Norepinephrine Transporter Density Research Articles

Levodopa responsive freezing of gait is associated with reduced norepinephrine transporter binding in Parkinson's disease

BackgroundFreezing of gait (FOG) is a major cause of falling in Parkinson's disease (PD) and can be responsive or unresponsive to levodopa. Pathophysiology is poorly understood. ObjectiveTo examine the link between noradrenergic systems, the development of FOG in PD and its responsiveness to levodopa. MethodsWe examined norepinephrine transporter (NET) binding via brain positron emission tomography (PET) to evaluate changes in NET density associated with FOG using the high affinity selective NET antagonist radioligand [11C]MeNER (2S,3S)(2-[α-(2-methoxyphenoxy)benzyl]morpholine) in 52 parkinsonian patients. We used a rigorous levodopa challenge paradigm to characterize PD patients as non-freezing (NO-FOG, N = 16), levodopa responsive freezing (OFF-FOG, N = 10), and levodopa-unresponsive freezing (ONOFF-FOG, N = 21), and also included a non-PD FOG group, primary progressive freezing of gait (PP-FOG, N = 5). ResultsLinear mixed models identified significant reductions in whole brain NET binding in the OFF-FOG group compared to the NO-FOG group (−16.8%, P = 0.021) and regionally in the frontal lobe, left and right thalamus, temporal lobe, and locus coeruleus, with the strongest effect in right thalamus (P = 0.038). Additional regions examined in a post hoc secondary analysis including the left and right amygdalae confirmed the contrast between OFF-FOG and NO-FOG (P = 0.003). A linear regression analysis identified an association between reduced NET binding in the right thalamus and more severe New FOG Questionnaire (N-FOG-Q) score only in the OFF-FOG group (P = 0.022). ConclusionThis is the first study to examine brain noradrenergic innervation using NET-PET in PD patients with and without FOG. Based on the normal regional distribution of noradrenergic innervation and pathological studies in the thalamus of PD patients, the implications of our findings suggest that noradrenergic limbic pathways may play a key role in OFF-FOG in PD. This finding could have implications for clinical subtyping of FOG as well as development of therapies.

Reduced density of noradrenergic fibers without prominent neuron loss or tau pathology in the locus coeruleus in APP knock‐in mouse models of Aβ amyloidosis

AbstractBackgroundLocus coeruleus (LC) is a nucleus containing the cell bodies of noradrenergic neurons in the brain, and is one of the earliest regions affected by Alzheimer’s disease (AD). Norepinephrine (NE) is critical for cognitive functions as well as anti‐inflammatory and neuroprotective properties, both of which are implicated in AD. LC neurons are among the first to display tau pathology and that significant loss of forebrain noradrenergic projections is evident at prodromal stage of AD, suggesting that abnormality in LC‐NE system initiates the disease pathogenesis. However, mechanisms underlying neurodegeneration in LC‐NE system remain elusive. We have previously reported that AppNL‐G‐F/NL‐G‐F mice, which harbor three familial AD mutations (Swedish, Beyreuther/Iberian, and Arctic) exhibited cognitive deficits and severe neuroinflammation accompanied by massive amyloid‐β (Aβ) pathology. In this study, we asked whether and how LC‐NE system is altered in AppNL‐G‐F/NL‐G‐F mice.MethodTo ask whether AppNL‐G‐F/NL‐G‐F mice exhibit noradrenergic degeneration, we compared the density of norepinephrine transporter (NET)‐positive fibers in the cortex and hippocampus as well as the number of dopamine β‐hydroxylase (DBH)‐positive neurons in the LC between AppNL‐G‐F/NL‐G‐F and wild‐type (WT) C57BL/6J mice at 24 months of age. We also examined whether tau pathology occurs in the LC‐NE system in these mice.ResultHistochemical analyses revealed that 24‐month‐old AppNL‐G‐F/NL‐G‐F mice exhibited significant decreases in the density of NET‐positive fibers in the prefrontal and entorhinal cortices and the hippocampal CA1, the regions associated with learning and memory, compared to WT mice. In contrast, AppNL‐G‐F/NL‐G‐F mice did not display loss of DBH‐positive neurons in the LC even at 24 months of age. Furthermore, AppNL‐G‐F/NL‐G‐F mice did not develop prominent tau pathology either in the nerve terminals or in the cell bodies of LC neurons.ConclusionThis study demonstrates that Aβ pathology is sufficient to reduce the density of noradrenergic fibers independent of neuron loss and tau pathology in the LC. These results also suggest that age‐dependent accumulation of Aβ in the cortex or tau in the LC may additively and independently damage LC neurons from the nerve terminals or from the cell bodies, respectively.

Spatial Inhomogeneity of Cardiac Norepinephrine Transport Protein and Meta-[123I]Iodobenzylguanidine Uptake in Swine Myocardial Tissue.

The aim of the present study was to evaluate the expression of the cardiac norepinephrine transporter (NET) in the left ventricle (LV) of healthy pigs and its relationship with regional meta-[123I]iodobenzylguanidine ([123I]MIBG) myocardial uptake. Experiments were performed on animals injected with [123I]MIBG and acquired 2h later using an ultrafast CZT gamma camera to assess the regional myocardial uptake. After image acquisition, animals were euthanized; the heart was quickly excised and underwent to an ex vivo single photon emission tomography (SPECT) imaging. Four small samples of tissue were then harvested from mid-walls and apex of the left ventricle; NET densities were evaluated and further normalized for protein loading per cardiac region. Three variants of NET protein with different molecular weights were detected. The expression of NET was not homogenous in the LV, with the highest density in the inferior wall and the lowest one in the apical area. The regional in vivo [123I]MIBG uptake revealed an analogous trend, showing a good linear relationship with NET expression. Parallel results were obtained from the ex vivo study. This study elucidates the expression of three different variants of NET proteins into the left ventricular myocardium of a healthy pig. NET expression into the LV was not homogeneous and paralleled by differences in regional [123I]MIBG uptake. Moreover, the correlation and the agreement between measurements of regional expression of NET variants and [123I]MIBG uptake represent a relevant finding for inferences about NET expression in the context of clinical imaging.

Responses of PKCε to cardiac overloads on myocardial sympathetic innervation and NET expression.

Protein kinase C (PKC) is a key mediator of many diverse physiological and pathological responses. PKC activation play an important regulatory role of cardiac function. The present study was performed to investigate whether there were differential activations of the PKCε and how the activation coupled with norepinephrine transporter (NET) surface expression, sympathetic innervation pattern and extracellular matrix remodeling in different cardiac hemodynamic overloads induced by abdominal aortic constriction or aortocaval fistula. At 8weeks after the operations, heart failure were induced, accompanied with myocardial hypertrophy, which was more pronounced in pressure overload (POL) than that of volume overload (VOL) rats, left ventricular dysfunction and increased plasma norepinephrine (NE). In POL rats there was an increase in myocardial collagen deposition, in contrast, the amount decreased in VOL as compared with the sham rats. POL remarkably upregulated PKCε membrane-cytosol ratio and downregulated NET membrane fraction, whereas, in VOL induced opposite changes. Accompanied with the PKCε activation, nerve sprouting, evidenced by myocardial GAP43 protein increased, and different nerve phenotypes were found, in POL tyrosine hydroxylase (TH) positive nerve density increased with NET and choline acetyltransferase (ChAT) immunoreactivity density decreased, in contrast, in VOL NET and ChAT increased, TH did not change. The overloads did not induce alteration of NET mRNA expression, but resulted in different myocardial β1-AR mRNA expression, in POL β1-AR mRNAwas significantly downregulated, while in VOL rats unaltered. Conclusion, the present results suggested that the different cardiac hemodynamic overload could differentially activate a common signaling, PKCε intermediate and thereby generate biological diversity.

A Positron Emission Tomography Study of Norepinephrine Transporter Occupancy and Its Correlation with Symptom Response in Depressed Patients Treated with Quetiapine XR.

BackgroundQuetiapine is effective in treating depressive symptoms in major depressive disorder and bipolar disorder, but the mechanisms underlying its antidepressants effects are unknown. Norquetiapine, a metabolite of quetiapine, has high affinity for norepinephrine transporter, which might account for its therapeutic efficacy.MethodsIn this study, we used positron emission tomography with (S,S)-[11C]O-methyl reboxetine to estimate norepinephrine transporter density and assess the relationship between norepinephrine transporter occupancy by quetiapine XR and improvement in depression in patients with major depressive disorder (n=5) and bipolar disorder (n=5). After the baseline positron emission tomography scan, patients were treated with quetiapine XR with a target dose of 150 mg in major depressive disorder and 300 mg in bipolar disorder. Patients had a second positron emission tomography scan at the end of week 2 and a final scan at week 7.ResultsNorepinephrine transporter density was significantly lower in locus ceruleus in patients compared with healthy subjects. Further, there was a significant positive correlation between quetiapine XR dose and norepinephrine transporter occupancy in locus ceruleus at week 2. The norepinephrine transporter occupancy at week 2 in hypothalamus but not in other regions predicted improvement in depression as reflected by reduction in MADRS scores from baseline to week 7. The estimated dose of quetiapine XR associated with 50% norepinephrine transporter occupancy in hypothalamus at week 2 was 256 mg and the estimated plasma levels of norquetiapine to achieve 50% norepinephrine transporter occupancy was 36.8 µg/L.ConclusionThese data provide preliminary support for the hypothesis that norepinephrine transporter occupancy by norquetiapine may be a contributor to the antidepressant effects of quetiapine.

PET Quantification of the Norepinephrine Transporter in Human Brain with (S,S)-18F-FMeNER-D2.

Norepinephrine transporter (NET) in the brain plays important roles in human cognition and the pathophysiology of psychiatric disorders. Two radioligands, (S,S)-11C-MRB and (S,S)-18F-FMeNER-D2, have been used for imaging NETs in the thalamus and midbrain (including locus coeruleus) using PET in humans. However, NET density in the equally important cerebral cortex has not been well quantified because of unfavorable kinetics with (S,S)-11C-MRB and defluorination with (S,S)-18F-FMeNER-D2, which can complicate NET quantification in the cerebral cortex adjacent to the skull containing defluorinated 18F radioactivity. In this study, we have established analysis methods of quantification of NET density in the brain including the cerebral cortex using (S,S)-18F-FMeNER-D2 PET. Methods: We analyzed our previous (S,S)-18F-FMeNER-D2 PET data of 10 healthy volunteers dynamically acquired for 240 min with arterial blood sampling. The effects of defluorination on the NET quantification in the superficial cerebral cortex was evaluated by establishing a time stability of NET density estimations with an arterial input 2-tissue-compartment model, which guided the less-invasive reference tissue model and area under the time-activity curve methods to accurately quantify NET density in all brain regions including the cerebral cortex. Results: Defluorination of (S,S)-18F-FMeNER-D2 became prominent toward the latter half of the 240-min scan. Total distribution volumes in the superficial cerebral cortex increased with the scan duration beyond 120 min. We verified that 90-min dynamic scans provided a sufficient amount of data for quantification of NET density unaffected by defluorination. Reference tissue model binding potential values from the 90-min scan data and area under the time-activity curve ratios of 70- to 90-min data allowed for the accurate quantification of NET density in the cerebral cortex. Conclusion: We have established methods of quantification of NET densities in the brain including the cerebral cortex unaffected by defluorination using (S,S)-18F-FMeNER-D2 These results suggest that we can accurately quantify NET density with a 90-min (S,S)-18F-FMeNER-D2 scan in broad brain areas.

Relationship between new-onset atrial fibrillation and sympathetic neural remodeling in a canine acute myocardial infarction model

To establish the canine model of new-onset atrial fibrillation (AF) after acute myocardial infarction (AMI), and explore the relationship between new-onset AF and sympathetic neural remodeling in this model. Twenty four adult mongrel dogs were randomly divided into 4 groups by applying random number table. Group A (n=6): ligate the left circumflex artery (LCX). Group B (n=6): ligate the LCX and right atrial anterior artery and right atrial middle artery. Group C (n=6): ligate left anterior descending artery.Group D (n=6): sham operation.Sequential electrophysiology study was performed in all dogs to determine the AF induction rate, AF duration, effective refractory period (ERP), the density of tyrosine hydroxylase (TH) and norepinephrine transporter (NET) before AMI or sham operation, and at 30 min, 2 hours and 4 hours after AMI or sham operation. (1) The highest AF induction rate of right atrium and left auricle was 96.7%(58/60) and 95.0%(57/60) in group B, 81.7%(49/60) and 38.3%(23/60) in group A, 28.3%(17/60) and 35.0%(21/60) in group C, 20.0%(12/60) and 33.3%(20/60) in group D. (2) At 4 hours after AMI, AF duration was significantly prolonged in group B(193.50±54.67) s, compared with group A(53.83±9.37) s, group C(45.00±19.50) s, and group D(16.67±4.50) s (all P<0.05). (3) In group B, the ERP of AF was prolonged at 30 minutes after AMI and shortened at 2 hours and 4 hours after AMI compared with baseline level(all P<0.05). (4) The TH density of left atrium ((3 485±694) µm2/mm2) and left auricle((2 645±454) µm2/mm2) in group A and the TH density of left atrium ((7 873±1159) µm2/mm2) and left auricle((3 070±605) µm2/mm2) in group B were significantly higher than those in group C ((1 474±475) µm2/mm2, (1 177±277) µm2/mm2) and group D ((678±206) µm2/mm2, (489±125) µm2/mm2) (all P<0.05), and the TH density of right atrium and right auricle in group B were higher than group A (all P<0.05). The NET density of left atrium((476±75) µm2/mm2) and left auricle ((414±52) µm2/mm2) in group A and the NET density of left atrium((527±81) µm2/mm2) and left auricle((429±85) µm2/mm2) in group B were lower than that in group C ((1 044±105) µm2/mm2, (867±67) µm2/mm2) and group D ((1 438±60) µm2/mm2, (1 027±119) µm2/mm2) (all P<0.05). Ligating the LCX, right atrial anterior artery and right atrial middle artery at the same time can significantly increase the success rate in establishing the canine model of new-onset atrial fibrillation after acute myocardial infarction and can also increase the AF duration.Cardiac sympathetic remodeling after acute myocardial infarction is associated with induction and duration of AF.

Imaging human brown adipose tissue under room temperature conditions with 11C-MRB, a selective norepinephrine transporter PET ligand

IntroductionBrown adipose tissue (BAT) plays a critical role in adaptive thermogenesis and is tightly regulated by the sympathetic nervous system (SNS). However, current BAT imaging modalities require cold stimulation and are often unreliable to detect BAT in the basal state, at room temperature (RT). We have shown previously that BAT can be detected in rodents under both RT and cold conditions with 11C-MRB ((S,S)-11C-O-methylreboxetine), a highly selective ligand for the norepinephrine transporter (NET). Here, we evaluate this novel approach for BAT detection in adult humans under RT conditions. MethodsTen healthy, Caucasian subjects (5 M: age 24.6±2.6, BMI 21.6±2.7kg/m2; 5 F: age 25.4±2.1, BMI 22.1±1.0kg/m2) underwent 11C-MRB PET-CT imaging for cervical/supraclavicular BAT under RT and cold-stimulated conditions (RPCM Cool vest; enthalpy 15°C) compared to 18F-FDG PET-CT imaging. Uptake of 11C-MRB, was quantified as the distribution volume ratio (DVR) using the occipital cortex as a low NET density reference region. Total body fat and lean body mass were assessed via bioelectrical impedance analysis. ResultsAs expected, 18F-FDG uptake in BAT was difficult to identify at RT but easily detected with cold stimulation (p=0.01). In contrast, BAT 11C-MRB uptake (also normalized for muscle) was equally evident under both RT and cold conditions (BAT DVR: RT 1.0±0.3 vs. cold 1.1±0.3, p=0.31; BAT/muscle DVR: RT 2.3±0.7 vs. cold 2.5±0.5, p=0.61). Importantly, BAT DVR and BAT/muscle DVR of 11C-MRB at RT correlated positively with core body temperature (r=0.76, p=0.05 and r=0.92, p=0.004, respectively), a relationship not observed with 18F-FDG (p=0.63). Furthermore, there were gender differences in 11C-MRB uptake in response to cold (p=0.03), which reflected significant differences in the change in 11C-MRB as a function of both body composition and body temperature. ConclusionsUnlike 18F-FDG, the uptake of 11C-MRB in BAT offers a unique opportunity to investigate the role of BAT in humans under basal, room temperature conditions.

Both chronic treatments by epothilone D and fluoxetine increase the short‐term memory and differentially alter the mood status of STOP/MAP6 KO mice

Recent evidence underlines the crucial role of neuronal cytoskeleton in the pathophysiology of psychiatric diseases. In this line, the deletion of STOP/MAP6 (Stable Tubule Only Polypeptide), a microtubule-stabilizing protein, triggers various neurotransmission and behavioral defects, suggesting that STOP knockout (KO) mice could be a relevant experimental model for schizoaffective symptoms. To establish the predictive validity of such a mouse line, in which the brain serotonergic tone is dramatically imbalanced, the effects of a chronic fluoxetine treatment on the mood status of STOP KO mice were characterized. Moreover, we determined the impact, on mood, of a chronic treatment by epothilone D, a taxol-like microtubule-stabilizing compound that has previously been shown to improve the synaptic plasticity deficits of STOP KO mice. We demonstrated that chronic fluoxetine was either antidepressive and anxiolytic, or pro-depressive and anxiogenic, depending on the paradigm used to test treated mutant mice. Furthermore, control-treated STOP KO mice exhibited paradoxical behaviors, compared with their clear-cut basal mood status. Paradoxical fluoxetine effects and control-treated STOP KO behaviors could be because of their hyper-reactivity to acute and chronic stress. Interestingly, both epothilone D and fluoxetine chronic treatments improved the short-term memory of STOP KO mice. Such treatments did not affect the serotonin and norepinephrine transporter densities in cerebral areas of mice. Altogether, these data demonstrated that STOP KO mice could represent a useful model to study the relationship between cytoskeleton, mood, and stress, and to test innovative mood treatments, such as microtubule-stabilizing compounds.

Noradrenergic innervation of pyramidal cells in the rat basolateral amygdala

The basolateral nuclear complex of the amygdala (BLC) receives dense noradrenergic/norepinephrine (NE) inputs from the locus coeruleus that play a key role in modulating emotional memory consolidation. Knowledge of the extent of synapse formation by NE inputs to the BLC, as well as the cell types innervated, would contribute to an understanding of how NE modulates the activity of the BLC. To gain a better understanding of NE circuits in the BLC, dual-label immunohistochemistry was used at the light and electron microscopic levels in the present study to analyze NE axons and their innervation of pyramidal cells in the anterior subdivision of the basolateral amygdalar nucleus (BLa). NE axons and BLa pyramidal cells were labeled using antibodies to the norepinephrine transporter (NET) and Ca2+/calmodulin-dependent protein kinase (CaMK), respectively. Dual localization studies using antibodies to NET and dopamine-beta-hydroxylase (DBH) revealed that virtually all NE axons and varicosities expressed both proteins. The BLa exhibited a medium density of NET+ fibers. Ultrastructural analysis of serial section reconstructions of NET+ axons revealed that only about half of NET+ terminals formed synapses. The main postsynaptic targets were small-caliber CAMK+ dendritic shafts and spines of pyramidal cells. A smaller number of NET+ terminals formed synapses with unlabeled cell bodies and dendrites. These findings indicate that the distal dendritic domain of BLa pyramidal cells is the major target of NE terminals in the BLa, and the relatively low synaptic incidence suggests that diffusion from non-synaptic terminals may be important for noradrenergic modulation of the BLa.

PATHOPHYSIOLOGY OF DEPRESSION: DO WE HAVE ANY SOLID EVIDENCE OF INTEREST TO CLINICIANS?

Due to the clinical and etiological heterogeneity of major depressive disorder, it has been difficult to elucidate its pathophysiology. Current neurobiological theories with the most valid empirical foundation and the highest clinical relevance are reviewed with respect to their strengths and weaknesses. The selected theories are based on studies investigating psychosocial stress and stress hormones, neurotransmitters such as serotonin, norepinephrine, dopamine, glutamate and gamma-aminobutyric acid (GABA), neurocircuitry, neurotrophic factors, and circadian rhythms. Because all theories of depression apply to only some types of depressed patients but not others, and because depressive pathophysiology may vary considerably across the course of illness, the current extant knowledge argues against a unified hypothesis of depression. As a consequence, antidepressant treatments, including psychological and biological approaches, should be tailored for individual patients and disease states. Individual depression hypotheses based on neurobiological knowledge are discussed in terms of their interest to both clinicians in daily practice and clinical researchers developing novel therapies.

The norepinephrine transporter (NET) radioligand (S,S)-[ 18F]FMeNER-D 2 shows significant decreases in NET density in the human brain in Alzheimer's disease: A post-mortem autoradiographic study

Earlier post-mortem histological and autoradiographic studies have indicated a reduction of cell numbers in the locus coeruleus (LC) and a corresponding decrease in norepinephrine transporter (NET) in brains obtained from Alzheimer's disease (AD) patients as compared to age-matched healthy controls. In order to test the hypothesis that the regional decrease of NET is a disease specific biomarker in AD and as such, it can be used in PET imaging studies for diagnostic considerations, regional differences in the density of NET in various anatomical structures were measured in whole hemisphere human brain slices obtained from AD patients and age-matched control subjects in a series of autoradiographic experiments using the novel selective PET radioligand for NET (S,S)-[ 18F]FMeNER-D 2. (S,S)-[ 18F]FMeNER-D 2 appears to be a useful imaging biomarker for quantifying the density of NET in various brain structures, including the LC and the thalamus wherein the highest densities are found in physiological conditions. In AD significant decreases of NET densities can be demonstrated with the radioligand in both structures as compared to age-matched controls. The decreases in AD correlate with the progress of the disease as indicated by Braak grades. As the size of the LC is below the spatial resolution of the PET scanners, but the size of the thalamus can be detected with appropriate spatial accuracy in advanced scanners, the present findings confirm our earlier observations with PET that the in vivo imaging of NET with (S,S)-[ 18F]FMeNER-D 2 in the thalamus is viable. Nevertheless, further studies are warranted to assess the usefulness of such an imaging approach for the early detection of changes in thalamic NET densities as a disease-specific biomarker and the possible use of (S,S)-[ 18F]FMeNER-D 2 as a molecular imaging biomarker in AD.

Norepinephrine Transporter is Involved in Down-Regulation of β1-Adrenergic Receptors Caused by Adjuvant Arthritis

Inflammation in forms of rheumatoid and experimental arthritis cause not only joint pain but also excessive cardiovascular mortality. The condition also reduces response to calcium channel and beta-adrenergic (beta1-AR) antagonists. For calcium channel inhibitors, the reduced response is shown to be due to the reduced expression of target proteins. Hydroxymethylglutaryl CoA reductase inhibitors (statins) restore response to propranolol and verapamil. We tested the effect of adjuvant arthritis on the norepinephrine (NE) transporter (NET) density since altered sympathetic nervous system innervation has been observed in rheumatoid arthritis. Male Sprague-Dawley rats were divided into the following groups: Healthy/Placebo, Healthy/Statin, Pre-AA/Placebo, and Pre-AA/Statin (n=7-8/group). On Day 0, to the Pre-AA and Healthy groups, was injected Mycobacterium butyricum or saline, respectively. On Days 4-8, Statin and Placebo groups received either pravastatin (6 mg/kg) or placebo twice daily, respectively. On day 8, heart and blood samples were collected. The density of NET and 1-AR in heart homogenate; NE in plasma and heart and inflammatory mediators (nitrite and interferon-gamma) in serum were determined. Inflammation was associated with a significant reduction in both beta1-AR and NET density with a positive correlation between the two proteins (r=0.978, p<0.0001). The down-regulating effect of inflammation was not reversed by pravastatin. Inflammation had no significant effect on the plasma or heart NE concentration. The close relations of NET and beta1-AR implicates altered sympathetic innervation and/or local NE handling in pharmacotherapeutic desensitization observed in arthritis.

Whole-Body Evaluation of MIBG Tissue Extraction in a Mouse Model of Long-Lasting Type II Diabetes and Its Relationship with Norepinephrine Transport Protein Concentration

Accelerated cardiac washout of (123)I-metaiodobenzylguanidine (MIBG), which is clinically used as an index of cardiac neuropathy in diabetes, is ascribed to decreased norepinephrine reuptake into synaptic vesicles. However, accelerated washout frequently contrasts with preserved early tracer uptake, whose significance remains undetermined. The aim of this study was to investigate in a mouse model of long-lasting type II diabetes whether the mismatch between MIBG early uptake and washout is the consequence of a more generalized disorder of the autonomic nervous system. Nine mice were given low doses of streptozotocin by intraperitoneal injection for 5 consecutive days. At 7 mo after streptozotocin, MIBG kinetics were evaluated by heart and liver time-activity curves and by tracer accumulation in the bladder. Data were compared with those obtained in 10 sham mice and correlated with the cardiac and hepatic tissue expression of norepinephrine transporter (NET) as assessed with a (3)H-desipramine saturation binding assay. In diabetic mice, myocardial and liver MIBG retention was reduced at 2 h and was associated with both increased tracer washout and reduced NET density. The rate of myocardial washout correlated with the degree of urinary MIBG excretion. The paradoxic observation of preserved early uptake associated with accelerated washout of MIBG in diabetes seems to be explained by a generalized disorder in NET function leading to reduced whole-body tracer removal from the blood and increased tracer availability for early myocardial uptake.

Evaluation of radioiodinated (2S,αS)-2-(α-(2-iodophenoxy)benzyl)morpholine as a radioligand for imaging of norepinephrine transporter in the heart

The norepinephrine transporter (NET) is located presynaptically on noradrenergic nerve terminals and plays a critical role in the regulation of the synaptic norepinephrine (NE) concentration via the reuptake of NE. Changes in NET have been recently reported in several cardiac failures. Therefore, a NET-specific radioligand is useful for in vivo assessment of changes in NET density in various cardiac disorders. Recently, we developed a radioiodinated reboxetine analogue, (2S,alphaS)-2-(alpha-(2-iodophenoxy)benzyl)morpholine ((S,S)-IPBM), for NET imaging. In the current study, we assessed the applicability of radioiodinated (S,S)-IPBM to NET imaging in the heart. The NET affinity and selectivity were measured from the ability to displace specific [3H]nisoxetine and (S,S)-[125 I]IPBM binding to rat heart membrane, respectively. To evaluate the distribution of (S,S)-[125 I]IPBM in vivo, biodistribution experiment was performed in rats. With the use of several monoamine transporter binding agents, pharmacological blocking experiments were performed in rats. In vitro binding assays showed that the affinity of (S,S)-IPBM to NET was similar to those of the well-known NET-specific binding agents, nisoxetine and desipramine. Furthermore, (S,S)-[125 I]IPBM binding was inhibited by nisoxetine and desipramine, but not by dopamine or serotonin transporter binding agents. These data indicated that (S,S)-IPBM had high affinity and selectivity for NET in vitro. Biodistribution studies in rats showed rapid and high uptake of (S,S)-[125 I]IPBM by the heart and rapid clearance from the blood. The heart-to-blood ratio was 31.9 at 180 min after the injection. The administration of nisoxetine and desipramine decreased (S,S)-[125 I]IPBM accumulation in the heart, but injection of fluoxetine and GBR12909 had little influence. Radioiodinated (S,S)-IPBM is a potential radioligand for NET imaging in the heart.

Myocardial nerve fibers are preserved in MPTP-treated mice, despite cardiac sympathetic dysfunction

Recently, we reported a profound depletion of cardiac sympathetic nerve fibers in Parkinson's disease (PD). This cardiac sympathetic denervation is a characteristic hallmark of PD. Cardiac sympathetic dysfunction was also observed in 1-methyl-4-phenyl-1,2,3,6-tetrahydroxypyridine (MPTP)-treated mice, a model of PD. Although binding assay showed a decreased density of norepinephrine transporter (NET) in the hearts of the mice, their histopathological alterations have not been demonstrated. In this study, we investigated hearts of MPTP-treated mice with immunohistochemical method and Western blot analyses. MPTP-treated mice showed significant decreases in the contents of cardiac noradrenaline and dopamine, suggesting the sympathetic dysfunction. Synaptophysin-, tyrosine hydroxylase- or NET-immunoreactive nerve fibers were abundant in the hearts of control mice and MPTP-treated mice, without apparent differences between the two groups. Western blot analyses also showed no difference in the amounts of these proteins. Myocardial nerve fibers were well preserved in MPTP-treated mice, despite apparent cardiac sympathetic dysfunction.

Imaging the norepinephrine transporter in humans with (S,S)-[11C]O-methyl reboxetine and PET: problems and progress

Results from human studies with the PET radiotracer (S,S)-[(11)C]O-methyl reboxetine ([(11)C](S,S)-MRB), a ligand targeting the norepinephrine transporter (NET), are reported. Quantification methods were determined from test/retest studies, and sensitivity to pharmacological blockade was tested with different doses of atomoxetine (ATX), a drug that binds to the NET with high affinity (K(i)=2-5 nM). Twenty-four male subjects were divided into different groups for serial 90-min PET studies with [(11)C](S,S)-MRB to assess reproducibility and the effect of blocking with different doses of ATX (25, 50 and 100 mg, po). Region-of-interest uptake data and arterial plasma input were analyzed for the distribution volume (DV). Images were normalized to a template, and average parametric images for each group were formed. [(11)C](S,S)-MRB uptake was highest in the thalamus (THL) and the midbrain (MBR) [containing the locus coeruleus (LC)] and lowest for the caudate nucleus (CDT). The CDT, a region with low NET, showed the smallest change on ATX treatment and was used as a reference region for the DV ratio (DVR). The baseline average DVR was 1.48 for both the THL and MBR with lower values for other regions [cerebellum (CB), 1.09; cingulate gyrus (CNG) 1.07]. However, more accurate information about relative densities came from the blocking studies. MBR exhibited greater blocking than THL, indicating a transporter density approximately 40% greater than THL. No relationship was found between DVR change and plasma ATX level. Although the higher dose tended to induce a greater decrease than the lower dose for MBR (average decrease for 25 mg=24+/-7%; 100 mg=31+/-11%), these differences were not significant. The different blocking between MBR (average decrease=28+/-10%) and THL (average decrease=17+/-10%) given the same baseline DVR indicates that the CDT is not a good measure for non-NET binding in both regions. Threshold analysis of the difference between the average baseline DV image and the average blocked image showed the expected NET distribution with the MBR (LC) and hypothalamus>THL>CNG and CB, as well as a significant change in the supplementary motor area. DVR reproducibility for the different brain regions was approximately 10%, but intersubject variability was large. The highest density of NETs was found in the MBR where the LC is located, followed by THL, whereas the lowest density was found in basal ganglia (lowest in CDT), consistent with the regional localization of NETs in the nonhuman primate brain. While all three doses of ATX were found to block most regions, no significant differences between doses were found for any region, although the average percent change across subjects of the MBR did correlate with ATX dose. The lack of a dose effect could reflect a low signal-to-noise ratio coupled with the possibility that a sufficient number of transporters were blocked at the lowest dose and further differences could not be detected. However, since the lowest (25 mg) dose is less than the therapeutic doses used in children for the treatment of attention-deficit/hyperactivity disorder ( approximately 1.0 mg/kg/day), this would suggest that there may be additional targets for ATX's therapeutic actions.

Iron deficiency affects acoustic startle response and latency, but not prepulse inhibition in young adult rats

Iron deficiency is associated with alterations in dopamine and serotonin transporters as well as changes in dopamine receptor (DR) density, monoamine concentrations, and in vivo extracellular contents of monoamines in terminal fields. Human infants with iron deficiency have both delayed maturation as well as lengthened central conduction times in auditory evoked potential studies. The current study utilizes the magnitude of the acoustic startle response (ASR), prepulse inhibition (PPI), and mean latency to maximum startle response ( T max), to examine the functional integrity of response to environmental cues. Male and female rats consumed iron deficient (ID) or iron adequate (CN) diets from weaning until adulthood. ID rats of both sexes had 20–60% reductions in ASR when compared to CN rats but there was no effect on PPI. T max was significantly longer by 10–20% in females, but not males. Dopamine transporter density was significantly lower in putamen, nucleus accumbens, and olfactory tubercle in males, but not female rats while the serotonin transporter was significantly different from control animal density in five of 14 brain regions. Norepinephrine transporter density was lower in the locus ceruleus of ID male rats but was unaffected in ID female rats. Regression modeling of ASR with brain monoamine transporters and receptors showed hematocrit, norepinephrine transporter (NET) in dentate gyrus, and D 1R in the nucleus accumbens account for nearly 49% of the variance in ASR. T max was not significantly associated with any of the independent variables. We conclude that iron deficiency affects the startle response, but not the inhibitory circuits involved in prepulse inhibition. Importantly, sex also strongly influenced these behavioral responses. Future studies, perhaps pharmacologic in nature, are necessary to ascertain whether iron deficiency modifies the contribution of monoaminergic systems to responses to environmental stimuli.

Synthesis and evaluation of radioiodinated (S,S)-2-(α-(2-iodophenoxy)benzyl)morpholine for imaging brain norepinephrine transporter

Abnormality of the brain norepinephrine transporter (NET) has been reported in several psychiatric and neuronal disorders. Since NET is an important target for the diagnosis of these diseases, the development of radiopharmaceuticals for imaging of brain NET has been eagerly awaited. In this study, we synthesized (S,S)-2-(alpha-(2-iodophenoxy)benzyl)morpholine [(S,S)-IPBM], a derivative of reboxetine iodinated at position 2 of the phenoxy ring, and evaluated its potential as a radiopharmaceutical for imaging brain NET using SPECT. (S,S)-(123/125)I-IPBM was synthesized in a halogen exchange reaction. The affinity and selectivity of (S,S)-IPBM for NET was measured by assaying the displacement of (3)H-nisoxetine and (S,S)-(125)I-IPBM from the binding site in rat brain membrane, respectively. The biodistribution of (S,S)-(125)I-IPBM was also determined in rats. Furthermore, SPECT studies with (S,S)-(123)I-IPBM were carried out in the common marmoset. (S,S)-(125)I-IPBM was prepared with high radiochemical yields (65%) and high radiochemical purity (>98%). (S,S)-IPBM showed high affinity and selectivity for NET in the binding assay experiments. In biodistribution experiments, (S,S)-(125)I-IPBM showed rapid uptake in the brain, and the regional cerebral distribution was consistent with the density of NET. The administration of nisoxetine, a selective NET-binding agent, decreased the accumulation of (S,S)-(125)I-IPBM in the brain, but the administration of selective serotonin transporter and dopamine transporter binding agents caused no significant changes in the accumulation. Moreover, (S,S)-(123)I-IPBM allowed brain NET imaging in the common marmoset with SPECT. These results suggest that (S,S)-(123)I-IPBM is a potential SPECT radiopharmaceutical for imaging brain NET.

Reduced 125I- meta-iodobenzylguanidine uptake and norepinephrine transporter density in the hearts of mice with MPTP-induced parkinsonism

Uptake of 123I- meta-iodobenzylguanidine ( 123I-MIBG) is markedly reduced in the hearts of patients with Parkinson's disease. Although the mechanism of this reduction is unclear, 12 5I-MIBG uptake is similarly reduced in the hearts of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydroxypyridine (MPTP)-induced parkinsonism. Three groups of ten 15-week-old C57BL6 mice received intraperitoneal injections of (1) saline (control), (2) 10 mg/kg MPTP or (3) 40 mg/kg MPTP. After 0.185 MBq of 125I-MIBG was injected, the percent injected dose of 125I-MIBG per gram of tissue (%ID/g) was determined and cardiac concentrations of norepinephrine were measured. Cardiac concentrations of norepinephrine transporter (NET) were measured in three groups of twenty 15-week-old C57BL6 mice receiving these same treatments. The %ID/g in mice receiving 10 or 40 mg/kg MPTP (5.7±1.1 and 4.4±1.2%/g) was significantly lower than that in control mice (11.3±2.2%/g; P<.00001 and P<.0000001, respectively). The norepinephrine concentration in mice receiving 10 or 40 mg/kg MPTP (7.86±0.67×10 5 and 7.50±0.89×10 5 pg/wet g) was significantly lower than that in control mice (9.21±0.97×10 5 pg/wet g; P<.01 and P<.001, respectively). The NET density in mice receiving 10 or 40 mg/kg MPTP (81±12, 61±7 fmol/mg protein) was significantly lower than that in control mice (126±7 fmol/mg protein; P<.000001 and P<.0000001, respectively). The %ID/g of 125I-MIBG and NET density decreased as the dose of MPTP increased. This study clearly shows that reduced cardiac 12 5I-MIBG uptake in mice with MPTP-induced parkinsonism is closely related to the reduced NET density in postganglionic cardiac sympathetic nerve terminals.