exposure occurring more than 3 years before entry into the study). The remaining five were withdrawn from antipsychotic treatment prior to participation (days drug-free, mean ± SEM, 23.2 ± 7.2 days; range, 14–48 days). The control group was 12 healthy volunteers who were free of past and current psychiatric disorders as determined by a structured diagnostic interview. There were no significant differences (P > 0.4, mean ± SEM) between schizophrenics and controls, respectively, for age (32.4 ± 3.0 vs. 29.2 ± 2.6 years), gender (M/F, 8/3 vs. 9/3), and body weight (85.6 ± 3.8 vs. 80.0 ± 7.0 kg).

PET Scanning Protocol.

Clinical studies were conducted on a General Electric (GE) Advance scanner at the National Institutes of Health Clinical Center. Acquisitions were done with the interplane septa retracted and a wide axial acceptance angle. Each scan yielded 35 planes, 4.25 mm apart. The effective resolution of the reconstruction was 6 mm both axially and in-plane. A transmission scan was performed using two rotating ⁶⁸Ge sources and was used for attenuation correction.

Subjects were positioned in the scanner such that acquired planes would be parallel to the orbital-meatal line. Head movement was minimized with individually fitted thermoplaster masks, and patches were applied over the orbits to reduce incoming light. [¹¹C]raclopride (2–8 mCi) was administered as bolus/constant infusion over 2 hr. The bolus dose was 53% of the total amount administered. Beginning with the raclopride bolus, 29 scans were acquired over the 2-hr period every 3–5 min. Fifty minutes after commencement of raclopride administration, amphetamine (0.2 mg/kg i.v.) was infused over 60 sec. A 50-min baseline period (as opposed to 40 min used in experiment 1) was used because it provided a longer tracer equilibrium period. A longer scanning period was permitted by the enhanced sensitivity of the GE Advance scanner compared with the Scanditronix used in the monkey study. The dose of amphetamine was selected based on the nonhuman primate data that demonstrated robust increases in dopamine levels (see below) and because it was well tolerated in terms of cardiovascular and behavioral effects in schizophrenic patients. For ethical and safety reasons, we elected not to use higher doses so that the likelihood of inducing a psychotic exacerbation was minimized. Plasma samples for amphetamine levels were drawn 40 min after amphetamine administration.

Behavioral responses were examined with the Brief Psychiatric Rating Scale (BPRS; ref. 28). The BPRS is a commonly used clinical rating scale that is composed of 18 different psychiatric symptoms. Four of the items are psychotic symptoms (i.e., hallucinations, paranoia, delusions, and conceptual disorganization) and are grouped together as the BPRS psychosis subfactor. Both the BPRS total score, reflecting global symptomatology, and the BPRS psychosis subfactor score were used in this study. The scale was administered in a clinical interview by a research psychiatrist at baseline (before raclopride administration), 15 min after amphetamine administration (during-drug), and at the end of the study. The BPRS total score and the psychosis subfactor score were derived by summing each symptom (rated from 1, not present, to 7, severe). During-drug minus baseline change scores were used for correlative analyses with changes in raclopride binding ratios.

Data Processing and Analysis.

Image processing and PET data analysis were performed as described in the preclinical study with the following exceptions. Ratio data from five consecutive scans 30–50 min after injection and immediately before amphetamine administration (baseline) and five consecutive scans 75–100 min post-raclopride bolus injection (postamphetamine) were averaged (see Fig. 1). Individual group comparisons were conducted with paired and unpaired t tests where appropriate. Behavioral data (BPRS total score and psychosis subfactor score) were analyzed with a repeated-measures ANOVA with “time” (i.e., baseline, during-drug, and end-of-study ratings) and “group” (i.e., schizophrenics vs. controls) as factors. Change in behavior (from baseline to during-drug) were correlated with change in percent [¹¹C]raclopride striatal binding ratios using a Pearson’s correlation coefficient. All comparisons were two-tailed and group data were presented as mean ± SEM.

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The effects of amphetamine (0.2 mg/kg) on [¹¹C]raclopride binding in a patient with schizophrenia. Amphetamine was injected at 50 min. (Upper) “Sum” images of five [¹¹C]raclopride PET scans obtained before (Left) and after (Right) amphetamine injection. (Lower) Time-activated curves obtained in the striatum (green) and cerebellum (blue) with a bolus/constant infusion of [¹¹C]raclopride. This patient had a −20.6% reduction in pre- vs. post-[¹¹C]raclopride specific binding (striatum/cerebellum−1).