Serotonin 1B Receptor Imaging in Alcohol Dependence

Abstract

Background

Although animal models suggest that alcohol dependence (AD) is associated with elevations in the number of serotonin-1B receptors (5HT_1BR), 5HT_1BR levels have not been investigated in people with AD. The selective 5HT_1BR antagonist radioligand, [¹¹C]P943, permits in vivo assessment of central 5HT_1BR binding potential (BP_ND) using positron emission tomography (PET). Because of its central role in AD, we were particularly interested in ventral striatal 5HT_1BR BP_ND values.

Methods

Twelve medication-free, recently abstinent (at least 4 weeks) patients with AD (mean age 35.2±10.1 years, 5 women) and 12 healthy control subjects (HC) (mean age 30.6±9.2 years, 5 women) completed [¹¹C]P943 PET on a high resolution research tomograph (HRRT). Individual MRI scans were collected to exclude individuals with anatomical abnormalities and for co-registration. Imaging data were analyzed using a multilinear reference tissue model.

Results

Ventral striatal 5-HT_1BR BP_ND values (2.01±0.57 and 1.55±0.09, 29% between-group difference, p=.006) were increased in AD compared to HC subjects. No influence of demographic or clinical variables or amount of injected radiotracer was observed.

Conclusions

This study provides the first evidence that AD in humans, like in rodent models, is associated with increased levels of ventral striatal 5HT_1BRs.

Introduction

Disturbances in the regulation of brain serotonin (5HT) systems have been implicated diminished inhibitory control of behavior, including pathological alcohol use (1-3). However, the specific mechanisms through which 5HT systems are dysregulated in alcohol dependence (AD) remain unclear. For example, recent studies have yielded conflicting results regarding the regulation of 5HT transporter (4-6) and 5HT_1A receptor function (4) in AD. Growing evidence suggests that serotonin-1B receptors (5HT_1BRs) modify the reinforcing, intoxicating, and discriminative stimulus effects of alcohol, and regulate its voluntary intake (7). The ventral striatum (VST) including globus pallidus and the nucleus accumbens area may be particularly important regions for the modulatory role of 5HT_1BRs on alcohol-related behaviors (8). In rodents, increased expression of 5HT_1BR in the VST modulates drinking behavior, i.e. increases ethanol consumption and shifts the animal's preference for a stronger concentration of ethanol (7). In humans, polymorphisms in the 5HT_1BR gene have been linked to antisocial AD (9), although not unequivocally (10).

The purpose of the current study was to determine whether, as predicted by animal models, human AD would be associated with increased 5HT_1BR levels in the globus pallidus and the nucleus accumbens area. The development of the selective 5-HT_1BR radioligand, [¹¹C]P943, permitted in vivo assessment of central 5-HT_1BR binding during positron emission tomography (PET) imaging.

Methods

Subjects

Twelve alcohol dependent subjects (5F, mean age 35.2±10.1 years, range 22-48) meeting DSM-IV criteria for current AD, and 12 healthy control subjects (5F, mean age 30.6±9.2 years, range 19-44) were recruited through public advertisement (Table 1.). Individuals reporting abuse or dependence of any other substance other than alcohol or nicotine were excluded. The protocol was approved by the Yale University School of Medicine Human Investigation Committee, the Human Subjects Subcommittee of the Veterans Affairs Connecticut Healthcare System, the Magnetic Resonance Research Center and the Yale New Haven Hospital Radiation Safety Committee. Written informed consent was obtained from all participants after full explanation of study procedures. All participants were evaluated by physical examination, electrocardiogram, standard laboratory tests, urine analysis and toxicology. Subjects with significant medical or neurological conditions, and with history of head injury with loss of consciousness were excluded from the study. AD subjects were admitted to the Clinical Neuroscience Research Unit (CNRU) at Connecticut Mental Health Center (CMHC) for detoxification and to ensure their medication-free status for at least 4 weeks before imaging. Urine toxicology and breathalyzer were collected repeatedly during the inpatient admission to the CNRU preceding the imaging studies, and on the days of the MRI and PET scans.

Table 1.

Demographics and Clinical Characteristics

Group	Healthy Control (N = 12)	Alcohol Dependence (N = 12)	P
Age (Years)	30.6 ± 9.2	35.2 ± 10.1	0.25
	range: 18 – 49 years	range: 22 – 51 years	-
Gender	5F; 7M	5F; 7M	1
Race	1Mix/1AS/10C	1Mix/4AA/7C	-
BMI	25.2 ± 4.0	25.5 ± 5.0	0.87
Smoking status	10 N; 2 S	3 N; 9 S	0.01*
Age at First Drinking (years)	-	13.3 ± 4.3	-
Total Years of Drinking	-	15.3 ± 9.9	-
Injected Dose (MBQ)	668.4 ± 30.2	695.3 ± 51.4	0.17
Specific Activity (MBQ/nmol)	9.8 ± 3.4	8.7 ± 2.6	0.39
Injected Mass (μg)	1.6 ± 0.8	1.8 ± 0.6	0.32

^-AA, African-American; AS, Asian-American; BMI, body mass index; C, Caucasian; F, female; M, male; Mix, mixed; N, nonsmoker; S, smoker.

^-Data represent mean ± SD. p Values by independent unpaired T-tests, except Gender and smoking status by Fisher's Chi-square tests.

Scanning and Imaging Procedures

Subject preparation for the PET scan consisted of indwelling venous catheter placement. A transmission scan using a ¹³⁷Cs point source was obtained before the emission scan. The PET scans were acquired for 120 minutes at rest using a single intravenous injection of high-specific activity [¹¹C]P943, a selective 5HT_1B receptor antagonist radiotracer (11), on an HRRT PET scanner (207 slices, resolution less than 3 mm full-width at half-maximum in 3D acquisition mode). Dynamic scan data were reconstructed with corrections (attenuation, normalization, scatter, randoms, and deadtime). Motion correction of PET data was performed by coregistering each reconstructed frame to an early summed image (0-10 min postinjection) using a 6-parameter mutual information algorithm and FMRIB's Linear Image Registration Tool (FLIRT, FSL 3.2, Analysis Group, FMRIB, Oxford, UK).

Magnetic resonance (MR) images were obtained for each subject on a Siemens 3 T Trio system to exclude individuals with anatomical abnormalities and for co-registration. A second summed image (0-10 min postinjection) was created from the motion-corrected PET data and registered to the subject's MR image, which, in turn, was registered (12-parameter affine transformation) to an MR template (MNI space). The globus pallidus/nucleus accumbens region of interest was taken from the template (Anatomical Automatic Labeling (AAL, (12) for SPM2) and applied to the PET data to produce time-activity curves for cerebellum. Pixel by pixel analysis was performed using the multilinear reference tissue model, MRTM2 (13) to produce images of binding potential (BP_ND) (14). The interpretation of BP_ND is f_ND*Bavail/Kd where f_ND is the tracer free fraction in a region without specific binding, Bavail is the unoccupied receptor concentration, and Kd is dissociation equilibrium constant of the tracer. Cerebellum was used as the reference region since it is practically devoid of 5HT_1B receptors (15). Assuming that there is no change in affinity or non-specific binding between subject groups, changes in BP_ND were interpreted as changes in receptor concentration. BP_ND values from MRTM2 have provided highly comparable results to those obtained with arterial input functions (16).

Statistical Analysis

Unpaired t-tests were used to compare clinical and demographic variables and a Mann-Whitney U test was used to compare 5HT1_BBP_ND values in the VST between AD and control groups. All tests were performed two-tailed, with results considered significant at p<0.05. Data are presented as means±SD.

Results

5HT_1BBP_ND was significantly elevated in the AD group relative to the healthy control group in the globus pallidus/nucleus accumbens area (2.01±0.57 and 1.55±0.09, respectively; 29% between-group difference; U=119.0, z=2.71, p=.006) (Fig.1), with effects found bilaterally (left hemisphere 35% between-group difference and right 24% between-group difference). None of the clinical measures or injection parameters correlated with 5HT_1BBP_ND in either group.

Figure 1.

In the upper row, the boxplot showing significant differences in ventral striatum (VST), including the globus pallidus and nucleus accumbens area [¹¹C]P943 binding potential (BP_ND) between patients with alcohol dependence (AD) and healthy control subjects (HC) is presented. In the lower row, the average [¹¹C]P943 BP_ND co-registered positron emission tomography (PET) images illustrate increased VST [¹¹C]P943 BP_ND in AD (right) relative to HC (left).

Discussion

The principal observation of this study was that ventral striatal 5HT_1BR BP_ND was increased in AD subjects who were scanned during early abstinence relative to a group of HC subjects. This increase was not directly related to features of the history of alcohol consumption among patients in this study. Our data do not clarify whether these differences are a pre-existing condition in AD patients or alternatively a consequence of the disorder.

The mechanism underlying the increase in ligand binding to 5HT_1BRs is not evident from the current data. The increases in 5HT_1BR BP_ND might reflect an overexpression of 5HT_1BRs or a higher affinity of these receptors. Alternatively, it is possible that low synaptic 5HT concentrations could leave more unoccupied 5HT_1BRs for the radioligand, [¹¹C]P943 binding and ligands that are sensitive to endogenous 5HT may be developed in the future. In either case, alterations in 5HT_1BR function might contribute to AD by influencing 5HT input to the VST via their role as 5HT terminal autoreceptors, dopaminergic input to the striatum via the role of these receptors as heteroreceptors on GABA terminals within the ventral tegmental area, and glutamatergic activity within the VST via heteroreceptors on corticofugal projections (17). We acknowledge that PET imaging cannot discriminate between pre- and postsynaptic receptors, and auto- and heteroreceptors and only provides a measure of the total number of receptors in a ROI. Emerging evidence, however, suggests that 5HT_1BRs in the nucleus accumbens shell neurons specifically are involved in the drug rewarding processes of ethanol (7) and other drugs of abuse (18).

A limitation of the present study is the difference in nicotine use between groups. Given the high prevalence of nicotine use among AD patients, and in order to study a representative cohort of AD, we decided to accept smokers into the study. We acknowledge, however, that our sample size is currently too small to specifically address the issue of co-morbidity between smoking and AD, and therefore do not know whether it affects the outcome of the study.

Altogether, the presented preliminary data suggest an important role for ventral striatal 5HT_1BRs in AD. Our data do not clarify, however, whether an overexpression of 5HT_1BRs is a biomarker for AD specifically, or is associated with drug reward responses as well as increased vulnerability to substance use disorders in general (19). Future research will be needed to determine whether these alterations are pre-existing or a consequence of AD and whether 5HT_1BRs might be productively targeted to treat AD.