Dopamine Selectively Sensitizes Dopaminergic Neurons to Rotenone-Induced Apoptosis

Among various types of neurons affected in Parkinson’s disease, dopamine (DA) neurons of the substantia nigra undergo the most pronounced degeneration. Products of DA oxidation and consequent cellular damage have been hypothesized to contribute to neuronal death. To examine whether elevated intracellular DA will selectively predispose the dopaminergic subpopulation of nigral neurons to damage by an oxidative insult, we first cultured rat primary mesencephalic cells in the presence of rotenone to elevate reactive oxygen species. Although MAP2⁺ neurons were more sensitive to rotenone-induced toxicity than type 1 astrocytes, rotenone affected equally both DA (TH⁺) neurons and MAP2⁺ neurons. In contrast, when intracellular DA concentration was elevated, DA neurons became selectively sensitized to rotenone. Raising intracellular DA levels in primary DA neurons resulted in dopaminergic neuron death in the presence of subtoxic concentrations of rotenone. Furthermore, mitochondrial superoxide dismutase mimetic, manganese (III) meso-tetrakis (4-benzoic acid) porphyrin, blocked activation of caspase-3, and consequent cell death. Our results demonstrate that an inhibitor of mitochondrial complex I and increased cytosolic DA may cooperatively lead to conditions of elevated oxidative stress and thereby promote selective demise of dopaminergic neurons.     

Investigation of Combining Plant Genotypic Values and Molecular Marker Information for Constructing Core Subsets

In the present study, a strategy was proposed for constructing plant core subsets by clusters based on the combination of continuous data for genotypic values and discrete data for molecular marker InformaUon. A mixed linear model approach was used to predict genotyplc values for eliminating the environment effect. The ＂mixed genetic distance＂ was designed to solve the difficult problem of combining continuous and discrete data to construct a core subset by cluster. Four commonly used genetic distances for continuous data （Euclidean distance, standardized Euclidean distance, city block distance, and Mahalanobls distance） were used to assess the validity of the conUnuous data part of the mixed genetic distance; three commonly used genetic distances for discrete data （cosine distance, correlaUon distance, and Jaccard distance） were used to assess the validity of the discrete data part of the mixed genetic distance, A rice germplasm group with eight quantitative traits and information for 60 molecular markers was used to evaluate the validity of the new strategy. The results suggest that the validity of both parts of the mixed geneUc distance are equal to or higher than the common geneUc distance. The core subset constructed on the basis of a combination of data for genotyplc values and molecular marker information was more representative than that constructed on the basis of data from genotypic values or molecular marker informaUon alone. Moreover, the strategy of using combined data was able to treat dominant marker informaUon and could combine any other continuous data and discrete data together to perform cluster to construct a plant core subset.     

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson''s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.     

Dopaminergic Regulation of Septohippocampal Cholinergic Neurons

Abstract: The extent to which acetylcholine (ACh) release in the hippocampus is regulated by dopaminergic mechanisms was assessed using in vivo microdialysis in freely moving rats. Systemic administration of the dopamine (DA) receptor agonist apomorphine (1.0 mg/kg) or the specific D₁ agonist CY 208–243 (1.0 mg/kg) increased microdialysate concentrations of ACh in the hippocampus. The D₂ receptor agonist quinpirole (0.5 mg/kg) produced a small but statistically significant decrease in hippocampal ACh release. d -Amphetamine (2.0 mg/kg) increased ACh release, an effect that was blocked by the D₁ receptor antagonist SCH 23390 (0.3 mg/kg) but not by the D₂ antagonist raclopride (1.0 mg/kg). These findings suggest that endogenous DA stimulates septo-hippocampal cholinergic neurons primarily via actions at D₁ receptors. In addition, these results are similar to previous findings regarding the dopaminergic regulation of cortical ACh release, and suggest that the anatomical continuum formed by basal forebrain cholinergic neurons that project to the cortex and hippocampus acts as a functional unit, at least with respect to its regulation by DA.     

Destruction of Midbrain Dopaminergic Neurons by Using Immunotoxin to Dopamine Transporter

1. The ability to target specific neurons can be used to produce selective neural lesions and potentially to deliver therapeutically useful moieties for treatment of disease. In the present study, we sought to determine if a monoclonal antibody to the dopamine transporter (anti-DAT) could be used to target midbrain dopaminergic neurons.2. The monoclonal antibody recognizes the second, large extracellular loop of DAT. The antibody was conjugated to the ribosome-inactivating protein saporin, and stereotactically pressure microinjected into either the center of the striatum or the left lateral ventricle of adult, male Sprague-Dawley rats.3. Local intrastriatal injections produced destruction of dopaminergic neurons in the ipsilateral substantia nigra consistent with suicide transport of the immunotoxin. Intraventricular injections (i.c.v.) produced significant loss of dopaminergic neurons in the substantia nigra and ventral tegmental area bilaterally without evident damage to any other aminergic structures such as the locus coeruleus and raphé nuclei. To confirm the anatomic findings, binding of [³H]mazindol to DAT in the striatum and midbrain was assessed using densitometric analysis of autoradiograms. Anti-DAT-saporin injected i.c.v. at a dose of 21 g, but not 8 g, produced highly significant decreases in mazindol binding consistent with loss of the dopaminergic neurons.4. These results show that anti-DAT can be used to target midbrain dopaminergic neurons and that anti-DAT-saporin may be useful for producing a lesion very similar to the naturally occurring neural degeneration seen in Parkinson's disease. Anti-DAT-saporin joins the growing list of neural lesioning agents based on targeted cytotoxins.     

Orexinergic Input to Dopaminergic Neurons of the Human Ventral Tegmental Area

The mesolimbic reward pathway arising from dopaminergic (DA) neurons of the ventral tegmental area (VTA) has been strongly implicated in reward processing and drug abuse. In rodents, behaviors associated with this projection are profoundly influenced by an orexinergic input from the lateral hypothalamus to the VTA. Because the existence and significance of an analogous orexigenic regulatory mechanism acting in the human VTA have been elusive, here we addressed the possibility that orexinergic neurons provide direct input to DA neurons of the human VTA. Dual-label immunohistochemistry was used and orexinergic projections to the VTA and to DA neurons of the neighboring substantia nigra (SN) were analyzed comparatively in adult male humans and rats. Orexin B-immunoreactive (IR) axons apposed to tyrosine hydroxylase (TH)-IR DA and to non-DA neurons were scarce in the VTA and SN of both species. In the VTA, 15.0±2.8% of TH-IR perikarya in humans and 3.2±0.3% in rats received orexin B-IR afferent contacts. On average, 0.24±0.05 and 0.05±0.005 orexinergic appositions per TH-IR perikaryon were detected in humans and rats, respectively. The majority (86–88%) of randomly encountered orexinergic contacts targeted the dendritic compartment of DA neurons. Finally, DA neurons of the SN also received orexinergic innervation in both species. Based on the observation of five times heavier orexinergic input to TH-IR neurons of the human, compared with the rat, VTA, we propose that orexinergic mechanism acting in the VTA may play just as important roles in reward processing and drug abuse in humans, as already established well in rodents.     

Developmental Sex Differences in Nicotinic Currents of Prefrontal Layer VI Neurons in Mice and Rats

Background

There is a large sex difference in the prevalence of attention deficit disorder; yet, relatively little is known about sex differences in the development of prefrontal attention circuitry. In male rats, nicotinic acetylcholine receptors excite corticothalamic neurons in layer VI, which are thought to play an important role in attention by gating the sensitivity of thalamic neurons to incoming stimuli. These nicotinic currents in male rats are significantly larger during the first postnatal month when prefrontal circuitry is maturing. The present study was undertaken to investigate whether there are sex differences in the nicotinic currents in prefrontal layer VI neurons during development.

Methodology/Principal Findings

Using whole cell recording in prefrontal brain slice, we examined the inward currents elicited by nicotinic stimulation in male and female rats and two strains of mice. We found a prominent sex difference in the currents during the first postnatal month when males had significantly greater nicotinic currents in layer VI neurons compared to females. These differences were apparent with three agonists: acetylcholine, carbachol, and nicotine. Furthermore, the developmental sex difference in nicotinic currents occurred despite male and female rodents displaying a similar pattern and proportion of layer VI neurons possessing a key nicotinic receptor subunit.

Conclusions/Significance

This is the first illustration at a cellular level that prefrontal attention circuitry is differently affected by nicotinic receptor stimulation in males and females during development. This transient sex difference may help to define the cellular and circuit mechanisms that underlie vulnerability to attention deficit disorder.     

脂多糖对大鼠多巴胺能神经元毒性作用的研究

目的　建立新的帕金森病 (Parkinson’sdisease ,PD)动物模型 ,探讨其发病机制。方法　在大鼠脑黑质(substantianigra ,SN)内注射脂多糖 (Lipopolysaccharide ,LPS)后 ,按大鼠不同存活期用高效液相色谱 (HPLC)来测定脑内多巴胺 (Dopamine,DA)及其代谢产物的含量 ;用免疫组化法观察酪氨酸羟化酶 (Tyrosinehydroxylase ,TH)阳性神经细胞、小胶质细胞的形态及数量变化。结果　DA及其代谢产物的含量在LPS注射侧随时间不同有不同程度下降 ,于第 14天达到最低 (P <0 0 1) ;注射侧黑质TH阳性神经元可以达到全部消失 ,该处可见大量被激活并有形态改变的小胶质细胞。结论　LPS可导致大鼠黑质多巴胺能神经元的损害     

Experimental Strategy to Identify Genes Susceptible to Oxidative Stress in Nigral Dopaminergic Neurons

Neuropathological evidence from both human and experimental models of Parkinson's disease (PD) firmly supports a significant role for oxidative stress (OS) in the death of dopaminergic (DA) neurons in substantia nigra. Largely unknown are the genes underlying selective susceptibility of nigral DA neuron to OS and how they effect nigral DA cell death. The major barriers to high-throughput identification of candidate genes are the paucity of nigral DA neurons as well as the dilution effect of non-DA cells both in primary cultures and brain tissues. To overcome these barriers, we have developed a DA cell line model, SN4741, appropriate for cDNA microarray analysis. Candidate genes were selected from both the microarray analysis and the molecular implication of their pathological mechanisms (i.e., decreased mitochondrial complex I activity and proteasomal dysfunction) of PD. Subsequent secondary validation tests were devised to characterize genes including clone #45 that may underlie selective vulnerability of nigral DA neuron to OS.     

Gender Differences in Sustained Attentional Control Relate to Gender Inequality across Countries

Sustained attentional control is critical for everyday tasks and success in school and employment. Understanding gender differences in sustained attentional control, and their potential sources, is an important goal of psychology and neuroscience and of great relevance to society. We used a large web-based sample (n = 21,484, from testmybrain.org) to examine gender differences in sustained attentional control. Our sample included participants from 41 countries, allowing us to examine how gender differences in each country relate to national indices of gender equality. We found significant gender differences in certain aspects of sustained attentional control. Using indices of gender equality, we found that overall sustained attentional control performance was lower in countries with less equality and that there were greater gender differences in performance in countries with less equality. These findings suggest that creating sociocultural conditions which value women and men equally can improve a component of sustained attention and reduce gender disparities in cognition.     

Depletion of Intracellular Glutathione Increases Susceptibility to Nitric Oxide in Mesencephalic Dopaminergic Neurons

Using primary neuronal cultures, we investigated the effects of GSH depletion on the cytotoxic effects of glutamate and NO in dopaminergic neurons. Intracellular GSH was depleted by 24-h exposure to L-buthionine-[S,R]-sulfoximine (BSO), an irreversible inhibitor of GSH synthase. BSO exposure caused concentration-dependent reduction of the viability of both dopaminergic and nondopaminergic neurons. In contrast, 24-h exposure of cultures to glutamate or NOC18, an NO-releasing agent, significantly reduced the viability of nondopaminergic neurons without affecting that of dopaminergic neurons. Pretreatment with N-acetyl-L-cysteine for 24 h ameliorated the NOC18-induced toxicity in nondopaminergic neurons. In dopaminergic neurons, sublethal concentrations of BSO reduced intracellular GSH content and markedly potentiated glutamate- and NOC18-induced toxicity. These results suggested that glutamate toxicity was enhanced in dopaminergic neurons by suppression of defense mechanisms against NO toxicity under conditions of GSH depletion. Under such conditions, free iron plays an important role because BSO-enhanced NO toxicity was ameliorated by the iron-chelating agent, deferoxamine. These results suggest that GSH plays an important role in the expression of NO-mediated glutamate cytotoxicity in dopaminergic neurons. Free iron may be related to enhanced NO cytotoxicity under GSH depletion.     

Dopaminergic Neurons Respond to Iron-Induced Oxidative Stress by Modulating Lipid Acylation and Deacylation Cycles

Metal-imbalance has been reported as a contributor factor for the degeneration of dopaminergic neurons in Parkinson Disease (PD). Specifically, iron (Fe)-overload and copper (Cu) mis-compartmentalization have been reported to be involved in the injury of dopaminergic neurons in this pathology. The aim of this work was to characterize the mechanisms of membrane repair by studying lipid acylation and deacylation reactions and their role in oxidative injury in N27 dopaminergic neurons exposed to Fe-overload and Cu-supplementation. N27 dopaminergic neurons incubated with Fe (1mM) for 24 hs displayed increased levels of reactive oxygen species (ROS), lipid peroxidation and elevated plasma membrane permeability. Cu-supplemented neurons (10, 50 μM) showed no evidence of oxidative stress markers. A different lipid acylation profile was observed in N27 neurons pre-labeled with [³H] arachidonic acid (AA) or [³H] oleic acid (OA). In Fe-exposed neurons, AA uptake was increased in triacylglycerols (TAG) whereas its incorporation into the phospholipid (PL) fraction was diminished. TAG content was 40% higher in Fe-exposed neurons than in controls. This increase was accompanied by the appearance of Nile red positive lipid bodies. Contrariwise, OA incorporation increased in the PL fractions and showed no changes in TAG. Lipid acylation profile in Cu-supplemented neurons showed AA accumulation into phosphatidylserine and no changes in TAG. The inhibition of deacylation/acylation reactions prompted an increase in oxidative stress markers and mitochondrial dysfunction in Fe-overloaded neurons. These findings provide evidence about the participation of lipid acylation mechanisms against Fe-induced oxidative injury and postulate that dopaminergic neurons cleverly preserve AA in TAG in response to oxidative stress.     

Calcitriol Imparts Neuroprotection In Vitro to Midbrain Dopaminergic Neurons by Upregulating GDNF Expression

During development a tightly controlled signaling cascade dictates the differentiation, maturation and survival of developing neurons. Understanding this signaling mechanism is important for developing therapies for neurodegenerative illnesses. In previous work we have sought to understand the complex signaling pathways responsible for the development of midbrain dopamine neurons using a proteomic approach. One protein we have identified as being expressed in developing midbrain tissue is the vitamin D receptor. Therefore we investigated the effect of the biologically active vitamin D₃ metabolite, calcitriol, on primary fetal ventral mesencephalic cultures of dopamine neurons. We observed a dose responsive increase in numbers of rat primary dopamine neurons when calcitriol was added to culture media. Western blot data showed that calcitriol upregulated the expression of glial derived neurotrophic factor (GDNF). Blocking GDNF signaling could prevent calcitriol’s ability to increase numbers of dopamine neurons. An apoptosis assay and cell birth dating experiment revealed that calcitriol increases the number of dopamine neurons through neuroprotection and not increased differentiation. This could have implications for future neuroprotective PD therapies.     

Involvement of Ventral Periaqueductal Gray Dopaminergic Neurons in Propofol Anesthesia

It has been reported that central dopaminergic system is implicated in the mechanism underlying general anesthesia. Whether dopamine (DA) neurons in midbrain ventral periaqueductal gray (vPAG) are involved in general anesthesia and how general anesthetics affect these neurons remain sparsely documented. To determine the role of vPAG DA neurons in propofol-induced anesthesia, we performed microinjection of 6-hydroxydopamine (6-OHDA) into vPAG to damage DA neurons and investigated the alteration in somatosensory electroencephalogram (EEG), as well as the induction and recovery time of propofol anesthesia. Subsequently, we examined the effect of propofol on the electrophysiological activity of DA neurons in vPAG using whole-cell patch clamp. Two weeks after 6-OHDA microinfusion, DA neurons in the vPAG were markedly reduced by 63.6% in the 6-OHDA-treated rats compared with vehicle rats. This lesion significantly shortened the induction time (7.15?±?3.97 s vs. 11.18?±?2.83 s, P?<?0.05) and prolonged the recovery time of propofol anesthesia (780.26?±?150.86 s vs. 590.68?±?107.97 s, P?<?0.05). Meanwhile, EEG in somatosensory cortex revealed that delta power (0–4 Hz) was significantly higher in 6-OHDA-treated rats than vehicle rats. In the electrophysiological experiment, propofol decreased the frequency of spontaneous excitatory postsynaptic currents rather than the amplitude and decay time. In addition, propofol preferentially increased the frequency and prolonged the decay time of spontaneous inhibitory postsynaptic currents without affecting the amplitude. Significance: Propofol can promote presynaptic GABA release, inhibit presynaptic glutamate release and increase postsynaptic GABA_A receptor sensitivity, which eventually inhibits the activity of vPAG DA neurons and thereby influences the state of consciousness.