Isolation,Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson’s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.     

Photosynthesis in an invasive grass and native forb at elevated CO2 during an El Niño year in the Mojave Desert

Annual and short-lived perennial plant performance during wet years is important for long-term persistence in the Mojave Desert. Additionally, the effects of elevated CO₂ on desert plants may be relatively greater during years of high resource availability compared to dry years. Therefore, during an El Niño year at the Nevada Desert FACE Facility (a whole-ecosystem CO₂ manipulation), we characterized photosynthetic investment (by assimilation rate-internal CO₂ concentration relationships) and evaluated the seasonal pattern of net photosynthesis (A_net) and stomatal conductance (g_s) for an invasive annual grass, Bromus madritensis ssp. rubens and a native herbaceous perennial, Eriogonum inflatum. Prior to and following flowering, Bromus showed consistent increases in both the maximum rate of carboxylation by Rubisco (V_Cmax) and the light-saturated rate of electron flow (J_max) at elevated CO₂. This resulted in greater A_net at elevated CO₂ throughout most of the life cycle and a decrease in the seasonal decline of maximum midday A_net upon flowering as compared to ambient CO₂. Eriogonum showed significant photosynthetic down-regulation to elevated CO₂ late in the season, but the overall pattern of maximum midday A_net was not altered with respect to phenology. For Eriogonum, this resulted in similar levels of A_net on a leaf area basis as the season progressed between CO₂ treatments, but greater photosynthetic activity over a typical diurnal period. While g_s did not consistently vary with CO₂ in Bromus, it did decrease in Eriogonum at elevated CO₂ throughout much of the season. Since the biomass of both plants increased significantly at elevated CO₂, these patterns of gas exchange highlight the differential mechanisms for increased plant growth. The species-specific interaction between CO₂ and phenology in different growth forms suggests that important plant strategies may be altered by elevated CO₂ in natural settings. These results indicate the importance of evaluating the effects of elevated CO₂ at all life cycle stages to better understand the effects of elevated CO₂ on whole-plant performance in natural ecosystems.     

NEWER METHODS FOR SELECTION OF PATIENTS FOR LUMBAR SYMPATHECTOMY

The degree of vasodilatation achieved by various diagnostic methods (arterial occlusion, ganglionic block established by certain agents, lumbar sympathetic block, spinal anesthesia, and indirect heating) was studied by means of the pneumo-plethysmogram as well as through readings of skin temperature and skin resistance. The data obtained were interpreted as to their value in determining the type of patient with vascular disease for whom lumbar sympathectomy would be of noticeable benefit. Arterial occlusion proved itself a rapid and simple method which in most patients produced satisfactory results. As a rule, lumbar sympathectomy was effective in patients who preoperatively had shown a positive response upon release of arterial occlusion. A negative response, that is, absence of significant increase in blood flow, does not necessarily indicate organic disease, and cannot be taken to mean that lumbar sympathectomy would always be ineffective. Ganglionic block, using 2.6 dimethyl piperidinium bromide or tetraethylammonium ion, was generally less reliable in indicating the probable results of sympathectomy than lumbar sympathetic block or indirect heating. Lumbar sympathetic block with procaine was followed by a greater increase in skin temperature and blood flow than spinal anesthesia, and permitted far more accurate conclusions as to the probable outcome of sympathectomy. Subsequent to indirect heating the plethysmogram showed characteristic differences depending on the degree of vascular disease present. From the effect of this simple, safe and painless method upon the relative blood flow to the toe it becomes possible to arrive at a comparatively accurate estimate of the clinical benefit to be expected from lumbar sympathectomy.     

Reduction of Brain Mitochondrial β-Oxidation Impairs Complex I and V in Chronic Alcohol Intake: The Underlying Mechanism for Neurodegeneration

Neuropathy and neurocognitive deficits are common among chronic alcohol users, which are believed to be associated with mitochondrial dysfunction in the brain. The specific type of brain mitochondrial respiratory chain complexes (mRCC) that are adversely affected by alcohol abuse has not been studied. Thus, we examined the alterations of mRCC in freshly isolated mitochondria from mice brain that were pair-fed the ethanol (4% v/v) and control liquid diets for 7–8 weeks. We observed that alcohol intake severely reduced the levels of complex I and V. A reduction in complex I was associated with a decrease in carnitine palmitoyltransferase 1 (cPT1) and cPT2 levels. The mitochondrial outer (cPT1) and inner (cPT2) membrane transporter enzymes are specialized in acylation of fatty acid from outer to inner membrane of mitochondria for ATP production. Thus, our results showed that alterations of cPT1 and cPT2 paralleled a decrease β-oxidation of palmitate and ATP production, suggesting that impairment of substrate entry step (complex I function) can cause a negative impact on ATP production (complex V function). Disruption of cPT1/cPT2 was accompanied by an increase in cytochrome C leakage, while reduction of complex I and V paralleled a decrease in depolarization of mitochondrial membrane potential (ΔΨ, monitored by JC-1 fluorescence) and ATP production in alcohol intake. We noted that acetyl-L-carnitine (ALC, a cofactor of cPT1 and cPT2) prevented the adverse effects of alcohol while coenzyme Q10 (CoQ10) was not very effective against alcohol insults. These results suggest that understanding the molecular, biochemical, and signaling mechanisms of the CNS mitochondrial β-oxidation such as ALC can mitigate alcohol related neurological disorders.     

A Highlights from MBoC Selection: Aurora B–mediated localized delays in nuclear envelope formation facilitate inclusion of late-segregating chromosome fragments

To determine how chromosome segregation is coordinated with nuclear envelope formation (NEF), we examined the dynamics of NEF in the presence of lagging acentric chromosomes in Drosophila neuroblasts. Acentric chromosomes often exhibit delayed but ultimately successful segregation and incorporation into daughter nuclei. However, it is unknown whether these late-segregating acentric fragments influence NEF to ensure their inclusion in daughter nuclei. Through live analysis, we show that acentric chromosomes induce highly localized delays in the reassembly of the nuclear envelope. These delays result in a gap in the nuclear envelope that facilitates the inclusion of lagging acentrics into telophase daughter nuclei. Localized delays of nuclear envelope reassembly require Aurora B kinase activity. In cells with reduced Aurora B activity, there is a decrease in the frequency of local nuclear envelope reassembly delays, resulting in an increase in the frequency of acentric-bearing, lamin-coated micronuclei. These studies reveal a novel role of Aurora B in maintaining genomic integrity by promoting the formation of a passageway in the nuclear envelope through which late-segregating acentric chromosomes enter the telophase daughter nucleus.