Effects of sleep disruption on stress, nigrostriatal markers, and behavior in a chronic/progressive MPTP male mouse model of parkinsonism.

Sleep complaints are an early clinical symptom of neurodegenerative disorders. Patients with Parkinson's disease (PD) experience sleep disruption (SD). The objective of this study was to determine if preexisting, chronic SD leads to a greater loss of tyrosine hydroxylase (TH) within the striatum and the substantia nigra following chronic/progressive exposure with the neurotoxin, 1-methyl-2-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Male mice underwent chronic SD for 4 weeks, then injected with vehicle (VEH) or increasing doses of MPTP for 4 weeks. There was a significant decrease in the plasma corticosterone levels in the MPTP group, an increase in the SD group, and a return to the VEH levels in the SD+MPTP group. Protein expression levels for TH in the striatum (terminals) and substantia nigra pars compacta (dopamine [DA] cell counts) revealed up to a 78% and 38% decrease, respectively, in the MPTP and SD+MPTP groups compared to their relevant VEH and SD groups. DA transporter protein expression increased in the striatum in the MPTP versus VEH group and in the SN/midbrain between the SD+MPTP and the VEH group. There was a main effect of MPTP on various gait measures (e.g., braking) relative to the SD or VEH groups. In the SD+MPTP group, there were no differences compared to the VEH group. Thus, SD, prior to administration of MPTP, has effects on serum corticosterone and gait but more importantly does not potentiate greater loss of TH within the nigrostriatal pathway compared to the MPTP group, suggesting that in PD patients with SD, there is no exacerbation of the DA cell loss.

Classics in Neuroimaging: Radioligands for the Vesicular Monoamine Transporter 2.

Positron emission tomography (PET) studies of the monoamine neurotransmitter systems in the human brain employ a variety of radiotracers targeting the many receptors, transporters, and enzymes present in monoaminergic neurons. One of these is the vesicular monoamine transporter 2 (VMAT2), the protein responsible for the energy-dependent accumulation of monoamines into synaptic vesicles. The development of in vivo imaging radiotracers for VMAT2 is a story of starting with a well-characterized clinically used drug (tetrabenazine) which had a pharmacologically active metabolite: that metabolite that was in stepwise fashion refined and modified to provide both carbon-11 and fluorine-18 labeled VMAT2 radiotracers that are now used for human PET studies of neurodegenerative and psychiatric diseases. The design approach taken, which involved understanding the metabolism of the radiotracers and identification of the optimal ligand stereochemistry, are representative of important steps in the general concepts behind successful in vivo radiotracer design for brain imaging agents.

Diagnostic accuracy of imaging brain vesicular monoamine transporter type 2 (VMAT2) in clinically uncertain parkinsonian syndrome (CUPS): a 3-year follow-up study in community patients.

OBJECTIVES: To further validate the diagnostic utility of 18F-AV-133 vesicular monoamine transporter type 2 (VMAT2) positron emission tomography (PET) in patients with clinically uncertain parkinsonian syndromes (CUPS) by comparison to clinical diagnosis at 3 years follow-up. DESIGN, SETTING AND PARTICIPANTS: In a previous study, we reported that 18F-AV-133 PET in community patients with CUPS changed diagnosis and management and increased diagnostic confidence. The current diagnosis of this cohort was obtained from the patient and treating specialist and compared with the diagnosis suggested 3 years earlier by the 18F-AV-133 PET. A second 18F-AV-133 PET was available in those with a discordant or inconclusive final diagnosis. STUDY OUTCOME MEASURES: The primary end point was the proportion of patients who had a follow-up clinical diagnosis, which was concordant with their initial 18F-AV-133 PET scan. Secondary end points were the proportion of patients who had the same diagnosis at follow-up as that reached after the initial scan and the stability of diagnostic changes made after the first scan. RESULTS: 81 of the 85 patients previously recruited to the CUPS study had follow-up of which 79 had a clinical diagnosis and 2 remained CUPS. The diagnosis was in agreement with the initial 18F-AV-133 PET scan result in 74 cases. Five patients had a discordant diagnosis; one patient with rubral tremor had a severely abnormal scan that had worsened when rescanned; four cases with normal initial and repeat scans had a clinical diagnosis of Parkinson's disease. Two patients with suspected genetic disorders remained classified as CUPS and both had normal scans. In the 24 CUPS cohort patients where 18F-AV-133 PET initially changed diagnosis, this change was supported by follow-up diagnosis in all but the one rubral tremor case. CONCLUSION: 18F-AV-133 PET is a useful tool in improving diagnostic accuracy in CUPS providing results and diagnostic changes that remain robust after 3 years follow-up.

Immunochemical localization of vesicular monoamine transporter 2 (VMAT2) in mouse brain.

Vesicular monoamine transporter 2 (VMAT2, SLC18A2) is a transmembrane transporter protein that packages dopamine, serotonin, norepinephrine, and histamine into vesicles in preparation for neurotransmitter release from the presynaptic neuron. VMAT2 function and related vesicle dynamics have been linked to susceptibility to oxidative stress, exogenous toxicants, and Parkinson's disease. To address a recent depletion of commonly used antibodies to VMAT2, we generated and characterized a novel rabbit polyclonal antibody generated against a 19 amino acid epitope corresponding to an antigenic sequence within the C-terminal tail of mouse VMAT2. We used genetic models of altered VMAT2 expression to demonstrate that the antibody specifically recognizes VMAT2 and localizes to synaptic vesicles. Furthermore, immunohistochemical labeling using this VMAT2 antibody produces immunoreactivity that is consistent with expected VMAT2 regional distribution. We show the distribution of VMAT2 in monoaminergic brain regions of mouse brain, notably the midbrain, striatum, olfactory tubercle, dopaminergic paraventricular nuclei, tuberomammillary nucleus, raphe nucleus, and locus coeruleus. Normal neurotransmitter vesicle dynamics are critical for proper health and functioning of the nervous system, and this well-characterized VMAT2 antibody will be a useful tool in studying neurodegenerative and neuropsychiatric conditions characterized by vesicular dysfunction.

ECL-cell carcinoids and carcinoma in patients homozygous for an inactivating mutation in the gastric H(+) K(+) ATPase alpha subunit.

A family with a missense variant of the ATP4A gene encoding the alpha subunit of the gastric proton pump (H(+) K(+) ATPase) has recently been described. Homozygous siblings were hypergastrinemic (median gastrin 486 pM) and had gastric tumours diagnosed at a median age of 33 years. In the current histopathological study, we further characterized the tumours found in the gastric corpus. The tumours had the histological appearance of carcinoids (NET G1 or G2) and were immunoreactive for the general neuroendocrine markers chromogranin A (CgA) and synaptophysin as well as the ECL-cell markers vesicular monoamine transporter 2 (VMAT2) and histidine decarbozylase (HDC). One of the tumours consisted of a NET G2 component, but also had a component with glandular growth, which morphologically was classified as an intestinal type adenocarcinoma. Many glands of the adenocarcinoma contained a large proportion of cells positive for neuroendocrine markers, especially the small vesicle marker synaptophysin and the cytoplasmic enzyme HDC. In conclusion, patients homozygous for an inactivating ATP4A mutation develop gastric ECL-cell carcinoids in their 3rd or 4th decade. The adenocarcinoma may be classified as neuroendocrine with ECL-cell differentiation.

Large-scale evaluation of SLC18A2 in prostate cancer reveals diagnostic and prognostic biomarker potential at three molecular levels.

Limitations of current diagnostic and prognostic tools for prostate cancer (PC) have led to over-diagnosis and over-treatment. Here, we investigate the biomarker potential of the SLC18A2 (VMAT2) gene for PC at three molecular levels. Thus, SLC18A2 promoter methylation was analyzed in 767 malignant and 78 benign radical prostatectomy (RP) samples using methylation-specific qPCR and Illumina 450K methylation microarray data. SLC18A2 transcript levels were assessed in 412 malignant and 45 benign RP samples using RNAseq data. SLC18A2 protein was evaluated by immunohistochemistry in 502 malignant and 305 benign RP samples. Cancer-specificity of molecular changes was tested using Mann-Whitney U tests and/or receiver operating characteristic (ROC) analyses. Log rank, uni- and multivariate Cox regression tests were used for survival analyses. We found that SLC18A2 promoter hypermethylation was highly cancer-specific (area under the curve (AUC): 0.923-0.976) and associated with biochemical recurrence (BCR) after RP in univariate analyses. SLC18A2 transcript levels were reduced in PC and had independent prognostic value for BCR after RP (multivariate HR 0.13, P < 0.05). Likewise, SLC18A2 protein was down-regulated in PC (AUC 0.898) and had independent prognostic value for BCR (multivariate HR 0.51, P < 0.05). Reduced SLC18A2 protein expression was also associated with poor overall survival in univariate analysis (HR 0.29, P < 0.05). Our results highlight SLC18A2 as a new promising methylation marker candidate for PC diagnosis. Furthermore, SLC18A2 expression (RNA and protein) showed promising prognostic potential beyond routine clinicopathological variables. Thus, novel SLC18A2-based molecular tests could have useful future applications for PC detection and identification of high-risk patients.

Histidine decarboxylase and urinary methylimidazoleacetic acid in gastric neuroendocrine cells and tumours.

AIM: To study histidine decarboxylase (HDC) expression in normal and neoplastic gastric neuroendocrine cells in relationship to the main histamine metabolite. METHODS: Control tissues from fundus (n = 3) and corpus (n = 3) mucosa of six patients undergoing operations for gastric adenocarcinoma, biopsy and/or gastric surgical specimens from 64 patients with primary gastric neuroendocrine tumours (GNETs), as well as metastases from 22 of these patients, were investigated using conventional immunohistochemistry and double immunofluorescence with commercial antibodies vs vesicular monoamine transporter 2 (VMAT-2), HDC and ghrelin. The urinary excretion of the main histamine metabolite methylimidazoleacetic acid (U-MeImAA) was determined using high-performance liquid chromatography in 27 of the 64 patients. RESULTS: In the gastric mucosa of the control tissues, co-localization studies identified neuroendocrine cells that showed immunoreactivity only to VMAT-2 and others with reactivity only to HDC. A third cell population co-expressed both antigens. There was no co-expression of HDC and ghrelin. Similar results were obtained in the foci of neuroendocrine cell hyperplasia associated with chronic atrophic gastritis type A and also in the tumours. The relative incidence of the three aforementioned markers varied in the tumours that were examined using conventional immunohistochemistry. All of these GNETs revealed both VMAT-2 and HDC immunoreactivity, and their metastases showed an immunohistochemical pattern and frequency similar to that of their primary tumours. In four patients, increased U-MeImAA excretion was detected, but only two of the patients exhibited related endocrine symptoms. CONCLUSION: Human enterochromaffin-like cells appear to partially co-express VMAT-2 and HDC. Co-expression of VMAT-2 and HDC might be required for increased histamine production in patients with GNETs.

Dopamine, vesicular transporters, and dopamine receptor expression in rat major salivary glands.

The localization of dopamine stores and the expression and localization of dopamine (DAT) and vesicular monoamine transporters (VMAT) type-1 and -2 and of dopamine D1-like and D2-like receptor subtypes were investigated in rat submandibular, sublingual, and parotid salivary glands by HPLC with electrochemical detection, as well as immunochemical and immunohistochemical techniques. Male Wistar rats of 2 mo of age were used. The highest dopamine levels were measured in the parotid gland, followed by the submandibular and sublingual glands. Western blot analysis revealed DAT, VMAT-1, VMAT-2, and dopamine receptors immunoreactivity in membrane preparations obtained from the three glands investigated. Immunostaining for dopamine and transporters was developed within striated ducts. Salivary glands processed for dopamine receptors immunohistochemistry developed an immunoreaction primarily in striated and excretory ducts. In the submandibular gland, acinar cells displayed strong immunoreactivity for the D2 receptor, while cells of the convoluted granular tubules were negative for both D1-like and D2-like receptors. Parotid glands acinar cells displayed the highest immunoreactivity for both D1 and D2 receptors compared with other salivary glands. The above localization of dopamine and dopaminergic markers investigated did not correspond closely with neuron-specific enolase (NSE) localization. This indicates that at least in part, catecholamine stores and dopaminergic markers are independent from glandular innervation. These findings suggest that rat major salivary glands express a dopaminergic system probably involved in salivary secretion. The stronger immunoreactivity for dopamine transporters and receptors in striated duct cells suggests that the dopaminergic system could regulate not only quality, but also volume and ionic concentration of saliva.

The gastric mucosa 25 years after proximal gastric vagotomy.

OBJECTIVE: Vagotomy causes inhibition of basal and post-prandial acid secretion in humans, but the knowledge about the trophic effect of the vagal nerves is limited. Vagotomy is known to induce hypergastrinemia and we aimed to study the long-term effects of proximal gastric vagotomy (PGV) on the oxyntic mucosa and the enterochromaffin-like (ECL) cell density in particular. MATERIAL AND METHODS: Eleven patients operated with PGV because of duodenal ulcer and age- and sex-matched controls were examined 26 to 29 years postoperatively by gastroscopy with biopsies from the antrum and oxyntic mucosa. Neuroendocrine cell volume densities were calculated after immunohistochemical labeling of gastrin, the general neuroendocrine cell marker chromogranin A (CgA) and the ECL cell marker vesicular monoamine transporter 2 (VMAT2). Gastritis was graded and Helicobacter pylori (H. pylori) status was determined by polymerase chain reaction of gastric biopsies. Fasting serum gastrin and CgA were measured. RESULTS: Serum gastrin was higher in the PGV group compared to controls (median 21.0 [interquartile range (IQR) = 22.0] pmol/L vs 13.0 [IQR = 4.0] pmol/L, p = 0.04). However, there was neither a significant difference in serum CgA or in CgA (neuroendocrine) nor VMAT2 (ECL cell) immunoreactive cell volume density in the oxyntic mucosa. There was significantly more inflammation and atrophy in H. pylori-positive patients, but PGV did not influence the grade of gastritis. CONCLUSION: Despite higher serum gastrin concentrations, patients operated with PGV did not have higher ECL cell mass or serum CgA. Vagotomy may prevent the development of ECL cell hyperplasia caused by a moderate hypergastrinemia.

GABA is localized in dopaminergic synaptic vesicles in the rodent striatum.

Recently, electrophysiological evidence was given for inhibitory postsynaptic responses at dopaminergic striatal synapses. These responses were independent of the vesicular GABA transporter, VGAT, but dependent on the vesicular dopamine transporter VMAT2. The identity and the exact source of the released molecule, as well as the presence of the putative inhibitory transmitter in VMAT2 containing synaptic vesicles remain to be shown. To clarify this, in particular to determine whether GABA is responsible for the inhibitory response at dopaminergic synapses, we used the electron microscopic immunogold method to label in vivo perfusion fixed striatal tissue with antibodies recognising GABA, VGAT, VMAT2 and tyrosine hydroxylase. We show that about 13 % of tyrosine hydroxylase positive and 11 % of VMAT2 axonal terminals in the caudo-putamen contain significant labelling for GABA. Immunogold signals for tyrosine hydroxylase and VGAT was totally segregated into different pools of nerve terminals. Quantitative analyses of the distance between gold particles signalling GABA and synaptic vesicles showed that GABA was as closely associated with synaptic vesicles in tyrosine hydroxylase positive as in tyrosine hydroxylase negative nerve terminals. Likewise, in dopaminergic terminals GABA and VMAT2 immunogold particles showed a close spatial localization, strongly suggesting the presence of GABA in VMAT2 positive synaptic vesicles. Our results suggest that GABA is exocytosed together with dopamine from dopaminergic nerve terminals in the caudo-putamen through VGAT negative and VMAT2 positive synaptic vesicles.

New fluorescent substrate enables quantitative and high-throughput examination of vesicular monoamine transporter 2 (VMAT2).

Vesicular monoamine transporter 2 (VMAT2) is an essential component of the monoaminergic neurotransmission system in the brain as it transports monoamine neurotransmitters from the neuronal cytosol into the synaptic vesicles and thus contributes to modulation of neurotransmitter release. Considering the continuing interest in VMAT2 as a drug target, as well as a target for the design of imaging probes, we have developed a fluorescent substrate well suited for the study of VMAT2 in cell culture. Herein, we report the synthesis and characterization of a new fluorescent probe, FFN206, as an excellent VMAT2 substrate capable of detecting VMAT2 activity in intact cells using fluorescence microscopy, with subcellular localization to VMAT2-expressing acidic compartments without apparent labeling of other organelles. VMAT2 activity can also be measured via microplate reader. The apparent Km of FFN206 at VMAT2 was found to be 1.16 ± 0.10 µM, similar to that of dopamine. We further report the development and validation of a cell-based fluorescence assay amenable to high-throughput screening (HTS) using VMAT2-transfected HEK cells (Z'-factor of 0.7-0.8), enabling rapid identification of VMAT2 inhibitors and measurement of their inhibition constants over a broad range of affinities. FFN206 thus represents a new tool for optical examination of VMAT2 function in cell culture.

T-cell subpopulations express a different pattern of dopaminergic markers in intra- and extra-thymic compartments.

An involvement of dopamine in regulation of the immune function has been assessed and dopaminergic system has been found widely represented in thymus. Nevertheless detail on the characterization of dopaminergic system in assisting thymocytes development and lymphocytes mature physiology are still lacking. The present study was designed to characterize dopamine plasma membrane transporter (DAT), vesicular dopamine transporters (VMAT)-1 and -2, and dopamine D1-like and D2-like receptors in rat thymocytes, splenocytes and peripheral blood mononuclear cells. Western blot and RT-PCR analyses, performed on these cells, showed an expression of dopamine transporters and receptors during thymocyte development (when of CD4 and CD8 markers are differently expressed). Furthermore FACS analysis, indicates that DAT and dopamine D1-like receptors are expressed at high levels in thymocytes, splenocytes, and peripheral lymphocytes. The percentage of CD4+ CD8+ (double-positive) thymocytes expressing dopaminergic markers was significantly higher compared to the percentage of double-negative ones. The percentage of CD8+ single positive cells expressing dopaminergic markers was significantly higher than that of CD4+ cells. The results suggest that the dopaminergic system plays a role in the thymus microenvironment during T-cell development. The more pronounced expression of dopaminergic markers in CD8+ subsets suggests that dopamine plays a role in development of cytotoxic T-cells. Our findings indicate dopaminergic system to have a role during the maturation and selection of lymphocytes, and support its involvement in the active phases of immune response.

Quantitative analysis of binding sites for 9-fluoropropyl-(+)-dihydrotetrabenazine ([¹8F]AV-133) in a MPTP-lesioned PD mouse model.

UNLABELLED: [¹8F]AV-133 is a novel PET tracer for targeting the vesicular monoamine transporter 2 (VMAT2). The aim of this study is to characterize and quantify the loss of monoamine neurons with [¹8F]AV-133 in the MPTP-lesioned PD mouse model using animal PET imaging and ex vivo quantitative autoradiography (QARG). METHODS: Optimal imaging time window of [¹8F]AV-133 was first determined in normal C57BL/6 mice (n = 3) with a 90-min dynamic scan. The reproducibility of [¹8F]AV-133 PET imaging was evaluated by performing a test-retest study within 1 week for the normal group (n = 6). For MPTP-lesioned studies, normal, and MPTP-treated [25 mg mg/kg once (Group A) and twice (Group B), respectively, daily for 5 days, i.p., groups of four normal and MPTP-treated] mice were used. PET imaging studies at baseline and at Day 4 post-MPTP injections were performed at the optimal time window after injection of 11.1 MBq [¹8F]AV-133. Specific uptake ratio (SUr) of [¹8F]AV-133 was calculated by [(target uptake-cerebellar uptake)/cerebellar uptake] with cerebellum as the reference region. Ex vitro QARG and immunohistochemistry (IHC) studies with tyrosine hydroxylase antibody were carried out to confirm the abundance of dopaminergic neurons. RESULTS: The variability between [¹8F]AV-133 test-retest striatal SUr was 6.60 ± 3.61% with less than 5% standard deviation between animals (intervariability). The percentages of MPTP lesions were Group A 0.94 ± 0.29, -42.1% and Group B 0.65 ± 0.09, -60.4%. By QARG, specific binding of [¹8F]AV-133 was reduced relative to the control groups by 50.6% and 60.7% in striatum and by 30.6% and 46.4% in substantia nigra (Groups A and B, respectively). Relatively small [¹8F]AV-133 SUr decline was noted in the serotonin and norepinephrine-enriched regions (7.9% and 9.4% in mid-brain). Results obtained from IHC consistently confirmed the sensitivity and selectivity of dopaminergic neuron loss after MPTP treatment. CONCLUSIONS: [¹8F]AV-133 PET SUr displayed a high test-retest stability. The SUr significantly declined in the caudate putamen but not in the hypothalamus and midbrain regions after MPTP treatment in the mouse brain. The results obtained for QARG and IHC were consistent and correlated well with the PET imaging studies. On the basis of these concordant results, we find that [¹8F]AV-133 should serve as a useful and reliable PET tracer for evaluating nigrostriatal degeneration.

Symptoms of rapid eye movement sleep behavior disorder are associated with cholinergic denervation in Parkinson disease.

OBJECTIVE: Rapid eye movement sleep behavior disorder (RBD) is common in Parkinson disease (PD), but its relationship to the varied neurotransmitter deficits of PD and prognostic significance remain incompletely understood. RBD and cholinergic system degeneration are identified independently as risk factors for cognitive impairment in PD. We aimed to assess the association between cholinergic denervation and symptoms of RBD in PD patients without dementia. METHODS: Eighty subjects with PD without dementia (age, 64.6 ± 7.0 years; range, 50-82 years; 60 males, 20 females; mean Montreal Cognitive Assessment Test [MoCA] score, 26.2 ± 2.1; range 21-30) underwent clinical assessment, neuropsychological testing, and [(11)C]methylpiperidyl propionate acetylcholinesterase and [(11)C]dihydrotetrabenazine (DTBZ) vesicular monoamine transporter type 2 positron emission tomography (PET) imaging. (11)C3-Amino-4-(2-dimethylaminomethyl-phenylsulfaryl)-benzonitrile (DASB) serotonin transporter PET imaging was performed in a subset of 35 subjects. The presence of RBD symptoms was determined using the Mayo Sleep Questionnaire. RESULTS: Twenty-seven of 80 subjects (33.8%) indicated a history of RBD symptoms. Subjects with and without RBD symptoms showed no significant differences in age, motor disease duration, MoCA, Unified Parkinson Disease Rating Scale motor scores, or striatal DTBZ binding. Subjects with RBD symptoms, in comparison to those without, exhibited decreased neocortical, limbic cortical, and thalamic cholinergic innervation (0.0213 ± 0.0018 vs 0.0236 ± 0.0022, t = 4.55, p < 0.0001; 0.0388 ± 0.0029 vs 0.0423 ± 0.0058, t = 2.85, p = 0.0056; 0.0388 ± 0.0025 vs 0.0427 ± 0.0042, t = 4.49, p < 0.0001, respectively). Brainstem and striatal DASB binding showed no significant differences between groups. INTERPRETATION: The presence of RBD symptoms in PD is associated with relative neocortical, limbic cortical, and thalamic cholinergic denervation although not with differential serotoninergic or nigrostriatal dopaminergic denervation. The presence of RBD symptoms may signal cholinergic system degeneration.

Effects of trimethyltin on hippocampal dopaminergic markers and cognitive behaviour.

The triorganotin compound trimethyltin (TMT) is a highly toxic molecule which has a great impact on human health. The aim of this study was to investigate the specific alteration of dopamine receptors and transporters in the hippocampus of TMT-treated rats. The TMT-treated group showed impaired spatial reference memory in a Morris water maze task compared to the control group, whereas memory consolidation tested 24 hours after the last training session was preserved. In the open field, TMT-treated rats showed a decrease in time spent in rearing episodes reflecting a lower interest to explore a novel environment. In the hippocampal area of the TMT-treated group, we observed a reduction in neuronal viability accompanied by a significant decrease in the expression of the dopamine receptors (D1 and D2), and dopamine transporters (DAT, VMAT1 and VMAT2). A less pronounced reduction was observed for D3 and D5 while D4 did not change. These data were confirmed by RT-PCR analysis. The present study on TMT-induced neurodegeneration highlights the link between hippocampal asset of dopamine receptors and transporters and the impaired performance of rats in a spatial reference memory task.

Elevated levels of the vesicular monoamine transporter and a novel repetitive behavior in the Drosophila model of fragile X syndrome.

Fragile X Syndrome (FXS) is characterized by mental impairment and autism in humans, and it often features hyperactivity and repetitive behaviors. The mechanisms for the disease, however, remain poorly understood. Here we report that the dfmr1 mutant in the Drosophila model of FXS grooms excessively, which may be regulated differentially by two signaling pathways. Blocking metabotropic glutamate receptor signaling enhances grooming in dfmr1 mutant flies, whereas blocking the vesicular monoamine transporter (VMAT) suppresses excessive grooming. dfmr1 mutant flies also exhibit elevated levels of VMAT mRNA and protein. These results suggest that enhanced monoamine signaling correlates with repetitive behaviors and hyperactivity associated with FXS.

VMAT2: a dynamic regulator of brain monoaminergic neuronal function interacting with drugs of abuse.

The monoaminergic neuron, in particular the dopaminergic neuron, is central to mediating the hedonic and addictive properties of drugs of abuse. The effects of amphetamine (AMPH) and cocaine (COC), for example, depend on the ability to increase dopamine in the synapse, by effects on either the plasma membrane transporter DAT or the vesicular transporter for monoamine storage, VMAT2. The potential role of DAT as a target for AMPH and COC has been reviewed extensively. Here, we present VMAT2 as a target that enables the rewarding and addictive actions of these drugs, based on imaging, neurochemical, biochemical, cell biological, genetic, and immunohistochemical evidence. The presence of VMAT2 in noradrenergic, serotoninergic, histaminergic, and potentially trace aminergic neurons invites consideration of a wider role for aminergic neurotransmission in AMPH and COC abuse and addiction.

Maternal high-fat intake alters presynaptic regulation of dopamine in the nucleus accumbens and increases motivation for fat rewards in the offspring.

High caloric intake during early postnatal development can have long term consequences for the offspring. We previously reported that the adult offspring of dams fed a high-fat diet during the last week of gestation and throughout lactation display blunted locomotor response to amphetamine (AMP) and reduced sensitization to the drug compared to offspring of control diet dams. Here, we report that the subsensitivity of high-fat offspring to AMP's locomotor stimulant action reflects, at least in part, altered regulation of nucleus accumbens (NAc) dopamine (DA) transmission. When compared to controls, the DA response of high-fat animals to AMP, as measured with microdialysis, was attenuated in the NAc, but unaffected in the prefrontal cortex (PFC). A relatively higher activity of NAc synaptosomal DA transporter sites without changes in vesicular monoamine transporter (VMAT) uptake capacity was also observed in high-fat offspring. Moreover, ventral tegmental area (VTA) D(2) receptor mRNA levels were decreased in high-fat offspring, suggesting a reduction in DA release-regulating D(2) autoreceptors in terminal regions such as the NAc. The magnitude of locomotor response to D(2/3) receptor activation (with quinpirole) was greater in high-fat than in control animals despite having comparable postsynaptic D(2) mRNA levels in the NAc. Finally, while operant responding for a sugar-enriched food reward did not differ between diet groups, high-fat offspring displayed increased operant responding for a fat-enriched reward compared to controls. These findings add to mounting evidence that early life exposure to elevated dietary maternal fat can lead to long lasting changes in DA-mediated behavioral responses to stimulant drugs and fat-enriched foods.

Measurement of plasmalemmal dopamine transport, vesicular dopamine transport, and K(+)-stimulated dopamine release in frozen rat brain tissue.

This report describes experiments designed to (1) establish the specificity of dopamine (DA) transporter (DAT)-mediated plasmalemmal DA transport, vesicular monoamine transporter-2 (VMAT-2)-mediated vesicular DA transport, and K+-stimulated DA release in samples prepared from frozen rat striata, and (2) characterize the time-course of the effects of freezing on these processes. The procedure described herein uses a simple method of freezing brain tissue (first cooling in ice-cold buffer and then freezing at -80 degrees C) that allows for the storage of rat striata followed by the assay of DA transport and K+-stimulated DA release using rotating disk electrode voltammetry. Plasmalemmal DA transport into samples prepared from frozen striata was blocked by the DAT inhibitor, cocaine, and vesicular DA transport was blocked by the VMAT-2 inhibitor, dihydrotetrabenazine. Additionally, K+-stimulated DA release was Ca+2-dependent. Freezing decreases DAT-mediated DA transport, VMAT-2-mediated DA transport, and K+-stimulated DA release. However activity is still measurable even after 3 weeks of storage. These results suggest that rat striata retain some DA transport and DA release activity even when stored frozen for a few weeks. Frozen storage of rat striata may thus be valuable for experiments requiring lengthy assays, the accumulation of material, or the transport of samples from one laboratory to another for analysis. These results may also be applicable to the study of frozen human brain tissue.

Striatal [11C]dihydrotetrabenazine and [11C]methylphenidate binding in Tourette syndrome.

OBJECTIVE: Tourette syndrome (TS) is a common neurodevelopmental disorder marked by tics and behavioral comorbidities. Clinical pharmacology suggests that dopaminergic signaling abnormalities are part of the pathophysiology of TS. Prior molecular imaging studies of nigrostriatal dopaminergic terminal markers report conflicting results. Our goal was to characterize the distribution of nigrostriatal dopaminergic terminals in subjects with TS. METHODS: Thirty-three adult subjects with TS were studied with PET using [11C]dihydrotetrabenazine (DTBZ), a ligand for the type 2 vesicular monoamine transporter, and with [11C] methylphenidate (MP), a ligand for the plasmalemmal dopamine transporter. Subjects were characterized with standard rating instruments for tic severity, obsessive-compulsive behaviors, and attentional deficits. RESULTS: We found no differences between subjects with TS and control subjects in DTBZ and MP binding in any striatal region. There was no correlation between binding measures and clinical variables. Ventral striatal DTBZ and MP binding distributions in subjects with TS were normal. CONCLUSIONS: We found no evidence of increased striatal dopaminergic innervation in Tourette syndrome (TS). Discrepancy between our present results and those of other studies may be explained by heterogeneity of TS.