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NIMH SPECT Lab., CBDB/NIMH/NIH, 10 Center Dr. Rm 4C216, Bethesda, MD 20792-1364, USA
Semple et al (1999) report a reduction in vivo of 123I-labelled 2ß-carbomethoxy-3ß-(4-iodophenyl)tropane (ß-CIT) uptake in the cerebral cortex of 3,4-methylenedioxymethamphetamine (MDMA, ‘ecstasy’) users. They interpret this observation to be an indication of a decrease in serotonin transporters in the cortex of MDMA users. However, there are serious methodological concerns with this interpretation of their data.
It has been demonstrated that the radioligand [123I]ß-CIT binds with high affinity to dopamine, serotonin and noradrenaline transporters in human brain (Farde et al, 1994; Laruelle et al, 1994). In the case of the serotonin transporter, in vivo displacement of ß-CIT binding by selective serotonin reuptake inhibitors (SSRIs) has established that specific (displaceable) binding occurs in brainstem and thalamus (Laruelle et al, 1993; Pirker et al, 1995; Tauscher et al, 1999). However, this is not true for the cerebral cortex. Indeed, Laruelle et al (1993) observed that [123I]ß-CIT uptake in cortical areas was unaffected by citalopram administration in non-human primates. Similarly, recent SSRI displacement studies of [11C]McN-5652, a selective serotonin transporter radioligand for positron emission tomography (PET) imaging, failed to observe specific binding in the cerebral cortex (Parsey et al, 1999). The lack of evidence for specific binding to serotonin transporters in the cerebral cortex in vivo is not surprising when one considers the paucity of these transporters in primate cortex (Jagust et al, 1996). We are aware of only a single report of apparent displacement of ß-CIT by citalopram in primate cortex: a PET study of [11C]ß-CIT uptake in two cynomolgus monkeys (Farde et al, 1994). However, the shape and time-scale of the binding curves for the cynomolgus monkeys are strikingly different from those observed in other non-human primate species (Laruelle et al, 1993) and in humans (Farde et al, 1994; Laruelle et al, 1994; Pirker et al, 1995). This discrepancy is particularly pronounced for the cortical curve, and one wonders to what extent these data may be relevant to human studies. Be that as it may, the bulk of the evidence indicates that serotonin transporters are present in sufficient density to be measured reliably with [123I]ß-CIT only in the thalamus and brainstem, and not the cerebral cortex. The region of choice is the raphe area of the brainstem because the thalamus may have a substantial admixture of noradrenaline transporters (Farde et al, 1994) and because it is difficult to avoid scattered radiation from the much greater accumulation of activity in the striatum in a thalamic region of interest. We have found this to be true in our studies of serotonin transporters with [123I]ß-CIT, and we have observed that uptake in cortical regions does not differ significantly from the non-displaceable (non-specific) uptake seen in the cerebellum (Heinz et al, 1998). At extended times (>4 hours post-injection in humans), when specific binding to serotonin transporters in the brainstem approaches a near-equilibrium plateau and non-specific uptake continues to washout throughout the brain, it becomes clear that cortical uptake is ‘tracking’ that of the cerebellum.
This latter point raises a further methodological concern. Semple et al (1999) imaged [123I]ß-CIT uptake at 90 minutes post-injection hoping to assess radioligand binding to serotonin transporters. However, near-equilibrium conditions for ß-CIT at serotonin transporters are not established in human brain earlier than about four hours post-injection (Laruelle et al, 1994; Pirker et al, 1995). Once near-equilibrium has been established, [123I]ß-CIT binding to serotonin transporters in the brainstem is quite stable and persists well into the following day (Laruelle et al, 1994; Pirker et al, 1995). Measurements at extended times of [123I]ß-CIT activity in human brainstem (following decay correction and subtraction of non-specific uptake) are simply proportional to the density of serotonin transporters (Laruelle et al, 1994). Unfortunately, this is not the case for the measurements of Semple et al (1999) at 90 minutes post-injection. At times this early, the system is not near equilibrium, and factors related to radioligand delivery and washout, rather than transporter binding per se, play a prevalent role in determining the appearance of [123I]ß-CIT images. Thus, it seems likely that factors such as blood flow, blood-brain barrier integrity, tissue permeability, etc. have confounded the cortical measurements that Semple et al (1999) have assumed to be due to serotonin transporters.
In summary, although Semple et al (1999) report an interesting reduction in ß-CIT uptake in the cerebral cortex of MDMA users, there is no scientifically sound basis for ascribing this observation to a decrease in cortical serotonin transporters.
REFERENCES
Farde, L., Halldin, C., Mueller, L., et al (1994) PET study of [C-II]ß-CIT binding to monoamine transporters in the monkey and human brain. Synapse, 16, 93-103.[CrossRef][Medline]
Heinz, A., Ragan, P., Jones, D. W., et al
(1998) Reduced serotonin transporters in alcoholism.
American Journal of Psychiatry,
155,
1544-1549.
Jagust, W.J., Eberling, J. L., Biegon, A., et al
(1996) Iodine-123-5-iodo-6-nitroquipazine: SPECT radiotracer
to image the serotonin transporter. Journal of Nuclear
Medicine, 37,
1207-1214.
Laruelle, M., Baldwin, R., Malison, R., et al (1993) SPECT imaging of dopamine and serotonin transporters with [123]ß-CIT: Pharmacological characterization of brain uptake in non-human primates. Synapse, 13, 295-309.[CrossRef][Medline]
Laruelle, M., Wallace, A., Seibyl, J. P., et al (1994) Graphical, kinetic, and equilibrium analyses of in vivo [123-1]ß-CIT binding to dopamine transporters in healthy human subjects. Journal of Cerebral Blood Flow and Metabolism, 14, 982-994.[Medline]
Parsey, R. V., Simpson, N., Hwang, D. R., et al (1999) Kinetic modelling of [C-II]McN5652, a serotonin transporter radioligand, in human volunteers. Journal of Nuclear Medicine, 40 (suppl.), 28P-29P.
Pirker, W., Asenbaum, S., Kasper, S., et al (1995) ß-CIT SPECT demonstrates blockade of 5HT-uptake sites by citalopram in the human brain in vivo. Journal of Neural Transmission, 100, 247-256.[CrossRef][Medline]
Semple, D. H., Ebmeier, K. P., Glabus, M. F., et al
(1999) Reduced in vivo binding to the serotonin
transporter in the cerebral cortex of MDMA (‘ecstasy’) users.
British Journal of Psychiatry,
175, 63-69.
Tauscher, J., Pirker, W., de Zwaan, M., et al (1999) In vivo visualization of serotonin transporters in the human brain during fluoxetine treatment. European Neuropsychopharmacology, 9, 177-179.[Medline]
MRC Brain Metabolism Unit, Department of Psychiatry, Royal Edinburgh Hospital, Morningside Park, Edinburgh EH10 5HF
By necessity, the discussion of methodological constraints had to be very concise in the published version of our paper (it was more detailed and included some of the arguments raised by Heinz & Jones in the originally submitted manuscript). We are therefore glad to have this opportunity to respond to the constructive comments of Heinz & Jones. They essentially make two claims: that ß-CIT does not reliably label cortical serotonin transporters, so that our observed group difference must be due to an alternative mechanism; and at 90 minutes after tracer injection there is a significant admixture of other effects, such as blood flow, blood-brain barrier integrity and tissue permeability, with the same result.
The first claim is supported by some, but not all, displacement studies with SSRIs in monkeys, but inter-species comparisons of brain measures have to be judged with reserve, as Heinz & Jones point out. They also cite a very recently published abstract of a study in six humans, using an alternative (PET-) ligand. We look forward to the full paper; if the initially reported claim survives peer review, it may certainly call into question our interpretation. Moreover, it will specifically weaken the McCann et al (1998) paper, whose authors used the same PET tracer and found cortical reductions in serotonin transporter labelling. The design of our study was based on Kuikka et al's (1995) original report. They examined relatively large numbers of healthy volunteers (28) and patients (9) at one and two hours after injection of ß-CIT. They reported significant tracer washout with 20 mg citalopram from medial prefrontal cortex (Brodmann's area 12) in 25 subjects, 1-2 hours after injection. They also found significant specific binding of serotonin transporters in occipital cortex. Both are regions that showed activity reductions in our MDMA users. They further described reduced medial prefrontal cortex ß-CIT activity at 1 hour in five (alcoholic) patients compared with controls, in the absence of perfusion differences measured with the single photon emission computed tomography (SPECT) ligand 99mTc-ethyl cysteinate dimer.
The second (weaker) claim made by Heinz & Jones is correct in the sense that group differences in ß-CIT binding at 90 minutes do not necessarily reflect a difference in serotonin transporter binding. However, in the absence of a priori hypotheses about generalised cell loss, reductions in blood flow, increased blood-brain barrier integrity or reduced tissue permeability, our results are at least consistent with our interpretation. We think the writers over-state their point if they claim that at one hour non-specific factors are ‘prevalent’ in determining binding (Kuikka, 1995). Our ß-CIT images clearly show activity patterns that parallel the known distribution of serotonin transporters, with relatively high activity in midbrain (Fig. 1 in Semple et al, 1999).
An important experiment that has not yet been performed is the displacement of ß-CIT binding by ‘cold’ serotonin transporter ligands (e.g. citalopram) in areas that are found to be abnormal. It needs to be emphasised, however, that the more specific investigations also tend to be more invasive (e.g. PET with arterial blood sampling) or more of a burden to the subject (e.g. dynamic SPECT scan 4-24 hours after tracer injection with citalopram, resulting in corresponding increases in radiation dose or scan time). This can potentially increase measurement error and aggravate the selection bias of the study, thereby reducing its validity. What is gained in theoretical experimental power may well be lost in spurious or biased sampling, if subjects have to be paid to participate (ours were not) or if subjects are self-selected on the basis of some perceived problem. It behoves the reader to be sceptical about any claims based on small samples, as well as non-specific methodologies, and to scan the medical literature for replicable results, keeping in mind that there is publication bias in favour of positive findings. As far as MDMA-induced damage to human serotonin neurons is concerned, the jury is clearly still out.
REFERENCES
Kuikka, J.T., Tijhonen, J., Bergström, K. A., et al (1995) Imaging of serotonin and dopamine transporters in the living human brain. European Journal of Nuclear Medicine, 22, 346-350.[CrossRef][Medline]
McCann, U. D., Szabo, Z., Scheffel, U., et al (1998) Positron emission tomographic evidence of the toxic effect of MDMA (‘ecstasy’) on brain serotonin neurons in human beings. Lancet, 352, 1433-1437.[CrossRef][Medline]
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