Norepinephrine Transporter Blocker Research Articles

Effects of Atomoxetine for the Treatment of Neurogenic Orthostatic Hypotension in Patients With Alpha-synucleinopathies: A Systematic Review of Randomized Controlled Trials and a Focus-Group Discussion.

Neurogenic orthostatic hypotension (nOH) is one of the most important nonmotor symptoms in patients with α-synucleinopathies. Atomoxetine is a selective norepinephrine transporter blocker that is a treatment option for nOH. This systematic review and expert focus-group study was designed to obtain evidence from published data and clinical experiences of Korean movement-disorder specialists about the efficacy and safety of atomoxetine for the pharmacological treatment of nOH in patients with α-synucleinopathies. The study comprised a systematic review and a focus-group discussion with clinicians. For the systematic review, multiple comprehensive databases including MEDLINE, Embase, Cochrane Library, CINAHL, PsycInfo, and KoreaMed were searched to retrieve articles that assessed the outcomes of atomoxetine therapy. A focus-group discussion was additionally performed to solicit opinions from experts with experience in managing nOH. The literature review process yielded only four randomized controlled trials on atomoxetine matching the inclusion criteria. Atomoxetine effectively increased systolic blood pressure and improved OH-related symptoms as monotherapy or in combination with other drugs. Its effects were pronounced in cases with central autonomic failure, including multiple-system atrophy (MSA). Atomoxetine might be a safe monotherapy regarding the risk of supine hypertension. Atomoxetine is an effective and safe option for short-term nOH management, which could be more evident in patients with central autonomic dysfunction such as MSA. However, there is a paucity of evidence in the literature, and data from the focus-group discussion were inadequate, and so further investigation is warranted.

Characterization of D3 Autoreceptor Function in Whole Zebrafish Brain with Fast-Scan Cyclic Voltammetry.

Zebrafish (Danio rerio) are ideal model organisms for investigating nervous system function, both in health and disease. Nevertheless, functional characteristics of dopamine (DA) release and uptake regulation are still not well-understood in zebrafish. In this study, we assessed D3 autoreceptor function in the telencephalon of whole zebrafish brains ex vivo by measuring the electrically stimulated DA release ([DA]max) and uptake at carbon fiber microelectrodes with fast-scan cyclic voltammetry. Treatment with pramipexole and 7-OH-DPAT, selective D3 autoreceptor agonists, sharply decreased [DA]max. Conversely, SB277011A, a selective D3 antagonist, nearly doubled [DA]max and decreased k, the first-order rate constant for the DA uptake, to about 20% of its original value. Treatment with desipramine, a selective norepinephrine transporter blocker, failed to increase current, suggesting that our electrochemical signal arises solely from the release of DA. Furthermore, blockage of DA uptake with nomifensine-reversed 7-OH-DPAT induced decreases in [DA]max. Collectively, our data show that, as in mammals, D3 autoreceptors regulate DA release, likely by inhibiting uptake. The results of this study are useful in the further development of zebrafish as a model organism for DA-related neurological disorders such as Parkinson's disease, schizophrenia, and drug addiction.

Effect of concurrent organic cation transporter blockade on norepinephrine clearance inhibiting- and antidepressant-like actions of desipramine and venlafaxine

Depression is a major health problem for which most patients are not effectively treated. This underscores a need to identify new targets for the development of antidepressants with improved efficacy. Studies have shown that blockade of low-affinity/high-capacity transporters, such as organic cation transporters (OCTs) and the plasma membrane monoamine transporter (PMAT), with decynium-22 can produce antidepressant-like effects and inhibit serotonin clearance in brain when the serotonin transporter is pharmacologically or genetically compromised. In vitro studies show that OCTs/PMAT are also capable of norepinephrine transport, raising the possibility that decynium-22 might enhance the antidepressant-like effects of norepinephrine transporter inhibitors. Using in vivo electrochemistry, we show that local administration of decynium-22 into dentate gyrus of hippocampus enhanced the ability of the norepinephrine transporter blocker, desipramine, but not the dual norepinephrine/serotonin transporter blocker venlafaxine, to inhibit norepinephrine clearance. In parallel, systemic administration of decynium-22 (0.32 mg/kg) enhanced the antidepressant-like effects of desipramine (32 mg/kg), but not those of venlafaxine, in the tail suspension test, underscoring the heterogeneous response of mice to antidepressants, including those that share similar mechanisms of action. Systemic administration of normetanephrine, a potent blocker of OCT3, failed to potentiate the antidepressant-like effects of desipramine, suggesting that the actions of decynium-22 to augment the antidepressant-like effects of desipramine are likely mediated by another OCT isoform and/or PMAT. Taken together with existing literature, concurrent blockade of OCTs and/or PMAT merits further investigation as an adjunctive therapeutic for desipramine-like antidepressant drugs.

Cocaine and desipramine elicit distinct striatal noradrenergic and behavioral responses in selectively bred obesity-resistant and obesity-prone rats

Previous studies have demonstrated a role for norepinephrine (NE) in energy regulation and feeding, and basal differences have been observed in hypothalamic NE systems in obesity-prone vs. obesity-resistant rats. Differences in the function of brain reward circuits, including in the nucleus accumbens (NAc), have been shown in obesity-prone vs. obesity-resistant populations, leading many researchers to explore the role of striatal dopamine in obesity. However, alterations in NE transmission also affect NAc mediated behaviors. Therefore, here we examined differences in striatal NE and the response to norepinephrine transporter blockers in obesity-prone and obesity-resistant rats. We found that striatal NE levels increase following systemic cocaine administration in obesity-prone, but not obesity-resistant rats. This could result from either blockade of striatal norepinephrine transporters (NET) by cocaine leading to reduced NE reuptake, or circuit-based responses following cocaine administration resulting in increased NE release. Retrodialysis of the NET inhibitor, desipramine, into the ventral striatum did not cause selective increases in striatal NE levels in obesity-prone rats, suggesting that circuit-based mechanisms underlie NE increases following systemic cocaine administration. Consistent with this, systemic desipramine treatment decreased locomotor activity in obesity-prone, but not obesity-resistant rats. Furthermore, obesity-prone rats were also more sensitive to desipramine-induced reductions in food intake compared to obesity-resistant rats. Taken together, these data expand our understanding of differences in NE systems of obesity-prone vs. resistant rats, and provide new insights into basal differences in striatal systems that may influence feeding behavior.

The effect of atomoxetine on random and directed exploration in humans.

The adaptive regulation of the trade-off between pursuing a known reward (exploitation) and sampling lesser-known options in search of something better (exploration) is critical for optimal performance. Theory and recent empirical work suggest that humans use at least two strategies for solving this dilemma: a directed strategy in which choices are explicitly biased toward information seeking, and a random strategy in which decision noise leads to exploration by chance. Here we examined the hypothesis that random exploration is governed by the neuromodulatory locus coeruleus-norepinephrine system. We administered atomoxetine, a norepinephrine transporter blocker that increases extracellular levels of norepinephrine throughout the cortex, to 22 healthy human participants in a double-blind crossover design. We examined the effect of treatment on performance in a gambling task designed to produce distinct measures of directed exploration and random exploration. In line with our hypothesis we found an effect of atomoxetine on random, but not directed exploration. However, contrary to expectation, atomoxetine reduced rather than increased random exploration. We offer three potential explanations of our findings, involving the non-linear relationship between tonic NE and cognitive performance, the interaction of atomoxetine with other neuromodulators, and the possibility that atomoxetine affected phasic norepinephrine activity more so than tonic norepinephrine activity.

Norepinephrine transporter blocker atomoxetine increases salivary alpha amylase

It has been suggested that central norepinephrine (NE) activity may be inferred from increases in salivary alpha-amylase (SAA), but data in favor of this proposition are limited. We administered 40mg of atomoxetine, a selective NE transporter blocker that increases central NE levels, to 24 healthy adult participants in a double-blind, placebo-controlled cross-over design. Atomoxetine administration significantly increased SAA secretion and concentrations at 75–180min after treatment (more than doubling baseline levels). Consistent with evidence that elevation in central NE is a co-determinant of hypothalamic-pituitary-adrenal axis activity, salivary cortisol also approximately doubled at the same time points. Moreover, changes in salivary cortisol positively correlated with SAA (0.44<rho<0.56), bolstering the position that the origin of the changes in SAA reflect central NE. This work points toward the potential value of SAA as an inexpensive and non-invasive procedure to obtain information about activation of the central NE system.

Catecholamine-Mediated Increases in Gain Enhance the Precision of Cortical Representations.

Neurophysiological evidence suggests that neuromodulators, such as norepinephrine and dopamine, increase neural gain in target brain areas. Computational models and prominent theoretical frameworks indicate that this should enhance the precision of neural representations, but direct empirical evidence for this hypothesis is lacking. In two functional MRI studies, we examine the effect of baseline catecholamine levels (as indexed by pupil diameter and manipulated pharmacologically) on the precision of object representations in the human ventral temporal cortex using angular dispersion, a powerful, multivariate metric of representational similarity (precision). We first report the results of computational model simulations indicating that increasing catecholaminergic gain should reduce the angular dispersion, and thus increase the precision, of object representations from the same category, as well as reduce the angular dispersion of object representations from distinct categories when distinct-category representations overlap. In Study 1 (N = 24), we show that angular dispersion covaries with pupil diameter, an index of baseline catecholamine levels. In Study 2 (N = 24), we manipulate catecholamine levels and neural gain using the norepinephrine transporter blocker atomoxetine and demonstrate consistent, causal effects on angular dispersion and brain-wide functional connectivity. Despite the use of very different methods of examining the effect of baseline catecholamine levels, our results show a striking convergence and demonstrate that catecholamines increase the precision of neural representations. Norepinephrine and dopamine are among the most widely distributed and ubiquitous neuromodulators in the mammalian brain and have a profound and pervasive impact on cognition. Baseline catecholamine levels tend to increase with increasing task engagement in tasks involving perceptual decisions, yet there is currently no direct evidence of the specific impact of these increases in catecholamine levels on perceptual encoding. Our results fill this void by showing that catecholamines enhance the precision of encoding cortical object representations, and by suggesting that this effect is mediated by increases in neural gain, thus offering a mechanistic account of our key finding.

Responding for a conditioned reinforcer or unconditioned sensory reinforcer in mice: interactions with environmental enrichment, social isolation, and monoamine reuptake inhibitors.

Environmental factors influence the etiology of many psychiatric disorders. Likewise, environmental factors can alter processes central to motivation. Therefore, motivational deficits present in many disorders may be influenced by early life environmental conditions. We examined whether housing animals in different environmental conditions influenced the ability of sensory stimuli to acquire incentive value and whether elevated monoamine activity altered responsing for these stimuli. Isolation-housed (IH), pair-housed (PH), and environmentally enriched (EE) male C57BL/6N mice were examined in tests of responding for a conditioned reinforcer (CRf) or an unconditioned sensory reinforcer (USRf). The CRf was previously paired with saccharin delivery through Pavlovian conditioning, while the USRf was not conditioned with a reward. Following baseline tests of responding for the CRf or USRf, the effects of elevated monoamine activity were examined. At baseline, PH and EE mice respondedsimilarly for the CRf or USRf. IH mice responded more for the CRf but exhibited slower acquisition of responding for the USRf. Administration of citalopram, a serotonin transporter blocker, or atomoxetine, a norepinephrine transporter blocker, decreased responding for the CRf and USRf in all groups. The dopamine transporter blocker GBR 12909 generally increased responding for the CRf and USRf, but further analysis revealed enhanced responding for both reinforcers only in EE mice. Baseline incentive motivation is strongly influenced by the social component of housing conditions. Furthermore, environmental enrichment increased the sensitivity to elevated dopamine activity, while acute elevations in serotonin and norepinephrine inhibit incentive motivation irrespective of housing condition.

Initial Assessment of β3-Adrenoceptor-Activated Brown Adipose Tissue in Streptozotocin-Induced Type 1 Diabetes Rodent Model Using [18F]Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography.

Metabolic activity of brown adipose tissue (BAT) is activated by β3-adrenoceptor agonists and norepinephrine transporter (NET) blockers and is measurable using [(18)F]fluorodeoxyglucose ([(18)F]FDG) positron emission tomography/computed tomography (PET/CT) in rats. Using the streptozotocin (STZ)-treated rat model of type 1 diabetes mellitus (T1DM), we investigated BAT activity in this rat model under fasting and nonfasting conditions using [(18)F]FDG PET/CT. Drugs that enhance BAT activity may have a potential for therapeutic development in lowering blood sugar in insulin-resistant diabetes. Rats were rendered diabetic by administration of STZ and confirmed by glucose measures. [(18)F]FDG was injected in the rats (fasted or nonfasted) pretreated with either saline or β3-adrenoceptor agonist CL316,243 or the NET blocker atomoxetine for PET/CT scans. [(18)F]FDG metabolic activity was computed as standard uptake values (SUVs) in interscapular brown adipose tissue (IBAT) and compared across the different drug treatment conditions. Blood glucose levels > 500 mg/dL were established for the STZ-treated diabetic rats. Under fasting conditions, average uptake of [(18)F]FDG in the IBAT of STZ-treated diabetic rats was approximately 70% lower compared to that of normal rats. Both CL316,243 and atomoxetine activated IBAT in normal rats had an SUV > 5, whereas activation in STZ-treated rats was significantly lower. The agonist CL316,243 activated IBAT up to threefold compared to saline in the fasted STZ-treated rat. In the nonfasted rat, the IBAT activation was up by twofold by CL316243. Atomoxetine had a greater effect on lowering blood sugar levels compared to CL316,243 in the nonfasted rats. A significant reduction in metabolic activity was observed in the STZ-treated diabetic rodent model. Increased IBAT activity in the STZ-treated diabetic rat under nonfasted conditions using the β3-adrenoceptor agonist CL316,243 suggests a potential role of BAT in modulating blood sugar levels. Further studies are needed to evaluate the therapeutic role of β3-adrenoceptor agonists in insulin-resistant T1DM.

Antidepressant potential of organic cation transporter blockade: the role of norepinephrine (1144.5)

Depression is a major health problem for which most patients are not effectively treated. This underscores a need to identify new targets for the development of antidepressants with improved efficacy. Our previous studies have shown that blockade of organic cation transporters (OCTs) and the plasma membrane monoamine transporter (PMAT) with decynium‐22 (D‐22) can produce antidepressant‐like effects, which are correlated with the ability of D‐22 to inhibit serotonin (5‐HT) clearance in brain, when the 5‐HT transporter (SERT) is pharmacologically or genetically compromised. In vitro studies show that OCT3 takes up norepinephrine (NE) more efficiently than 5‐HT, which raises the possibility that D‐22 might enhance the antidepressant‐like actions of drugs that block NE uptake via the NE transporter (NET). Using in vivo chronoamperometry, an electrochemical technique which allows second by second recording of extracellular biogenic amine concentrations, we show D‐22 increases time to clear NE from the extracellular space, and show D‐22 potentiates the effects of NET blockers venlafaxine (VEN) and desipramine (DMI) to increase clearance time of NE in the dentate gyrus and CA3 of the hippocampus. We next investigated if D‐22 can enhance the antidepressant‐like effects of VEN/DMI. Both VEN and DMI reduced immobility on the tail suspension test (TST), a test commonly used to assay antidepressant‐like activity of drugs in mice. Unlike the enhancing effect of D‐22 on increasing clearance time of NE, D‐22 did not enhance the antidepressant‐like effect of VEN/DMI in wild‐type mice. Ongoing studies are investigating the antidepressant‐like effect of D22 in SERT KO and heterozygous mice.Grant Funding Source: Supported by MH093320 (LD &amp; WK), MH064489 (LD), NARSAD (LD) and UL1RR025767 (R25 START UP).

Dissociations between cognitive and motor effects of psychostimulants and atomoxetine in hyperactive DAT-KO mice

Psychostimulants such as amphetamine and methylphenidate, which target the dopamine transporter (DAT), are the most frequently used drugs for the treatment of hyperactivity and cognitive deficits in humans with attention deficit hyperactivity disorder (ADHD). While psychostimulants can increase activity in healthy subjects, they exert a "paradoxical" calming effect in humans with ADHD as well as in hyperactive mice lacking the dopamine transporter (DAT-KO mice). However, the mechanism of action of these drugs and their impact on cognition in the absence of DAT remain poorly understood. This study was conducted to investigate the effects of psychostimulants and noradrenergic and serotonergic drugs on cognition in DAT-KO mice and normal (WT) littermates. We used a recently developed behavioral apparatus, the automated H-maze. The H-maze involves the consecutive learning of three different rules: delayed alternation, nonalternation, and reversal tasks. Treatment of WT animals with the psychostimulants replicated the behavior observed in untreated DAT-KO mice while "paradoxically" restoring cognitive performances in DAT-KO mice. Further investigation of the potential involvement of other monoamine systems in the regulation of cognitive functions showed that the norepinephrine transporter blocker atomoxetine restored cognitive performances in DAT-KO mice without affecting hyperactivity. In contrast, the nonselective serotonin receptor agonist 5CT, which antagonizes hyperactivity in DAT-KO mice, had no effect on cognitive functions. Taken together, these data allow dissociation of the locomotor and cognitive effects of ADHD drugs and suggest that the combination of DAT-KO mice with the automated H-maze can constitute a powerful experimental paradigm for the preclinical development of therapeutic approaches for ADHD.

Yohimbine Depresses Excitatory Transmission in BNST and Impairs Extinction of Cocaine Place Preference Through Orexin-Dependent, Norepinephrine-Independent Processes

The alpha2 adrenergic receptor (α(2)-AR) antagonist yohimbine is a widely used tool for the study of anxiogenesis and stress-induced drug-seeking behavior. We previously demonstrated that yohimbine paradoxically depresses excitatory transmission in the bed nucleus of the stria terminalis (BNST), a region critical to the integration of stress and reward pathways, and produces an impairment of extinction of cocaine-conditioned place preference (cocaine-CPP) independent of α(2)-AR signaling. Recent studies show yohimbine-induced drug-seeking behavior is attenuated by orexin receptor 1 (OX(1)R) antagonists. Moreover, yohimbine-induced cocaine-seeking behavior is BNST-dependent. Here, we investigated yohimbine-orexin interactions. Our results demonstrate yohimbine-induced depression of excitatory transmission in the BNST is unaffected by alpha1-AR and corticotropin-releasing factor receptor-1 (CRFR(1)) antagonists, but is (1) blocked by OxR antagonists and (2) absent in brain slices from orexin knockout mice. Although the actions of yohimbine were not mimicked by the norepinephrine transporter blocker reboxetine, they were by exogenously applied orexin A. We find that, as with yohimbine, orexin A depression of excitatory transmission in BNST is OX(1)R-dependent. Finally, we find these ex vivo effects are paralleled in vivo, as yohimbine-induced impairment of cocaine-CPP extinction is blocked by a systemically administered OX(1)R antagonist. These data highlight a new mechanism for orexin on excitatory anxiety circuits and demonstrate that some of the actions of yohimbine may be directly dependent upon orexin signaling and independent of norepinephrine and CRF in the BNST.

Activation of adenosine 2A receptors (A2ARs) enhances norepinephrine (NE) transport into perivenous sympathetic nerves in normotensive, but not DOCA‐salt hypertensive rats

NE and ATP are released from perivascular sympathetic nerves to control peripheral resistance and blood pressure. NE is cleared from the neuroeffector junction by the NE transporter (NET). Adenosine, a product of ATP hydrolysis, inhibits NE release via an action at adenosine A1 receptors (A1Rs). A2ARs are also expressed on perivascular sympathetic nerves, but their function is unclear. We determined the role of A2ARs in modulating NE release and tested if A2AR function is impaired in DOCA‐salt rats. Electrically‐evoked NE release was measured in vitro using amperometry. The amplitude, rise slope, and decay slope of NE oxidation currents were measured in the absence or presence of drugs. CGS21680 (1–100 nM, A2AR agonist) increased the decay slope in mesenteric veins, but not arteries. The effect was antagonized by an A2AR antagonist (SCH58261 400 nM), and a NET blocker (cocaine 10 μM). This effect was mimicked by adenylate cyclase activation (forkolin, 1μM), but not PKC activation (PDBu 100 nM, or PMA 100 nM). A PKA inhibitor, H‐89 (1 μM) blocked the CGS‐induced effect. In mesenteric veins from DOCA‐salt rats, forskolin but not CGS21680 increased the decay slope of NE current. These data indicate that A2AR activation enhances NE transport into perivenous sympathetic nerves through activation of adenylate cyclase/cAMP/PKA pathway and A2AR function is impaired in DOCA‐salt veins.

Methylphenidate enhances extinction of contextual fear

Methylphenidate (MPH, Ritalin) is a norepinephrine and dopamine transporter blocker that is widely used in humans for treatment of attention deficit disorder and narcolepsy. Although there is some evidence that targeted microinjections of MPH may enhance fear acquisition, little is known about the effect of MPH on fear extinction. Here, we show that MPH, administered before or immediately following extinction of contextual fear, will enhance extinction retention in C57BL/6 mice. Animals that received MPH (2.5-10 mg/kg) before an extinction session showed decreased freezing response during extinction, and the effect of the 10 mg/kg dose on freezing persisted to the next day. When MPH (2.5-40 mg/kg) was administered immediately following an extinction session, mice that received MPH showed dose-dependent decreases in freezing during subsequent tests. MPH administered immediately after a 3-min extinction session or 4 h following the first extinction session did not cause significant differences in freezing. Together, these findings demonstrate that MPH can enhance extinction of fear and that this effect is sensitive to dose, time of injection, and duration of the extinction session. Because MPH is widely used in clinical treatments, these experiments suggest that the drug could be used in combination with behavioral therapies for patients with fear disorders.

A greater role for the norepinephrine transporter than the serotonin transporter in murine nociception

Norepinephrine and serotonin involvement in nociceptive functions is supported by observations of analgesic effects of norepinephrine transporter (NET) and serotonin transporter (SERT) inhibitors such as amitriptyline. However, the relative contribution of NET and SERT to baseline nociception, as well as amitriptyline analgesia, is unclear. Amitriptyline and morphine analgesia in wild-type (WT) mice and littermates with gene knockout (KO) of SERT, NET or both transporters was conducted using the hotplate and tail-flick tests. Hypoalgesia was observed in NET KO mice, and to a lesser extent in SERT KO mice. The magnitude of this hypoalgesia in NET KO mice was so profound that it limited the assessment of drug-induced analgesia. Nonetheless, the necessary exclusion of these subjects because of profound baseline hypoalgesia strongly supports the role of norepinephrine and NET in basal nociceptive behavior while indicating a much smaller role for serotonin and SERT. To further clarify the role of NET and SERT in basal nociceptive sensitivity further experiments were conducted in SERT KO and NET KO mice across a range of temperatures. NET KO mice were again found to have pronounced thermal hypoalgesia compared to WT mice in both the hotplate and tail-flick tests, while only limited effects were observed in SERT KO mice. Furthermore, in the acetic acid writhing test of visceral nociception pronounced hypoalgesia was again found in NET KO mice, but no change in SERT KO mice. As some of these effects may have resulted from developmental consequences of NET KO, the effects of the selective NET blocker nisoxetine and the selective SERT blocker fluoxetine were also examined in WT mice: only nisoxetine produced analgesia in these mice. Collectively these data suggest that NET has a far greater role in determining baseline analgesia, and perhaps other analgesic effects, than SERT in mice.

Slow and incomplete sympathetic reinnervation of rat tail artery restores the amplitude of nerve-evoked contractions provided a perivascular plexus is present

We have investigated the recovery of sympathetic control following reinnervation of denervated rat tail arteries by relating the reappearance of noradrenergic terminals to the amplitude of nerve-evoked contractions of isometrically mounted artery segments in vitro. We have also assessed reactivity to vasoconstrictor agonists. Freezing the collector nerves near the base of the tail in adult rats denervated the artery from ∼40 mm along the tail. Restoration of the perivascular plexus declined along the length of the tail, remaining incomplete for >6 mo. After 4 mo, nerve-evoked contractions were prolonged but of comparable amplitude to control at ∼60 mm along the tail; they were smaller at ∼110 mm. At ∼60 mm, facilitation of contractions to short trains of stimuli by the norepinephrine transporter blocker, desmethylimipramine, and by the α2-adrenoceptor antagonist, idazoxan, was reduced in reinnervated arteries. Blockade of nerve-evoked contractions by the α1-adrenoceptor antagonist, prazosin, was less and by idazoxan greater than control after 8 wk but similar to control after 16 wk. Sensitivity of reinnervated arteries to the α1-adrenoceptor agonist, phenylephrine, was raised in the absence but not in the presence of desmethylimipramine. Sensitivity to the α2-adrenoceptor agonist, clonidine, was maintained in 16-wk reinnervated arteries when it had declined in controls. Thus regenerating sympathetic axons have a limited capacity to reinnervate the rat tail artery, but nerve-evoked contractions match control once a relatively sparse perivascular plexus is reestablished. Functional recovery involves prolongation of contractions and deficits in both clearance of released norepinephrine and autoinhibition of norepinephrine release.

In vivo voltammetric monitoring of catecholamine release in subterritories of the nucleus accumbens shell

Fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes has been used to demonstrate that sub-second changes in catecholamine concentration occur within the nucleus accumbens (NAc) shell during motivated behaviors, and these fluctuations have been attributed to rapid dopamine signaling. However, FSCV cannot distinguish between dopamine and norepinephrine, and caudal regions of the NAc shell receive noradrenergic projections. Therefore, in the present study, we examined the degree to which norepinephrine contributes to catecholamine release within rostral and caudal portion of NAc shell. Analysis of tissue content revealed that dopamine was the major catecholamine detectable in the rostral NAc shell, whereas both dopamine and norepinephrine were found in the caudal subregion. To examine releasable catecholamines, electrical stimulation was used to evoke release in anesthetized rats with either stimulation of the medial forebrain bundle, a pathway containing both dopaminergic and noradrenergic projections to the NAc, or the ventral tegmental area/substantia nigra, the origin of dopaminergic projections. The catecholamines were distinguished by their responses to different pharmacological agents. The dopamine autoreceptor blocker, raclopride, as well as the monoamine and dopamine transporter blockers, cocaine and GBR 12909, increased evoked catecholamine overflow in both the rostral and caudal NAc shell. The norepinephrine autoreceptor blocker, yohimbine, and the norepinephrine transporter blocker, desipramine, increased catecholamine overflow in the caudal NAc shell without significant alteration of evoked responses in the rostral NAc shell. Thus, the neurochemical and pharmacological results show that norepinephrine signaling is restricted to caudal portions of the NAc shell. Following raclopride and cocaine or raclopride and GBR 12909, robust catecholamine transients were observed within the rostral shell but these were far less apparent in the caudal NAc shell, and they did not occur following yohimbine and desipramine. Taken together, the data demonstrate that catecholamine signals in the rostral NAc shell detected by FSCV are due to change in dopamine transmission.

SPECT imaging with the serotonin transporter radiotracer [123I]p ZIENT in nonhuman primate brain

Serotonin dysfunction has been linked to a variety of psychiatric diseases; however, an adequate SPECT radioligand to probe the serotonin transporter system has not been successfully developed. The purpose of this study was to characterize and determine the in vivo selectivity of iodine-123-labeled 2beta-carbomethoxy-3beta-(4'-((Z)-2-iodoethenyl)phenyl)nortropane, [(123)I]p ZIENT, in nonhuman primate brain. Two ovariohysterectomized female baboons participated in nine studies (one bolus and eight bolus to constant infusion at a ratio of 9.0 h) to evaluate [(123)I]p ZIENT. To evaluate the selectivity of [(123)I]p ZIENT, the serotonin transporter blockers fenfluramine (1.5, 2.5 mg/kg) and citalopram (5 mg/kg), the dopamine transporter blocker methylphenidate (0.5 mg/kg) and the norepinephrine transporter blocker nisoxetine (1 mg/kg) were given at 8 h post-radiotracer injection. In the bolus to constant infusion studies, equilibrium was established by 4-8 h. [(123)I]p ZIENT was 93% and 90% protein bound in the two baboons and there was no detection of lipophilic radiolabeled metabolites entering the brain. In the high-density serotonin transporter regions (diencephalon and brainstem), fenfluramine and citalopram resulted in 35-71% and 129-151% displacement, respectively, whereas methylphenidate and nisoxetine did not produce significant changes (<10%). These findings suggest that [(123)I]p ZIENT is a favorable compound for in vivo SPECT imaging of serotonin transporters with negligible binding to norepinephrine and dopamine transporters.

Differential Alteration of Sympathetic Norepinephrine Transporter (NET) in Mesenteric Arteries and Veins in DOCA‐salt hypertensive rats

Increased sympathetic nervous system activity contributes to DOCA‐salt hypertension in rats. Sympathetic nerves release NE that acts on the postjunctional α1 receptors causing vasoconstriction. NE is cleared by NET. We tested the hypothesis that NET function is impaired in DOCA‐salt rats. Video microscopy and amperometry were used to measure neurogenic vasoconstriction and real time NE release in mesenteric arteries (MA) and veins (MV). Neurogenic constriction was larger in DOCA‐salt MA compared to control MA but vascular reactivity to NE was similar in DOCA‐salt and control MA. Cocaine, a NET blocker, increased NE peak current in arteries but not veins. Cocaine caused less increase in NE current in DOCA‐salt MA artery compared to control. Guanethidine caused concentration dependent (10–300 nM) inhibition of NE currents release in control MA, MV and DOCA‐salt MV, but not DOCA‐salt MA. These data suggest NET function is more prominent in MA compared to MV. Increased NE currents in DOCA‐salt MA are due partly to impaired NET function. (Supported by HL70687)

A Radioiodinated MIBG–Octreotate Conjugate Exhibiting Enhanced Uptake and Retention in SSTR2-Expressing Tumor Cells

Several neuroendocrine tumors are known to express both the somatostatin receptor subtype 2 (SSTR2) and the norepinephrine transporter (NET), and radiopharmaceuticals directed toward both these targets such as MIBG and octreotide derivatives are routinely used in the clinic. To investigate the possibility of targeting both NET and SSTR2 conjointly, a conjugate of radioiodinated MIBG and octreotate was synthesized. Attempts to synthesize the radioiodinated target compound (MIBG-octreotate; [ (131)I] 12a) from a tin precursor were futile; however, it could be accomplished from a bromo precursor by exchange radioiodination in 3-36% ( n = 10) radiochemical yields. The total uptake of [ (131)I] 12a in SK-N-SH human neuroblastoma cells transfected to express SSTR2 (SK-N-SHsst2) was similar to that for [ (125)I]MIBG at all time points (34.9 +/- 2.4% vs 43.8 +/- 1.2% at 4 h; p < 0.05), while it was substantially lower (5.4 +/- 0.3% vs 35.9 +/- 1.2%) in the SH-SY5Y cell line, a subclone of SK-N-SH line that is known to express SSTR2. The NET blocker desipramine reduced the uptake of [ (131)I] 12a only to a small extent, further suggesting a limited role of NET in its binding and accumulation. Uptake of [ (131)I] 12a in SK-N-SHsst2 cells was 8-10-fold higher ( p < 0.05) than that of [ (125)I]I-Gluc-TOCA, an octreotide analogue, at all time points over a 4 h period and was reduced to about 20% by 10 muM octreotide demonstrating that the uptake of [ (131)I] 12a in this cell line is predominantly mediated by SSTR2. The intracellularly trapped radioactivity in SK-N-SHsst2 cells was substantially higher for [ (131)I] 12a compared to that for [ (125)I]OIBG-octreotate, an isomeric congener of 12a. Because MIBG has more specific NET-mediated uptake than OIBG, this suggests at least a partial role for NET-mediated uptake of [ (131)I] 12a in this cell line. While further refinement in the structure of the conjugate-probably interposition of a flexible and/or cleavable linker between the MIBG and octreotate moieties-may be necessary to make it a substrate/ligand for both NET and SSTR2, this conjugate is demonstrated to be much superior than I-Gluc-TOCA with respect to the uptake in SSTR2-expressing cells.