Proneural bHLH neurogenin 2 differentially regulates Nurr1-induced dopamine neuron differentiation in rat and mouse neural precursor cells in vitro

Roles of Nurr1 and neurogenin 2 (Ngn2) have been shown in midbrain dopamine (DA) neuron development. We present here rat and mouse species-dependent differences of Nurr1 and Ngn2 actions in DA neuron differentiation. Nurr1 exogene expression caused an efficient generation of tyrosine hydroxylase (TH)-positive DA cells from rat neural precursor cells (NPCs). Nurr1-induced TH+ cell yields were low and highly variable depending on the origins of NPCs in mouse cultures. Coexpression of Ngn2 repressed Nurr1-induced generation of TH+ cells in rat cultures. In clear contrast, a robust enhancement in Nurr1-induced DA cell yields was observed in mouse NPCs by Ngn2. These findings imply that DA neurons may develop differently in the midbrains of these two species.     

Galanin inhibits tyrosine hydroxylase expression in midbrain dopaminergic neurons

Galanin (GAL) inhibits midbrain dopamine (DA) activity in several experimental paradigms, yet the mechanism underlying this inhibition is unclear. We examined the effects of GAL on the expression of tyrosine hydroxylase (TH) in primary cultures of rat embryonic (E14) ventral mesencephalon (VM). One micromolar GAL had no effect on the number of TH-immunoreactive (ir) neurons in VM cultures. However, 1 micro m GAL reduced an approximately 100% increase in TH-ir neurons in 1 mm dibutyryl cAMP (dbcAMP)-treated cultures by approximately 50%. TH-ir neuron number in dbcAMP-treated VM cultures was dose-responsive to GAL and the GAL receptor antagonist M40 blocked GAL effects. Semi-quantitative RT-PCR and quantitative immunoblotting experiments revealed that GAL had no effect on TH mRNA levels in VM cultures but reduced TH protein. VM cultures expressed GALR1, GALR2, and GALR3 receptor mRNA. However, dbcAMP treatment resulted in a specific approximately 200% increase in GALR1 mRNA. GALR1 activity is linked to a pertussis toxin (PTX)-sensitive opening of G protein-gated K+ channels (GIRKs). GAL reduction of TH-ir neuron number in dbcAMP + GAL-treated cultures was sensitive to both PTX and tertiapin, a GIRK inhibitor. GAL inhibition of midbrain DA activity may involve a GALR1- mediated reduction of TH in midbrain dopaminergic neurons.     

Dopaminergic neuronal differentiation from rat embryonic neural precursors by Nurr1 overexpression

In vitro expanded CNS precursors could provide a renewable source of dopamine (DA) neurons for cell therapy in Parkinson's disease. Functional DA neurons have been derived previously from early midbrain precursors. Here we demonstrate the ability of Nurr1, a nuclear orphan receptor essential for midbrain DA neuron development in vivo, to induce dopaminergic differentiation in naïve CNS precursors in vitro. Independent of gestational age or brain region of origin, Nurr1‐induced precursors expressed dopaminergic markers and exhibited depolarization‐evoked DA release in vitro. However, these cells were less mature and secreted lower levels of DA than those derived from mesencephalic precursors. Transplantation of Nurr1‐induced DA neuron precursors resulted in limited survival and in vivo differentiation. No behavioral improvement in apomorphine‐induced rotation scores was observed. These results demonstrate that Nurr1 induces dopaminergic features in naïve CNS precursors in vitro. However, additional factors will be required to achieve in vivo function and to unravel the full potential of neural precursors for cell therapy in Parkinson's disease.     

Ngn2 and Nurr1 act in synergy to induce midbrain dopaminergic neurons from expanded neural stem and progenitor cells

Parkinson's Disease (PD) is a debilitating motor function disorder due primarily to a loss of midbrain dopaminergic neurons and a subsequent reduction in dopaminergic innervation of the striatum. Several attempts have been made to generate dopaminergic neurons from progenitor cell populations in vitro for potential use in cell replacement therapy for PD. However, expanding cells from fetal brain with retained potential for dopaminergic differentiation has proven to be difficult. In this study, we sought to generate mesencephalic dopaminergic (mesDA) neurons from an expanded population of fetal mouse ventral midbrain (VM) progenitors through the use of retroviral gene delivery. We over-expressed Ngn2 and Nurr1, two genes present in the ventral midbrain and important for normal development of mesDA neurons, in multi-passaged neurosphere-expanded midbrain progenitors. We show that over-expression of Ngn2 in these progenitors results in increased neuronal differentiation but does not promote mesDA formation. We also show that over-expression of Nurr1 alone is sufficient to generate tyrosine hydroxylase (TH) expressing cells with an immature morphology, however the cells do not express any additional markers of mesDA neurons. Over-expression of Nurr1 and Ngn2 in combination generates morphologically mature TH-expressing neurons that also express additional mesencephalic markers.     

Hub and switches: endocannabinoid signalling in midbrain dopamine neurons

The last decade has provided a wealth of experimental data on the role played by lipids belonging to the endocannabinoid family in several facets of physiopathology of dopamine neurons. We currently suggest that these molecules, being intimately connected with diverse metabolic and signalling pathways, might differently affect various functions of dopamine neurons through activation not only of surface receptors, but also of nuclear receptors. It is now emerging how dopamine neurons can regulate their constituent biomolecules to compensate for changes in either internal functions or external conditions. Consequently, dopamine neurons use these lipid molecules as metabolic and homeostatic signal detectors, which can dynamically impact cell function and fitness. Because dysfunctions of the dopamine system underlie diverse neuropsychiatric disorders, including schizophrenia and drug addiction, the importance of better understanding the correlation between an unbalanced endocannabinoid signal and the dopamine system is even greater. Particularly, because dopamine neurons are critical in controlling incentive-motivated behaviours, the involvement of endocannabinoid molecules in fine-tuning dopamine cell activity opened new avenues in both understanding and treating drug addiction. Here, we review recent advances that have shed new light on the understanding of differential roles of endocannabinoids and their cognate molecules in the regulation of the reward circuit, and discuss their anti-addicting properties, particularly with a focus on their potential engagement in the prevention of relapse.     

SKF83959 selectively regulates phosphatidylinositol-linked D1 dopamine receptors in rat brain

Previously a distinct D1-like dopamine receptor (DAR) that selectively couples to phospholipase C/phosphatidylinositol (PLC/PI) was proposed. However, lack of a selective agonist has limited efforts aimed at characterizing this receptor. We characterized the in vitro and in vivo effects of SKF83959 in regulating PI metabolism. SKF83959 stimulates (EC50, 8 micro m) phosphatidylinositol 4,5-biphosphate hydrolysis in membranes of frontal cortex (FC) but not in membranes from PC12 cells expressing classical D1A DARs. Stimulation of FC PI metabolism was attenuated by the D1 antagonist, SCH23390, indicating that SKF83959 activates a D1-like DAR. The PI-linked DAR is located in hippocampus, cerebellum, striatum and FC. Most significantly, administration of SKF83959 induced accumulations of IP3 in striatum and hippocampus. In contrast to other D1 DAR agonists, SKF83959 did not increase cAMP production in brain or in D1A DAR-expressing PC12 cell membranes. However, SKF83959 inhibited cAMP elevation elicited by the D1A DAR agonist, SKF81297, indicating that the compound is an antagonist of the classical D1A DAR. Lastly, we demonstrated that SKF83959 enhances [35S]guanosine 5'-O-(3-thiotriphosphate) binding to membrane Galphaq and Galphai proteins, suggesting that PI stimulation is mediated by activation of these guanine nucleotide-binding regulatory proteins. Results indicate that SKF83959 is a selective agonist for the PI-linked D1-like DAR, providing a unique tool for investigating the functions of this brain D1 DAR subtype.     

Functional diversity of ventral midbrain dopamine and GABAergic neurons

Recent findings indicate that VTA and SN dopaminergic (DA) and GABAergic neurons form subpopulations that are divergent in their electrophysiological features, vulnerability to neurodegeneration, and regulation by neuropeptides. This diversity can be correlated with the anatomical organization of the VTA and SN and their inputs and outputs. In this review we describe the heterogeneity in ion channels and firing patterns, especially burst firing, in subpopulations of dopamine neurons. We go on to describe variations in vulnerability to neurotoxic damage in models of Parkinson’s disease in subgroups of DA neurons and its possible relationship to developmental gene regulation, the expression of different ion channels, and the expression of different protein markers, such as the neuroprotective marker calbindin. The electrophysiological properties of subgroups of GABAergic midbrain neurons, patterns of expression of protein markers and receptors, possible involvement of GABAergic neurons in a number of processes that are usually attributed exclusively to dopaminergic neurons, and the characteristics of a subgroup of neurons that contains both dopamine and GABA are also discussed.     

Dopamine D1 receptor signaling system regulates ryanodine receptor expression after intermittent exposure to methamphetamine in primary cultures of midbrain and cerebral cortical neurons

Regulatory mechanisms of ryanodine receptor (RyR) expression are not well known, although methamphetamine (METH) has been reported to up-regulate RyRs in mouse brain. This study investigate regulatory mechanisms of RyR expression by dopaminergic system using the midbrain and cerebral cortical neurons in primary culture intermittently exposed to METH and dopamine receptor (DR) agonists (1 h/day, for 3 days). Intermittent METH (10 μM) exposure enhanced RyR-1 and -2 proteins and their mRNA, but not RyR-3 expression in the both types of the neurons. These METH-induced increases of RyR proteins and their mRNA were dose-dependently blocked by SCH23390 (a selective D(1) DR antagonist), but not a D(2)DR antagonist sulpiride, suggesting a regulatory role of D(1)DRs in RyR expression by METH in these neurons. In cerebral cortical neurons, intermittent SKF82958 (a selective D(1)DR agonist) exposure increased RyR-1 and -2 proteins and their mRNA, whereas quinpirole (a selective D(2)DR agonist) showed no effects. KT5720, a protein kinase A inhibitor, dose-dependently attenuated the METH-stimulated RyR-1 and -2 expressions in cerebral cortical neurons. METH significantly increased phosphorylation of cAMP-response element-binding protein, which was completely suppressed by SCH23390. These results indicate that RyR-1 and -2 expressions are regulated by D(1)DRs via the signal transduction linked to D(1)DRs.     

Development and function of the midbrain dopamine system: what we know and what we need to

The past two decades have seen an explosion in our understanding of the origin and development of the midbrain dopamine system. Much of this work has been focused on the aspects of dopamine neuron development related to the onset of movement disorders such as Parkinson's disease, with the intent of hopefully delaying, preventing or fixing symptoms. While midbrain dopamine degeneration is a major focus for treatment and research, many other human disorders are impacted by abnormal dopamine, including drug addiction, autism and schizophrenia. Understanding dopamine neuron ontogeny and how dopamine connections and circuitry develops may provide us with key insights into potentially important avenues of research for other dopamine‐related disorders. This review will provide a brief overview of the major molecular and genetic players throughout the development of midbrain dopamine neurons and what we know about the behavioral‐ and disease‐related implications associated with perturbations to midbrain dopamine neuron development. We intend to combine the knowledge of two broad fields of neuroscience, both developmental and behavioral, with the intent on fostering greater discussion between branches of neuroscience in the service of addressing complex cognitive questions from a developmental perspective and identifying important gaps in our knowledge for future study.