Functional characterization of vesicular excitatory amino acid transport by human sialin.

Sialin, the protein coded by SLC17A5, is responsible for membrane potential (Δψ)-driven aspartate and glutamate transport into synaptic vesicles in addition to H+/sialic acid co-transport in lysosomes. Rodent sialin mutants harboring the mutations associated with Salla disease in humans did not transport aspartate and glutamate whereas H+/sialic acid co-transport activity was about one-third of the wild-type protein. In this study, we investigate the effects of various mutations on the transport activities of human sialin. Proteoliposomes containing purified heterologously expressed human sialin exhibited both Δψ-driven aspartate and glutamate transport activity and H+/sialic acid co-transport activity. Aspartate and glutamate transport was not detected in the R39C and K136E mutant forms of SLC17A5 protein associated with Salla disease, whereas H+/sialic acid co-transport activity corresponded to 30-50% of the recombinant wild-type protein. In contrast, SLC17A5 protein harboring the mutations associated with infantile sialic acid storage disease, H183R and Δ268SSLRN272 still showed normal levels of Δψ-driven aspartate and glutamate transport even though H+/sialic acid co-transport activity was absent. Human sialin carrying the G328E mutation that causes both phenotypes, and P334R and G378V mutations that cause infantile sialic acid storage disease showed no transport activity. These results support the idea that people suffering from Salla disease have been defective in aspartergic and glutamatergic neurotransmissions.

GM1 enhances dopaminergic markers in the brain of aged rats.

A number of presynaptic markers are compromised in the dopaminergic neurons of aged Sprague-Dawley rats (22 months old) compared with young rats (3 months old). Indeed, in the striatum of the aged rats there is a diminished capacity to transport dopamine (DA), to bind the dopamine transporter (DAT) marker mazindol, to bind the vesicular monoamine transporter 2 (VMAT2) marker dihydrotetrabenazine, and to release DA under basal conditions or after induction by K(+) or amphetamine. Furthermore, the expression of DAT and VMAT2 mRNA in the midbrain is suppressed. GM1 ganglioside, 30 mg/kg ip daily, administered for 30 days, restores the afore-mentioned markers to values approaching those for young rats. Taken together with our published observations that GM1 partially restores tyrosine hydroxylase activity and DA metabolism in aged nigrostriatal and mesoaccumbal neurons and improves their morphology, our work suggests that GM1 might act as a dopaminergic neurotrophic factor in the aged brain and be a useful adjuvant for treating age-associated dopaminergic deficits.

Apparent opposite effects of tetrabenazine and reserpine on the toxic effects of 1-methyl-4-phenylpyridinium or 6-hydroxydopamine on nigro-striatal dopaminergic neurons.

It is well documented that VMAT2 protects nigrostriatal DA neurons against MPP(+) by sequestering it inside vesicles away from its mitochondrial site of neurotoxic action. However, the implication of the VMAT2 in the mechanism of action exerted by 6-OHDA has received little attention. Therefore, the aim of the present study was to determine whether the vesicular sequestration of 6-OHDA would protect dopaminergic neurons from its toxicity similarly to what is observed with MPP(+). We injected mice with 6-OHDA 90 min after TBZ treatment. Since, unexpectedly, TBZ pretreatment prevented 6-OHDA neurotoxicity, we performed a similar experience replacing 6-OHDA with MPP(+) in order to check our experimental protocol. TBZ pretreatment similarly prevented MPP(+) neurotoxicity. This discrepancy with what is commonly describe in the literature, led us to use reserpine. Indeed, the long lasting VMAT2 inhibition induced by reserpine allowed us to inject neurotoxins while mice no longer presented hypothermia. Contrary to TBZ pretreatment, reserpine pretreatment potentiated both 6-OHDA and MPP(+) toxicity on dopaminergic neurons. Hypothermia elicited by TBZ appeared to be responsible, at least in part, for the neuroprotective effect observed. To verify this hypothesis, we investigated the influence of hypothermia on the toxic activity of both neurotoxins. A hypothermia similar to that induced by TBZ was obtained by a forced swimming test of putting mice into cool water (23 degrees C). The hypothermia prevented both 6-OHDA and MPP(+)-induced neurotoxicity. We finally reported that VMAT2 inhibition potentiates both MPP(+) and 6-OHDA neurotoxicity.

Obstructive sleep apnea is related to a thalamic cholinergic deficit in MSA.

OBJECTIVE: To explore the neurochemical basis of obstructive sleep apnea (OSA) in multiple-system atrophy (MSA). METHODS: In 13 patients with probable MSA, nocturnal, laboratory-based polysomnography was used to rate the severity of OSA using the apnea-hypopnea index during sleep. SPECT with (-)-5-[123I]iodobenzovesamicol ([123I]IBVM) was utilized to measure the density of thalamic cholinergic terminals, which project from the brainstem pedunculopontine and laterodorsal tegmental nuclei. PET with (+)-[11C]dihydrotetrabenazine ([11C]DTBZ) was also used to measure the density of striatal monoaminergic terminals, which project from the brainstem. Findings in the patient group were compared with data from 12 normal control subjects scanned utilizing [123I]IBVM and 15 normal control subjects utilizing [11C]DTBZ. RESULTS: Age and gender distributions were similar in patient and control groups. The MSA subjects showed decreased [123I]IBVM binding in the thalamus (p < 0.001) and decreased mean [11C]DTBZ binding in the striatum (p < 0.0001) in comparison with the control subjects. In the MSA group, thalamic [123I]IBVM binding was inversely correlated with the severity of OSA (p = 0.011). Striatal [11C]DTBZ binding was not correlated with the severity of OSA (p = 0.19). CONCLUSION: Decreased pontine cholinergic projections may contribute to OSA in MSA.

Methamphetamine rapidly decreases mouse vesicular dopamine uptake: role of hyperthermia and dopamine D2 receptors.

Multiple high-dose administrations of the dopamine-releasing agent, methamphetamine, rapidly and persistently decrease vesicular dopamine uptake in purified vesicles prepared from striata of treated rats. Because important differences in the neurotoxic effects of stimulants have been documented in rats and mice, the purpose of this study was to determine if methamphetamine-induced effects in rats occur in mice and to elucidate mechanisms underlying these effects. Results reveal methamphetamine treatment rapidly decreased mouse striatal vesicular dopamine uptake; a phenomenon associated with a subcellular redistribution of vesicular monoamine transporter-2 (VMAT-2) immunoreactivity. Both methamphetamine-induced hyperthermia and dopamine D2 receptor activation contributed to the stimulant-induced deficits in vesicular dopamine uptake. Unlike methamphetamine, the dopamine reuptake inhibitors, methylphenidate and cocaine, rapidly increased vesicular dopamine uptake. The implications of these phenomena are discussed.

PACAP and NGF regulate common and distinct traits of the sympathoadrenal lineage: effects on electrical properties, gene markers and transcription factors in differentiating PC12 cells.

To determine the possible role of pituitary adenylate cyclase-activating polypeptide (PACAP) in the development of the sympathoadrenal cell lineage, we have examined the effects of this neurotrophic peptide, in comparison to nerve growth factor (NGF), on the morphology, electrophysiological properties, expression of neuronal and neuroendocrine marker genes, and activity of transcription factors during differentiation of sympathoadrenal-derived cells, using the rat pheochromocytoma PC12 cell model. Both PACAP and NGF elicited rapid neurite outgrowth, which was accompanied by induction of cell excitability and the development of both sodium and calcium currents. Concurrently, PACAP and NGF increased the expression of a marker of synaptic vesicles. By contrast, PACAP, but not NGF, regulated the expression of different constituents of neuroendocrine large dense core vesicles in PC12 cells. Furthermore, PACAP and NGF differentially regulated the expression of mammalian achaete-scute homologue and paired homeobox 2b genes, transcription factors instrumental for sympathoadrenal development. To compare downstream effectors activated by PACAP and NGF, we studied the effects of these factors on the binding activity of consensus 12-O-tetradecanoylphorbol-13-acetate- and cAMP-responsive elements to nuclear extracts of differentiating PC12 cells. We found that both PACAP and NGF markedly increase the binding activity of these cis-regulatory sequences and that PACAP preferentially recruits activator protein-1-like transcription factors to these elements. Taken together, these results show that PACAP and NGF exert common as well as different effects on neuronal and neuroendocrine traits in differentiating PC12 cells, strongly suggesting that these two trophic factors could play complementary roles in the development of the sympathoadrenal cell lineage.

Plasma extravasation induced by dietary supplemented histamine in histamine-free mice.

Histidine decarboxylase (HDC) synthesizes endogenous histamine from histidine in mammals. To evaluate the role of histamine in skin allergic reaction, we used HDC gene knockout mice lacking histamine. No plasma extravasation reaction was observed in HDC-/- mice after passive cutaneous anaphylaxis (PCA) test. Compound 48/80, a mast cell granule depletor, produced plasma extravasation inHDC+/+ mice but no extravasation in HDC-/- mice. Interestingly, orally administered histamine was distributed in the skin in HDC-/- mice and in these histamine-supplemented mice the plasma extravasation reaction was observed after the injection of compound 48/80 and the PCA test. Cultured bone marrow-derived mast cells of HDC-/- mice took up histamine from the histamine-supplemented medium into the secretory granules. The absorbed histamine was released in response to the same antigen and antibody combination used as in PCA test. In contrast to the immediate-type response, the delayed-type hypersensitive response, observed as a thickening of the ear skin after trinitrochlorobenzene challenge (following sensitization), showed no differences between HDC+/+ and HDC-/- mice. Therefore, among the allergic skin reactions, histamine is revealed to be an important mediator especially for the plasma extravasation in an immediate-type allergy model.

The role of zinc ions in reverse transport mediated by monoamine transporters.

The human dopamine transporter (hDAT) contains an endogenous high affinity Zn2+ binding site with three coordinating residues on its extracellular face (His193, His375, and Glu396). Upon binding to this site, Zn2+ causes inhibition of [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) uptake. We investigated the effect of Zn2+ on outward transport by superfusing hDAT-expressing HEK-293 cells preloaded with [3H]MPP+. Although Zn2+ inhibited uptake, Zn2+ facilitated [3H]MPP+ release induced by amphetamine, MPP+, or K+-induced depolarization specifically at hDAT but not at the human serotonin and the norepinephrine transporter (hNET). Mutation of the Zn2+ coordinating residue His(193) to Lys (the corresponding residue in hNET) eliminated the effect of Zn2+ on efflux. Conversely, the reciprocal mutation (K189H) conferred Zn2+ sensitivity to hNET. The intracellular [3H]MPP+ concentration was varied to generate saturation isotherms; these showed that Zn2+ increased V(max) for efflux (rather than K(M-Efflux-intracellular)). Thus, blockage of inward transport by Zn2+ is not due to a simple inhibition of the transporter turnover rate. The observations provide evidence against the model of facilitated exchange-diffusion and support the concept that inward and outward transport represent discrete operational modes of the transporter. In addition, they indicate a physiological role of Zn2+, because Zn2+ also facilitated transport reversal of DAT in rat striatal slices.

Barrel pattern formation requires serotonin uptake by thalamocortical afferents, and not vesicular monoamine release.

Thalamocortical neurons innervating the barrel cortex in neonatal rodents transiently store serotonin (5-HT) in synaptic vesicles by expressing the plasma membrane serotonin transporter (5-HTT) and the vesicular monoamine transporter (VMAT2). 5-HTT knock-out (ko) mice reveal a nearly complete absence of 5-HT in the cerebral cortex by immunohistochemistry, and of barrels, both at P7 and adulthood. Quantitative electron microscopy reveals that 5-HTT ko affects neither the density of synapses nor the length of synaptic contacts in layer IV. VMAT2 ko mice, completely lacking activity-dependent vesicular release of monoamines including 5-HT, also show a complete lack of 5-HT in the cortex but display largely normal barrel fields, despite sometimes markedly reduced postnatal growth. Transient 5-HTT expression is thus required for barrel pattern formation, whereas activity-dependent vesicular 5-HT release is not.

High vesicular monoamine transporter binding in asymptomatic bipolar I disorder: sex differences and cognitive correlates.

OBJECTIVE: It has been hypothesized that anomalies in monoaminergic function underlie some of the manifestations of bipolar disorder. In this study the authors examined the possibility that trait-related abnormalities in the concentration of monoaminergic synaptic terminals may be present in patients with asymptomatic bipolar disorder type I. METHOD: The concentration of a stable presynaptic marker, the vesicular monoamine transporter protein (VMAT2), was quantified with (+)[(11)C]dihydrotetrabenazine (DTBZ) and positron emission tomography. Sixteen asymptomatic patients with bipolar I disorder who had a prior history of mania with psychosis (nine men and seven women) and individually matched healthy subjects were studied. Correlational analyses were conducted to examine the relationship between regional VMAT2 binding, cognitive function, and clinical variables. RESULTS: VMAT2 binding in the thalamus and ventral brainstem of the bipolar patients was higher than that in the comparison subjects. VMAT2 concentrations in these regions correlated with performance on measures of frontal, executive function. In addition, sex differences in VMAT2 binding were detected in the thalamus of the bipolar patients; the male patients had higher binding than the women. No sex differences in binding were observed in the healthy comparison group. CONCLUSIONS: These initial results suggest that higher than normal VMAT2 expression and, by extension, concentration of monoaminergic synaptic terminals, may represent a trait-related abnormality in patients with bipolar I disorder and that male and female patients show different patterns. Also, VMAT2 concentrations may be associated with some of the cognitive deficits encountered in euthymic bipolar disorder.

Rapid and differential losses of in vivo dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) radioligand binding in MPTP-treated mice.

The dose- and time-dependent changes of in vivo radioligand binding to the neuronal membrane dopamine transporter (DAT) and vesicular monoamine transporter type 2 (VMAT2) were examined in mouse brain after MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) administrations. Regional brain distribution studies were done in male C57BL/6 mice using simultaneous injections of d-threo-[(3)H]methylphenidate (DAT) and (+)-alpha-[(11)C]dihydrotetrabenazine (VMAT2). Single (55 mg/kg i.p. ) or multiple (4 x 10 mg/kg i.p., 1-hour intervals) administration of MPTP caused significant reductions in [(3)H]methylphenidate and [(11)C]dihydrotetrabenazine specific striatal binding, measured 14 days later. The single high dose of MPTP produced greater losses of [(11)C]dihydrotetrabenazine binding than did the multiple MPTP dosing regimen. Using the single high dose of MPTP, changes of in vivo binding of the two radioligands were determined at 1, 3, and 14 days after neurotoxin injection. At 1 day, there are large losses of [(3)H]methylphenidate binding (DAT) but no changes in [(11)C]dihydrotetrabenazine binding to the VMAT2 site in the striatum. At 3 and 14 days, there were >50% losses of binding of both bot radioligands, but significantly (P < 0.001) greater losses of VMAT2 binding of [(11)C]dihydrotetrabenazine. These studies indicate that the losses of the neuronal membrane and vesicular transporters are not always equal, and do not occur in the same time frame, after administration of the neurotoxin MPTP.

Selective substrates for non-neuronal monoamine transporters.

The recently identified transport proteins organic cation transporter 1 (OCT1), OCT2, and extraneuronal monoamine transporter (EMT) accept dopamine, noradrenaline, adrenaline, and 5-hydroxytryptamine as substrates and hence qualify as non-neuronal monoamine transporters. In the present study, selective transport substrates were identified that allow, by analogy to receptor agonists, functional discrimination of these transporters. To contrast efficiency of solute transport, stably transfected 293 cell lines, each expressing a single transporter, were examined side by side in uptake experiments with radiolabeled substrates. Normalized uptake rates indicate that tetraethylammonium, with a rate of about 0.5 relative to 1-methyl-4-phenylpyridinium (MPP+), is a good substrate for OCT1 and OCT2. It was not, however, accepted as substrate by EMT. Choline was transported exclusively by OCT1, with a rate of about 0.5 relative to MPP+. Histamine was a good substrate with a rate of about 0.6 relative to MPP+ for OCT2 and EMT, but was not transported by OCT1. Guanidine was an excellent substrate for OCT2, with a rate as high as that of MPP+. Transport of guanidine by OCT1 was low, and transport by EMT was negligible. With the guanidine derivatives cimetidine and creatinine, a pattern strikingly similar to guanidine was observed. Collectively, these substrates reveal key differences in solute recognition and turnover and thus challenge the concept of "polyspecific" organic cation transporters. In addition, our data, when compared with previous studies, suggest that OCT2 corresponds to the organic cation/H+ antiport mechanism in renal brush-border membrane vesicles, and that EMT corresponds to the guanidine/H+ antiport mechanism in membrane vesicles from placenta and intestine.

Acute interactions between L-DOPA and the neurotoxic effects of 1-methyl-4-phenylpyridinium or 6-hydroxydopamine in mice.

We have compared the effects of an i.p. pretreatment with L-DOPA (200 mg/kg) associated with benserazide (25 mg/kg) on neurotoxic effects of either 6-hydroxydopamine (6-OHDA) (50 microg, 10 microl per mouse) or 1-methyl-4-phenylpyridinium (MPP+) (17.5 microg, 10 microl per mouse). The striatal dopamine (DA) content, the vesicular monoamine transporter (VMAT2) density, as well as the hypothalamic norepinephrine (NE) content were measured 8 days after treatments. The L-DOPA-benserazide pretreatment worsened by 65% the 6-OHDA-induced depletion in striatal DA. On the contrary, it reduced by 42% the MPP+-induced depletion in striatal DA and by 54% the MPP+-induced decrease in VMAT2 density. It was noticed that the L-DOPA-benserazide pretreatment did not modify the marked decrease in hypothalamic NE content induced by 6-OHDA.

Transient developmental expression of monoamine transporters in the rodent forebrain.

Neurons in first-order sensory thalamic nuclei have been shown to express functional plasma membrane serotonin (SERT) and vesicular monoamine (VMAT2) transporters during early postnatal development. In the present study, we provide an extensive description of the spatial and the temporal patterns of VMAT2 and SERT expression, during early embryonic development and postnatal life, by using in situ hybridization and immunocytochemistry. VMAT2 and SERT genes are transiently expressed in a wide population of non-monoaminergic neurons in the central and peripheral nervous system with a large overlap in the temporal and spatial pattern of expression of both genes. A selective pattern of expression of both genes was observed in the thalamus with expression limited to the dorsal thalamus and more particularly to primary sensory relay nuclei that convey point to point projection maps. Transient expression of the transporters was also observed in sensory cranial nerves, in the hippocampus, cerebral cortex, septum, and amygdala. VMAT2 and SERT gene expression was not necessarily linked, as some neural populations expressed only VMAT2, while others only contained SERT. Since VMAT2 serves to transport catecholamines besides serotonin, we examined the developmental expression of the plasma membrane dopamine and norepinephrine transporters but found no transient expression of these genes. Despite minor temporal disparities, VMAT2 and SERT extinguished almost simultaneously during the second and third weeks of post-natal life. These expressions did not seem to be dependent on peripheral neural inputs, since monocular enucleations and infraorbital nerve cuts effected on the day of birth, did not modify the period of transporter expression or of extinction.

Transient expression of the vesicular monoamine transporter during development in the rat thalamus and cortex.

The postnatal developmental pattern of the central vesicular monoamine transporter-2 (VMAT2) was analyzed in the rat brain by means of quantitative autoradiography with a specific and high affinity ligand [3H]dihydrotetrabenazine ([3H]TBZOH). We show a dense expression of VMAT2 in the cortex (especially area 17) and thalamus (particularly the dorsal lateral geniculate nucleus) at postnatal days 1 and 8. This pattern of VMAT2 distribution was transient since it was no longer observed at day 20 or in the adult rat brain where VMAT2 density was weak and uniform in these regions. These data suggest that monoamine vesicular storage participates in the early postnatal maturation of thalamus and cortex.

Structural organization of the human vesicular monoamine transporter type-2 gene and promoter analysis using the jelly fish green fluorescent protein as a reporter.

The genomic structure of a human vesicle monoamine transporter, type-2 (hVMAT2) was determined from two overlapping cosmids, phVMAT2-cos1 and phVMT2-cos2, spanning more than 35 kb. The hVMAT2 open reading frame is encoded by 16 exons, with translation initiation and termination in exon 2 and exon 16, respectively. Several potential binding sites for transcriptional regulatory factors, including a cAMP response element (CRE) were identified in the 5'-upstream region of the gene. A promoter construct using the jellyfish green fluorescent protein (GFP) as reporter has been made and transfected into the human neuroblastoma cell line, SHSY-5Y. The cellular expression of the GFP was readily detected by fluorescence microscopy and cells expressing GFP could be sorted using a fluorescence-activated cell sorter (FACS), allowing the level of GFP expression in transfected SHSY-5Y cells to be quickly and reliably determined.

The neurotoxin 1-methyl-4-phenylpyridinium is a substrate for the canalicular organic cation/H+ exchanger.

Hepatic organic cation transport consists, in part, of carrier-mediated sinusoidal uptake stimulated by an inside-negative membrane potential and canalicular excretion driven by electroneutral organic cation/H+ exchange. Intracellular organic cation transport involves sequestration into acidified organelles, also mediated by organic cation/H+ exchange. A sinusoidal organic cation transporter has been cloned; however, canalicular organic cation transport has not been characterized at the molecular level. On the assumption that hepatic organic cation/H+ exchange resembles monoamine transport in synaptic vesicles, we examined, using canalicular rat liver plasma membrane vesicles, the transport of 1-methyl-4-phenylpyridinium (MPP+), a neurotoxin taken up by a synaptic vesicular monoamine transporter that has been cloned. Under voltage-clamped conditions, an outwardly directed H+ gradient stimulated [3H]MPP+ uptake, compared with uptake under pH-equilibrated conditions, consistent with electroneutral MPP+/H+ exchange. Substrates for canalicular organic cation/H+ exchange cis-inhibited pH-dependent MPP+ uptake. Equilibrium exchange of [14C]tetraethylammonium was inhibited by MPP+ in a concentration-dependent manner, consistent with a direct interaction of MPP+ with the organic cation carrier. Carrier-mediated MPP+ uptake exhibited saturability, with kinetic parameters similarto those described for canalicular tetraethylammonium+/H+ exchange. Canalicular [3H]MPP+ uptake was ATP-independent and, thus, distinct from P-glycoprotein-mediated efflux. The finding that MPP+ is a substrate for canalicular organic cation/H+ exchange is applicable to studies, using degenerate oligonucleotides complementary to sequences conserved in neurotransmitter transporters, aimed at cloning this transporter.

Transient uptake and storage of serotonin in developing thalamic neurons.

Serotonin (5-HT) has been shown to affect the development and patterning of the mouse barrelfield. We show that the dense transient 5-HT innervation of the somatosensory, visual, and auditory cortices originates in the thalamus rather than in the raphe: 5-HT is detected in thalamocortical fibers and most 5-HT cortical labeling disappears after thalamic lesions. Thalamic neurons do not synthesize 5-HT but take up exogenous 5-HT through 5-HT high affinity uptake sites located on thalamocortical axons and terminals. 3H-5-HT injected into the cortex is retrogradely transported to thalamic neurons. In situ hybridization shows a transient expression of the genes encoding the serotonin transporter and the vesicular monoamine transporter in thalamic sensory neurons. In these glutamatergic neurons, internalized 5-HT might thus be stored and used as a "borrowed transmitter" for extraneuronal signaling or could exert an intraneuronal control on thalamic maturation.

Binding of alpha-dihydrotetrabenazine to the vesicular monoamine transporter is stereospecific.

The two enantiomers of alpha-dihydrotetrabenazine were separated using chiral high performance liquid chromatography. The (+)-isomer showed high affinity in vitro (Ki = 0.97 +/- 0.48 nM) for the vesicular monoamine transporter (VMAT2) in rat brain striatum, whereas the (-)-isomer was inactive (Ki = 2.2 +/- 0.3 microM). Each isomer was then synthesized in carbon-11 labeled form, and regional brain biodistributions in mice determined after intravenous injection. Only (+)-alpha-dihydrotetrabenazine showed selective and specific accumulations in regions of dense monoaminergic innervation (e.g., striatum, hypothalamus), which could be blocked by coinjection of unlabeled tetrabenazine. Binding of alpha-dihydrotetrabenazine to the vesicular monoamine transporter is thus stereospecific.

PET study of [11C]beta-CIT binding to monoamine transporters in the monkey and human brain.

The cocaine congener beta-CIT has been labeled with 11C for positron emission tomographic (PET) studies of the dopamine transporter. In the present autoradiographic study on human brain sections and PET study on monkey and human [11C]beta-CIT accumulated markedly in the striatum. [11C]beta-CIT binding in the striatum was selective to the dopamine transporter. The binding in the thalamus was on an intermediate level and was displaced by compounds having affinity for norepinephrine and serotonin transporters. The neocortical binding was on a low level and could be displaced only by citalopram, a serotonin uptake inhibitor. A high dose of cocaine intravenously (7 mg/kg) induced a 50% occupancy of specific [11C]beta-CIT binding to the dopamine transporter in the striatum. This dose is much higher than the doses of 0.25-0.5 mg/kg i.v. for cocaine arousal in human subjects. The finding indicates that cocaine arousal may be induced at a low dopamine transporter occupancy of a few percent. [11C]beta-CIT should be a useful radioligand to explore cocaine actions in humans and to follow the pathophysiological process in vivo by PET in neurodegenerative diseases of the striatum.