DA Neurotransmission Research Articles

Motivational and Valence-Related Modulation of Sleep/Wake Behavior are Mediated by Midbrain Dopamine and Uncoupled from the Homeostatic and Circadian Processes.

Motivation and its hedonic valence are powerful modulators of sleep/wake behavior, yet its underlying mechanism is still poorly understood. Given the well‐established role of midbrain dopamine (mDA) neurons in encoding motivation and emotional valence, here, neuronal mechanisms mediating sleep/wake regulation are systematically investigated by DA neurotransmission. It is discovered that mDA mediates the strong modulation of sleep/wake states by motivational valence. Surprisingly, this modulation can be uncoupled from the classically employed measures of circadian and homeostatic processes of sleep regulation. These results establish the experimental foundation for an additional new factor of sleep regulation. Furthermore, an electroencephalographic marker during wakefulness at the theta range is identified that can be used to reliably track valence‐related modulation of sleep. Taken together, this study identifies mDA signaling as an important neural substrate mediating sleep modulation by motivational valence.

Dopamine Cell Therapy: From Cell Replacement to Circuitry Repair.

Cell therapy for Parkinson’s disease (PD) is aimed to replace the degenerated midbrain dopamine (mDA) neurons and restore DA neurotransmission in the denervated forebrain targets. A limitation of the intrastriatal grafting approach, which is currently used in clinical trials, is that the mDA neurons are implanted into the target area, in most cases the putamen, and not in the ventral midbrain where they normally reside. This ectopic location of the cells may limit their functionality due to the lack of appropriate afferent regulation from the host. Homotopic transplantation, into the substantia nigra, is now being pursued in rodent PD models as a way to achieve more complete circuitry repair. Intranigral grafts of mDA neurons, derived from human embryonic stem cells, have the capacity to re-establish the nigrostriatal and mesolimbic pathways in their entirety and restore dense functional innervations in striatal, limbic and cortical areas. Tracing of host afferent inputs using the rabies tracing technique shows that the afferent connectivity of grafts implanted in the nigra matches closely that of the intrinsic mDA system, suggesting a degree of circuitry reconstruction that exceeds what has been achieved before. This approach holds great promise, but to match the larger size of the human brain, and the 10 times greater distance between substantia nigra and its forebrain targets, it may be necessary to find ways to improve the growth capacity of the grafted mDA neurons, pointing to a combined approach where growth promoting factors are used to enhance the performance of mDA neuron grafts.

Abstract 327: Asphyxial Cardiac Arrest Induces Dynamic Changes in Dopamine Neurotransmission and Linked Behavioral Deficits

Introduction: Cardiac arrest survival has improved with advances in resuscitation care, but survivors face impairments from the resulting hypoxic-ischemic brain injury (HIBI). Given the popular clinical use of DA modulators, despite limited understanding of disturbances in DA neurotransmission after HIBI, we characterized striatal DA signaling and behavioral deficits in a rat model of asphyxial cardiac arrest (ACA). Hypothesis: ACA-induced HIBI alters DA neurotransmission linked to behavioral deficits. Methods: Adult male Sprague-Dawley rats (n=41) underwent either Sham procedures (n=10) or 5-min no-flow ACA (n=28) insult. Fast-scan cyclic voltammetry (FSCV) and maximal medial forebrain bundle stimulations (60Hz, 10s) were used to characterize presynaptic DA signaling in dorsal striatum (D-Str). FSCV findings were compared with sensorimotor processing [acoustic startle responses (ASR)], open field exploration (total distance & exploratory zone entries), myoclonus, and anhedonia (sucrose preference testing). Results: ACA increased maximum evoked overflow (fig 1) and several DA release-based kinetic metrics. ACA hindered sensorimotor processing via increased ASR %change, elicited myoclonic responses to auditory stimuli, reduced mobility & exploration, and increased anhedonia. Many behavioral measures correlated with D-Str neurotransmission ( fig 2 ). Conclusions: ACA causes early hypodopaminergia that evolves to a hyperdopaminergic state by 2 weeks that is associated with behavioral dysfunction. Future work should further characterize striatal pathology post-ACA and identify treatments to resolve altered DA signaling and behavioral deficits.

Preclinical Pharmacology of [2-(3-Fluoro-5-Methanesulfonyl-phenoxy)Ethyl](Propyl)amine (IRL790), a Novel Dopamine Transmission Modulator for the Treatment of Motor and Psychiatric Complications in Parkinson Disease.

IRL790 ([2-(3-fluoro-5-methanesulfonylphenoxy)ethyl](propyl)amine, mesdopetam) is a novel compound in development for the clinical management of motor and psychiatric disabilities in Parkinson disease. The discovery of IRL790 was made applying a systems pharmacology approach based on in vivo response profiling. The chemical design idea was to develop a new type of DA D3/D2 receptor type antagonist built on agonist rather than antagonist structural motifs. We hypothesized that such a dopamine antagonist with physicochemical properties similar to agonists would exert antidyskinetic and antipsychotic effects in states of dysregulated dopaminergic signaling while having little negative impact on physiologic dopamine transmission and, hence, minimal liability for side effects related to dopamine-dependent functions. At the level of in vivo pharmacology, IRL790 displays balancing effects on aberrant motor phenotypes, reducing l-DOPA-induced dyskinesias in the rodent 6-hydroxydopamine lesion model and reducing psychostimulant-induced locomotor hyperactivity elicited by pretreatment with either d-amphetamine or dizocilpine, without negatively impacting normal motor performance. Thus, IRL790 has the ability to normalize the behavioral phenotype in hyperdopaminergic as well as hypoglutamatergic states. Neurochemical and immediate early gene (IEG) response profiles suggest modulation of DA neurotransmission, with some features, such as increased DA metabolites and extracellular DA, shared by atypical antipsychotics and others, such as increased frontal cortex IEGs, unique to IRL790. IRL790 also increases extracellular levels of acetylcholine in the prefrontal cortex and ventral hippocampus. At the receptor level, IRL790 appears to act as a preferential DA D3 receptor antagonist. Computational docking studies support preferential affinity at D3 receptors with an agonist-like binding mode. SIGNIFICANCE STATEMENT: This paper reports preclinical pharmacology along with molecular modeling results on IRL790, a novel compound in clinical development for the treatment of motor and psychiatric complications in advanced Parkinson disease. IRL790 is active in models of perturbed dopaminergic and glutamatergic signaling, including rodent 6-hydroxydopamine l-DOPA-induced dyskinesias and psychostimulant-induced hyperactivity, in a dose range that does not impair normal behavior. This effect profile is attributed to interactions at dopamine D2/D3 receptors, with a 6- to 8-fold preference for the D3 subtype.

Regulator of G protein signaling 6 (RGS6) is a critical modulator of reward behavior and dopamine signaling in the mesolimbic circuit

Alcohol (EtOH) is the most commonly abused drug worldwide and chronic EtOH consumption often leads to development of dependence and abuse. EtOH dependence is a progressive, neurological disorder characterized by an accumulation of neuroadaptations resulting from chronic EtOH exposure. The neurobiological mechanisms underlying EtOH‐seeking behavior and dependence are not fully understood. As a result, there are limited therapeutics designed to reduce alcohol cravings or withdrawal symptomology, and abstinence remains the only effective way to prevent alcohol use disorders (AUDs). Previously, we showed that RGS6 deficiency in mice leads to reduced EtOH seeking and reward behaviors. Reduced EtOH seeking in RGS6−/− mice was partially reversed by GABAB and D2 autoreceptor (D2R) antagonism and completely reversed by singular inhibition of DAT. These findings, together with the observed reduction in striatal DA content in RGS6−/− mice, led us to hypothesize that RGS6 functions to promote mesolimbic DA transmission by suppressing GPCR‐Gαi/o‐mediated upregulation of DAT. To investigate this possibility, we examined the effects of RGS6 loss on DAT expression in VTA cell bodies and synaptic terminals in the nucleus accumbens (NAcc). In addition, we examined the effects of RGS6 loss on mesolimbic DA transmission using optogenetic stimulated release of DA and c‐Fos staining in the NAcc. We observed robust expression of RGS6 in VTA cell bodies and terminals where its loss led to upregulated expression of DAT. These findings suggest that RGS6 functions to promote EtOH seeking and reward behavior by promoting DA neurotransmission in the mesolimbic circuit by suppressing GPCR‐Gαi/o‐DAT upregulation.Support or Funding InformationNational Institutes of Health [AA025919]; the Michael J. Fox Foundation [1151]; and University of Iowa Pharmacological Sciences Training Grant [T32GM067795].

Translating the atypical dopamine uptake inhibitor hypothesis toward therapeutics for treatment of psychostimulant use disorders.

Medication-assisted treatments are unavailable to patients with cocaine use disorders. Efforts to develop potential pharmacotherapies have led to the identification of a promising lead molecule, JJC8-091, that demonstrates a novel binding mode at the dopamine transporter (DAT). Here, JJC8-091 and a structural analogue, JJC8-088, were extensively and comparatively assessed to elucidate neurochemical correlates to their divergent behavioral profiles. Despite sharing significant structural similarity, JJC8-088 was more cocaine-like, increasing extracellular DA concentrations in the nucleus accumbens shell (NAS) efficaciously and more potently than JJC8-091. In contrast, JJC8-091 was not self-administered and was effective in blocking cocaine-induced reinstatement to drug seeking. Electrophysiology experiments confirmed that JJC8-091 was more effective than JJC8-088 at inhibiting cocaine-mediated enhancement of DA neurotransmission. Further, when VTA DA neurons in DAT-cre mice were optically stimulated, JJC8-088 produced a significant leftward shift in the stimulation-response curve, similar to cocaine, while JJC8-091 shifted the curve downward, suggesting attenuation of DA-mediated brain reward. Computational models predicted that JJC8-088 binds in an outward facing conformation of DAT, similar to cocaine. Conversely, JJC8-091 steers DAT towards a more occluded conformation. Collectively, these data reveal the underlying molecular mechanism at DAT that may be leveraged to rationally optimize leads for the treatment of cocaine use disorders, with JJC8-091 representing a compelling candidate for development.

Deletion of Dlk1 increases the vulnerability to developing anxiety-like behaviors and ethanol consumption in mice

Anxiety and alcohol use disorders (AUD) often present together, constituting a significant public health problem worldwide. In this study, we investigated the role of DLK1, a ligand of the Delta/NOTCH epidermal growth factor (EGF)-like protein family, reported to play a role in DA neurons differentiation in the striatum, as a neurobiological factor involved in the mechanisms regulating this psychiatric comorbidity.We exposed Dlk1 knockout mice (Dlk1−/− mice) to the open-field (OF), the light-dark box (LBD) and the elevated plus maze (EPM) tests, evaluating motivation to drink and ethanol consumption using the oral ethanol self-administration (OEA) paradigm. Quantitative real time polymerase chain reaction (qPCR) studies were carried out to evaluate alterations in targets closely related to DA neurotransmission in the reward system, tyrosine hydroxylase (Th) in the ventral tegmental area (VTA), and μ-opioid receptor (Oprm1) in the nucleus accumbens (NAc).No differences were observed in the total or peripheral distances travelled by Dlk1−/− compared to wild-type (WT) mice in OF. However, central distance travelled significantly decreased in Dlk1−/− mice. Deletion of Dlk1 increased anxiety-like behaviors in the LDB and EPM, and, Dlk1−/− mice also presented higher ethanol intake and motivation to drink (number of effective responses) in the OEA. In addition, Th and Oprm1 gene expression was reduced in the VTA and NAc of Dlk1−/− mice.We conclude that deletion of Dlk1 increases anxiety-related behaviors and vulnerability to ethanol consumption and modifies the gene expression of key targets closely related with DA neurotransmission involved in the reinforcing actions of ethanol.

27. THE ROLE OF DOPAMINE IN SHAPING CIRCUITRY RELATED TO SCHIZOPHRENIA AND ADDICTION

Overall : Dopamine plays a central role in shaping circuitry within the brain, thus affecting learning and behavior. It also plays a central role in schizophrenia and addiction. This panel will examine the impact of dopaminergic signaling on specific circuits that may create special vulnerability for the emergence of comorbidity between schizophrenia and addiction. The talks will include two presentations in clinical samples using molecular and functional imaging and two presentations in animal models.Jared Van Snellenberg will discuss connectivity of striatal substructures to the rest of the brain in drug free patients with schizophrenia and their relationship to abnormal cortical D2 signaling and psychotic symptoms. He will present recent unpublished work, motivated by pre-clinical studies with a D2 receptor over-expressing mouse model, using simultaneous multi-slice (multiband) functional MR imaging in these patients. These results suggest that unmedicated patients have altered connectivity between specific basal ganglia subnuclei, consistent with the animal model.Nora Volkow will focus on the role of bidirectional interactions between dopamine reward system and prefrontal regions in the addicted brain with emphasis on the role of D2 receptor signaling in the striatum. She will discuss the functional impact of these interactions on reward/motivation and executive-function networks and will discuss the variables that influence D2 receptor function including genes, sleep and social stressors and how they interact with drug exposures to provide resilience or vulnerability to substance use disorders or schizophrenia. Finally, she will discuss how this knowledge can be used to tailor interventions to remediate or buffer neurocircuity dysfunction triggered by drugs and for prevention.Bita Moghaddam will present an animal model of behaviorally induced dysfunction in the mesocortical circuit by using a task where actions are consistently rewarded but probabilistically punished. Spike activity and local field potentials are recorded during this task simultaneously from VTA and mPFC, two reciprocally connected mesocortical regions. Under no risk of punishment, a synchronous interaction at multiple time scales between PFC and VTA dopamine neurons is observed. This synchrony collapsed as a function of punishment contingency during reward-seeking actions, with risk of punishment diminishing VTA-driven neural synchrony between the two regions. These data reveal a dynamic coding scheme in VTA-mPFC neural circuits in representing aversion-based modulation of rewarded actions. These data suggest that driving VTA dopamine neurons by drugs of abuse may reverse the diminished synchrony and serve as self-medication in comorbid conditions.Finally, Aurelio Galli will discuss the structural, functional, and behavioral insights into the dopamine dysfunction of comorbid conditions as modeled by a deletion of the SLC6A3 affecting the function of the human dopamine transporter (hDAT). Genetic variants in hDAT have been associated with neuropsychiatric disorders. An in-frame deletion in hDAT at N336 (∆N336) leads to abnormal DA homeostasis. He demonstrated these dysfunctions in brains of Drosophila melanogaster expressing hDAT ∆N336. Furthermore, these flies are hyperactive and display fear and impaired social interactions, traits associated with impaired DA neurotransmission. Insights from X-ray crystallography, electron paramagnetic resonance, molecular dynamic simulations, electrophysiology and behaviors describe how a genetic variation causes DA dysfunction resulting in combined behavioral alterations and psychostimulant use.

Local Administration of Oxytocin in The Nucleus Accumbens Shell Affects The Neurochemical Effects of Methylphenidate Related to Its Reinforcing Effects.

IntroductionDopamine (DA) transmission in mesolimbic areas is intimately involved in the mediation of natural‐ and drug‐reinforcing behavioral effects. However, oxytocin has been shown to interfere with psychostimulant‐induced behavioral effects, the mechanism for which is unknown. We have recently shown that systemic, peripheral oxytocin (10 min pretreatment) dose‐dependently decreased intravenous (i.v.) methylphenidate self‐administration behavior. Moreover, oxytocin did not significantly affect extracellular levels of DA in the nucleus accumbens shell (NAS), while it dose‐dependently enhanced methylphenidate‐stimulated extracellular NAS DA levels.MethodsIn order to better understand which brain area might be involved in the effects of oxytocin on neurochemistry related to reinforcing actions of methylphenidate, we implanted Sprague Dawley rats with microdialysis probes to assess methylphenidate's (0.1–1 mg/kg, i.v.) effects on levels of DA in the NAS and nucleus accumbens core (NAC); in the NAS, oxytocin was administered through the microdialysis probe by reverse dialysis, while systemic injections of oxytocin were administered to rats implanted with probes in the NAC. Intranasal administration of oxytocin was also tested in order to replicate the data obtained by its systemic peripheral administration before MPH doses from 0.1–1.0 mg/kg (i.v.).ResultsOxytocin did not elicit any change in dialysate DA levels when delivered either systemically in the NAC or intranasally or locally by reverse dialysis in the NAS, up to 2 hrs from the start of its administration. These effects confirm our previous data obtained after systemic oxytocin administration in the NAS. Similarly, dose‐dependent enhancements of i.v. methylphenidate‐stimulated DA levels were obtained when oxytocin was delivered intranasally or locally into the NAS, but no significant effects were obtained in the NAC. These results clearly indicate that these oxytocin actions are mediated by oxytocin receptors located in the NAS but not in the NAC. Our ongoing voltammetry studies will evaluate if oxytocin effects on methylphenidate are the results of changes in DA uptake or release.ConclusionsTogether with our previous studies, the present results suggest that oxytocin pretreatments might attenuate the psychostimulant‐like effects of methylphenidate, likely through changes in DA neurotransmission in the NAS, terminal area of the mesolimbic DA system, but not in the NAC. Thus, our results confirm and extend the potential translational value of oxytocin as a pharmacotherapy for psychostimulant use disorders.Support or Funding InformationResearch Funded by: MDP‐NIDA‐IRP, NIH/DHHS; Bench‐to‐Bedside Grant by the OBSSR/NIH; Intramural Grant ZIA AA000218‐01 (CPN Section) DICBR/NIAAA/NIH/DHHSThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of ketamine on the unconditioned and conditioned locomotor activity of preadolescent and adolescent rats: impact of age, sex, and drug dose.

Ketamine is used by preadolescent and adolescent humans for licit and illicit purposes. The goal of the present study was to determine the effects of acute and repeated ketamine treatment on the unconditioned behaviors and conditioned locomotor activity of preadolescent and adolescent rats. To assess unconditioned behaviors, female and male rats were injected with ketamine (5-40mg/kg), and distance traveled was measured on postnatal day (PD) 21-25 or PD 41-45. To assess conditioned activity, male and female rats were injected with saline or ketamine in either a novel test chamber or the home cage on PD 21-24 or PD 41-44. One day later, rats were injected with saline and conditioned activity was assessed. Ketamine produced a dose-dependent increase in the locomotor activity of preadolescent and adolescent rats. Preadolescent rats did not exhibit sex differences, but ketamine-induced locomotor activity was substantially stronger in adolescent females than males. Repeated ketamine treatment neither caused a day-dependent increase in locomotor activity nor produced conditioned activity in preadolescent or adolescent rats. The activity-enhancing effects of ketamine are consistent with the actions of an indirect dopamine agonist, while the inability of ketamine to induce conditioned activity is unlike what is observed after repeated cocaine or amphetamine treatment. This dichotomy could be due to ketamine's ability to both enhance DA neurotransmission and antagonize N-methyl-D-aspartate (NMDA) receptors. Additional research will be necessary to parse out the relative contributions of DA and NMDA system functioning when assessing the behavioral effects of ketamine during early ontogeny.

Non-parametric analysis of neurochemical effects and Arc expression in amphetamine-induced 50-kHz ultrasonic vocalization

A number of studies have identified the importance of dopaminergic, opioid, serotonergic, noradrenergic and glutamatergic neurotransmission in amphetamine-induced “50-kHz” ultrasonic vocalizations (USVs). Amphetamine became a topic of interest for many researchers interested in USVs due to its ability to induce 50-kHz USVs. To date, it has been difficult to identify the neurotransmitters responsible for this phenomenon. The aim of this study was to determine the following: (i) concentrations of neurotransmitters in selected structures of the rat brain after re-exposure of the rats to amphetamine administration; (ii) changes in Arc in the medial prefrontal cortex, striatum, nucleus accumbens core and shell, hippocampus, amygdala and ventral tegmental area; and (iii) a biological basis for differences in 50-kHz USV emissions in response to amphetamine administration. Re-exposure to amphetamine increased 50-kHz USVs. This parameter do not correlate with distance covered by the investigated animals. An increased concentration of noradrenaline in the nucleus accumbens (NAcc) strongly correlated with the number of 50-kHz USVs. We found that NAcc noradrenaline concentrations negatively correlated with the concentration of dopamine and dopamine metabolites and positively correlated with the concentration of GABA and 5-HIAA (serotonin metabolite) in this structure. We have also identified a positive correlation between striatal 3-MT (dopamine metabolite) concentrations and Arc expression in the hippocampal DG as well as a negative correlation between the concentration of GABA in the amygdala and Arc expression in the central amygdala. Thus, the relationship between the emission of 50-kHz USVs and the neurochemical changes that occur after re-exposure to amphetamine indicates cross-talk between NA, DA, 5-HT and GABA neurotransmission in the NAcc.

A PRIME Labeling Approach For Investigating Dopamine Transporter Endocytic Trafficking In Dopaminergic Cell Lines And Neurons

The presynaptic dopamine transporter (DAT) facilitates synaptic DA reuptake that spatially and temporally constrains DA neurotransmission. DAT is inhibited by addictive psychostimulants, and the rewarding properties of those psychostimulants require their direct binding to DAT. More recently, aberrant DAT function has been identified in Attention Deficit/Hyperactivity Disorder, Autism Spectrum Disorder and Infantile Parkinsonism, emphasizing the critical role DAT plays in maintaining normal DA tone. DAT robustly traffics to and from the cell surface via the endocytic pathway. Numerous stimuli, including protein kinase C (PKC) activation and amphetamine (AMPH) exposure acutely decrease DAT surface levels in dopaminergic terminals by promoting DAT endocytosis. However, DAT's post‐endocytic fate is unclear and conflicting studies report that DAT targets to either recycling or degradative endocytic compartments, or that DAT may not traffic in bona fide DAergic neurons. These discrepancies may stem from the various approaches used examine DAT trafficking, which rely primarily on either antibody or inhibitory ligand‐based methods that could potentially mistarget internalized DAT. In the current study, we take advantage of a novel PRIME (Probe Incorporation Mediated by Enzyme) labeling strategy to track the DAT endocytic itinerary under basal and stimulated conditions. This approach covalently couples fluorophore to surface proteins that encode a ligase acceptor peptide (LAP), thereby facilitating direct protein visualization without the need for inhibitory ligands or bulky antibodies. We engineered the LAP peptide into the DAT 2nd extracellular loop (LAP‐DAT) and observed highly specific fluorophore labeling at the cell surface in cells expressing LAP‐DAT, but not in cells expressing wildtype DAT. LAP‐DAT expresses, functions, and exhibits PKC‐mediated downregulation comparable to wildtype DAT. Moreover, LAP‐DAT robustly internalized in the immortalized mesencephalic cell line, AN27, allowing us to generate a dynamic flow profile that tracks DAT's movement through the endocytic pathway. Ongoing studies using AAV2‐expressed LAP‐DAT in the striatum of TH‐Cre mice, will permit a rigorous examination of DAT endocytic trafficking in bona fide native dopaminergic neurons in ex vivo tissue samples.Support or Funding InformationNIH R01DA035224 to H.E.M.

Angiotensin II promotes inflammation and DA sympathoexcitation in the Hypothalamic Paraventricular Nuclei through IGF‐1R upregulation

BackgroundAngiotensin II (Ang II)‐induced hypertension is accompanied by an increase in inflammatory proteins coupled with the depletion of neurotrophic factors in the paraventricular nucleus (PVN) of the hypothalamus. Similarly, changes in the expression pattern of receptors associated with the inflammatory molecules have been described extensively. However, the effect of Ang II on neurotropin receptor (insulin‐like growth factor 1 receptor; IGF‐1R) expression pattern and how it mediates neural and inflammatory changes in the PVN during hypertension remains underexplored. This study examines the hypothesis that changes to PVN IGF‐1R expression is a possible mechanism through which Ang II promotes inflammation and PVN dopaminergic synaptic activity in the progression of hypertension.MethodRetrograde tracer (Rhodamine B+ Amine, 10,000MW) was injected into the rostral ventrolateral medulla (RVLM) of C57BL/6 mice to identify the sympathoexcitatory PVN projection neurons. After a survival period of 7 days, in vitro coronal PVN slices were prepared in oxygenated artificial cerebrospinal fluid (ACSF) and the retrogradely labelled neurons were identified via fluorescence microscopy. Using patch‐clamping electrophysiology, whole cell parameters were recorded in the labelled PVN neurons following which 200μM Ang II was administered through the ACSF bath. Subsequent changes in neuronal activity were then measured in voltage and current clamp modes. In a separate set of experiments, cortical slices were incubated acutely in either ACSF or ACSF containing 200μM Ang II for 120 minutes. Coronal sections were processed for immunofluorescence to demonstrate the distribution of post‐synaptic protein (PSD‐95), DA activity (Tyrosine hydroxylase; THx), IGF‐1 Receptor and inflammatory markers; high‐mobility group box protein 1 (HMGB‐1) and nuclear factor Kappa Beta (NF‐κβ).Results and DiscussionAng II induced depolarization of the membrane potential (−35 to −40mV), resulting in spontaneous bursting potentials in PVN neurons when compared with untreated PVN neurons (−59.5 to −62mV). The observed increase in neuronal activity was also associated with a change in post‐synaptic morphology which was characterized by an increase in PSD‐95 expression in the PVN. Subsequent analysis of DA neuronal marker suggests an increase DA neurotransmission associated with the up‐regulation of PVN tyrosine hydroxylase. Furthermore, Ang II treatment caused an increase in the expression of HMGB‐1 and NF‐κβ, proteins linked with the danger associated molecular patterns (DAMP), in PVN neurons. Interestingly, the described increase in tyrosine hydroxylase/PSD‐95 and HMGB‐1/NF‐κβ in the PVN was also characterized by a corresponding up‐regulation of PVN IGF‐1R expression. This represents a physiological response to IGF‐1 depletion, known to promote inflammation and DA synaptic dysfunction in the brain. Ultimately, these outcomes suggest that Ang II mediated PVN‐driven systemic cardiovascular dysfunction involves the simultaneous progression of inflammation and DA sympathoexcitation which are intrinsically linked to IGF‐1R expression pattern of PVN neurons.ConclusionTaken together, our results show that Ang II‐mediated hypertension is a result of both PVN inflammation and dopaminergic sympathoexcitation associated with changes in the expression of neurotropin receptor IGF‐1R.Support or Funding InformationI currently have no support to attend the conference

Membrane potential shapes regulation of dopamine transporter trafficking at the plasma membrane.

The dopaminergic system is essential for cognitive processes, including reward, attention and motor control. In addition to DA release and availability of synaptic DA receptors, timing and magnitude of DA neurotransmission depend on extracellular DA-level regulation by the dopamine transporter (DAT), the membrane expression and trafficking of which are highly dynamic. Data presented here from real-time TIRF (TIRFM) and confocal microscopy coupled with surface biotinylation and electrophysiology suggest that changes in the membrane potential alone, a universal yet dynamic cellular property, rapidly alter trafficking of DAT to and from the surface membrane. Broadly, these findings suggest that cell-surface DAT levels are sensitive to membrane potential changes, which can rapidly drive DAT internalization from and insertion into the cell membrane, thus having an impact on the capacity for DAT to regulate extracellular DA levels.

Fast-scan cyclic voltammetry demonstrates that L-DOPA produces dose-dependent, regionally selective bimodal effects on striatal dopamine kinetics invivo.

Parkinson's disease (PD) is a debilitating condition that is caused by a relatively specific degeneration of dopaminergic (DAergic) neurons of the substantia nigra pars compacta. L-DOPA was introduced as a viable treatment option for PD over 40years ago and still remains the most common and effective therapy for PD. Though the effects of L-DOPA to augment striatal DA production are well known, little is actually known about how L-DOPA alters the kinetics of DA neurotransmission that contribute to its beneficial and adverse effects. In this study, we examined the effects of L-DOPA administration (50mg/kg carbidopa+0, 100, and 250mg/kg L-DOPA) on regional electrically stimulated DA response kinetics using fast-scan cyclic voltammetry in anesthetized rats. We demonstrate that L-DOPA enhances DA release in both the dorsal striatum (D-STR) and nucleus accumbens (NAc), but surprisingly causes a delayed inhibition of release in the D-STR. In both regions, L-DOPA progressively attenuated reuptake kinetics, predominantly through a decrease in Vmax . These findings have important implications on understanding the pharmacodynamics of L-DOPA, which may be informative for understanding its therapeutic effects and also common side effects like L-DOPA-induced dyskinesias (LID). L-DOPA is commonly used to treat Parkinsonian symptoms, but little is known about how it affects presynaptic DA neurotransmission. Using invivo fast-scan cyclic voltammetry, we show L-DOPA inhibits DA reuptake in a region-specific and dose-dependent manner, and L-DOPA has paradoxical effects on release. These findings may be important when considering mechanisms for L-DOPA's therapeutic benefits and adverse side-effects.

Impaired mTORC2 signaling in catecholaminergic neurons exaggerates high fat diet-induced hyperphagia.

ObjectiveFood intake is highly regulated by central homeostatic and hedonic mechanisms in response to peripheral and environmental cues. Neutral energy balance stems from proper integration of homeostatic signals with those “sensing” the rewarding properties of food. Impairments in brain insulin signaling causes dysregulation of feeding behaviors and, as a consequence, hyperphagia. Here, we sought to determine how the mammalian target of rapamycin complex 2 (mTORC2), a complex involved in insulin signaling, influences high fat feeding.MethodsRictor is a subunit of mTORC2, and its genetic deletion impairs mTORC2 activity. We used Cre-LoxP technology to delete Rictorin tyrosine hydroxylase (TH) expressing neurons (TH Rictor KO). We assessed food intake, body weight, body composition and DA dependent behaviors.ResultsTH Rictor KO mice display a high-fat diet specific hyperphagia, yet, when on low-fat diet, their food intake is indistinguishable from controls. Consistently, TH Rictor KO become obese only while consuming high-fat diet. This is paralleled by reduced brain DA content, and disruption of DA dependent behaviors including increased novelty-induced hyperactivity and exaggerated response to the psycho stimulant amphetamine (AMPH).ConclusionsOur data support a model in which mTORC2 signaling within catecholaminergic neurons constrains consumption of a high-fat diet, while disruption causes high-fat diet-specific exaggerated hyperphagia. In parallel, impaired mTORC2 signaling leads to aberrant striatal DA neurotransmission, which has been associated with obesity in human and animal models, as well as with escalating substance abuse. These data suggest that defects localized to the catecholaminergic pathways are capable of overriding homeostatic circuits, leading to obesity, metabolic impairment, and aberrant DA-dependent behaviors.

Fluoxetine Administration Exacerbates Oral Tremor and Striatal Dopamine Depletion in a Rodent Pharmacological Model of Parkinsonism.

The cardinal motor symptoms of Parkinson's disease (PD) include resting tremor, akinesia, bradykinesia, and rigidity, and these motor abnormalities can be modeled in rodents by administration of the VMAT-2 (type-2 vesicular monoamine transporter) inhibitor tetrabenazine (9,10-dimethoxy-3-(2-methylpropyl)-1,3,4,6,7, 11b hexahydrobenzo[a]quinolizin-2-one; TBZ). Depression is also commonly associated with PD, and clinical data indicate that selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine ((±)-N-methyl-γ-[4-(trifluoromethyl)phenoxy]benzenepropanamine hydrochloride; FLX) are frequently used to treat depression in PD patients. The aim of the present study was to characterize the effect of FLX on the motor dysfunctions induced by a low dose of TBZ (0.75 mg/kg), and investigate the neural mechanisms involved. This low dose of TBZ was selected based on studies with rat models of depressive symptoms. In rats, coadministration of FLX (2.5, 5.0, and 10.0 mg/kg) increased TBZ-induced oral tremor (tremulous jaw movements), and decreased locomotor activity compared with administration of TBZ alone. Coadministration of the serotonin 5-HT2A/2C antagonist mianserin (2.5 and 5.0 mg/kg) attenuated the increase in oral tremor induced by coadministration of TBZ (0.75 mg/kg) with FLX (5.0 mg/kg). Consistent with these behavioral data, coadministration of TBZ and FLX decreased DA tissue levels in the rat ventrolateral neostriatum compared with TBZ alone, and coadministration of mianserin with TBZ and FLX attenuated this effect, increasing DA tissue levels compared with the TBZ/FLX condition. These data suggest that SSRI administration in PD patients may result in worsening of motor symptoms, at least in part, by exacerbating existing DA depletions through 5-HT2A/2C-mediated modulation of DA neurotransmission.

Combined Nurr1 and Foxa2 roles in the therapy of Parkinson's disease.

Use of the physiological mechanisms promoting midbrain DA (mDA) neuron survival seems an appropriate option for developing treatments for Parkinson's disease (PD). mDA neurons are specifically marked by expression of the transcription factors Nurr1 and Foxa2. We show herein that Nurr1 and Foxa2 interact to protect mDA neurons against various toxic insults, but their expression is lost during aging and degenerative processes. In addition to their proposed cell-autonomous actions in mDA neurons, forced expression of these factors in neighboring glia synergistically protects degenerating mDA neurons in a paracrine mode. As a consequence of these bimodal actions, adeno-associated virus (AAV)-mediated gene delivery of Nurr1 and Foxa2 in a PD mouse model markedly protected mDA neurons and motor behaviors associated with nigrostriatal DA neurotransmission. The effects of the combined gene delivery were dramatic, highly reproducible, and sustained for at least 1 year, suggesting that expression of these factors is a promising approach in PD therapy.

Attenuated response to methamphetamine sensitization and deficits in motor learning and memory after selective deletion of β-catenin in dopamine neurons

In the present study, we analyzed mice with a targeted deletion of β-catenin in DA neurons (DA-βcat KO mice) to address the functional significance of this molecule in the shaping of synaptic responses associated with motor learning and following exposure to drugs of abuse. Relative to controls, DA-βcat KO mice showed significant deficits in their ability to form long-term memories and displayed reduced expression of methamphetamine-induced behavioral sensitization after subsequent challenge doses with this drug, suggesting that motor learning and drug-induced learning plasticity are altered in these mice. Morphological analyses showed no changes in the number or distribution of tyrosine hydroxylase-labeled neurons in the ventral midbrain. While electrochemical measurements in the striatum determined no changes in acute DA release and uptake, a small but significant decrease in DA release was detected in mutant animals after prolonged repetitive stimulation, suggesting a possible deficit in the DA neurotransmitter vesicle reserve pool. However, electron microscopy analyses did not reveal significant differences in the content of synaptic vesicles per terminal, and striatal DA levels were unchanged in DA-βcat KO animals. In contrast, striatal mRNA levels for several markers known to regulate synaptic plasticity and DA neurotransmission were altered in DA-βcat KO mice. This study demonstrates that ablation of β-catenin in DA neurons leads to alterations of motor and reward-associated memories and to adaptations of the DA neurotransmitter system and suggests that β-catenin signaling in DA neurons is required to facilitate the synaptic remodeling underlying the consolidation of long-term memories.

Contrasting effects of d-methamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxypyrovalerone, and 4-methylmethcathinone on wheel activity in rats

BackgroundReports from US, UK and European drug policy entities, and ongoing media accounts, show increasing recreational use of 4-methylmethcathinone (4-MMC, mephedrone) and 3,4-methylenedioxypyrovalerone (MDPV). Severe sympathomimetic symptoms, hallucinations, psychoses, and even deaths have been reported, yet little scientific information is available on the effects of these compounds in laboratory models. Available studies on the neurochemistry of these drugs show that 4-MMC and MDPV enhance DA neurotransmission, while 4-MMC additionally enhances 5-HT neurotransmission – a pattern much like that reported for methamphetamine versus 3,4-methylenedioxymethamphetamine (MDMA). As is the case for designer amphetamines, these neurochemical distinctions may predict differential potential for repetitive versus episodic abuse and distinct lasting toxicities. MethodsThis study determined relative locomotor stimulant effects of 4-MMC (1–10mg/kg, s.c.) and MDPV (0.5–5.6mg/kg, s.c.), in comparison with d-methamphetamine (MA; 0.5–5.6mg/kg, s.c.) and MDMA (1–7.5mg/kg, s.c.) on a measure of locomotor activity – voluntary wheel running – in male Wistar rats (N=8). ResultsCompared to counts of wheel rotations after saline, a biphasic change in the pattern of counts was observed after injections of MA and MDPV, with relatively higher counts following lower doses and lower counts following the highest dose. However, monophasic, dose-dependent reductions in counts were observed in response to injections of MDMA and 4-MMC. ConclusionThus, voluntary wheel running yielded the same categorical distinctions for these drugs as did prior experiments testing the effects of these drugs on monoaminergic neurotransmission. These data indicate that MDPV produces prototypical locomotor stimulant effects whereas 4-MMC is more similar to the entactogen MDMA.