Enhanced Astrocytic Nitric Oxide Production and Neuronal Modifications in the Neocortex of a NOS2 Mutant Mouse

BACKGROUND: It has been well accepted that glial cells in the central nervous system (CNS) produce nitric oxide (NO) through the induction of a nitric oxide synthase isoform (NOS2) only in response to various insults. Recently we described rapid astroglial, NOS2-dependent, NO production in the neocortex of healthy mice on a time scale relevant to neuronal activity. To explore a possible role for astroglial NOS2 in normal brain function we investigated a NOS2 knockout mouse (B6;129P2-Nos2(tm1Lau)/J, Jackson Laboratory). Previous studies of this mouse strain revealed mainly altered immune responses, but no compensatory pathways and no CNS abnormalities have been reported. METHODOLOGY/PRINCIPAL FINDINGS: To our surprise, using NO imaging in brain slices in combination with biochemical methods we uncovered robust NO production by neocortical astrocytes of the NOS2 mutant. These findings indicate the existence of an alternative pathway that increases basal NOS activity. In addition, the astroglial mutation instigated modifications of neuronal attributes, shown by changes in the membrane properties of pyramidal neurons, and revealed in distinct behavioral abnormalities characterized by an increase in stress-related parameters. CONCLUSIONS/SIGNIFICANCE: The results strongly indicate the involvement of astrocytic-derived NO in modifying the activity of neuronal networks. In addition, the findings corroborate data linking NO signaling with stress-related behavior, and highlight the potential use of this genetic model for studies of stress-susceptibility. Lastly, our results beg re-examination of previous studies that used this mouse strain to examine the pathophysiology of brain insults, assuming lack of astrocytic nitrosative reaction

Re-assembled casein micelles improve in vitro bioavailability of vitamin D in a Caco-2 cell model

The pandemic of vitamin D (VD) deficiency, and the global rise in obesity stimulate a need for staple low-fat foods and beverages enriched with VD. In light of consumer demand for a clean label, the use of natural endogenous food ingredients as delivery vehicles is of great interest. To this end, re-assembled casein micelles (rCM) have been shown to help retain VD during processing and shelf life and provide high bioavailability in low-fat milk and non-fat yoghurt. This follow-up study focused on the performance of VD-loaded rCM after drying and reconstitution, considering VD retention during simulated digestion, and the subsequent in vitro bioavailability of the vitamin. rCM conferred great protection to VD3 during simulated digestion with a significant increase in vitamin retention for 1 h under gastric conditions. This observation is believed to arise from the vitamin-casein binding and the system's natural gelation (curd formation) near the casein isoelectric point that seclude the vitamin from environmental stressors and couple its release with digestive proteolysis of the rCM matrix. Vitamin absorption by Caco-2 cells from digested rCM was not significantly different from the absorption of the digested free VD. However, thanks to the highly protective effect of the rCM, against VD gastric degradation, the overall effect of the rCM was a 4-fold higher bioavailability, compared to the free VD

NOS proteins are conserved in the mutant neocortex, but NOS activity is increased.

<p>(A) Example of western blot analysis from 3 control and 3 mutant mice neocortex, demonstrating tight conservation of all 3 NOS proteins. (B) The results of NOS radioenzymatic assay reveal an increase in the total NOS activity in mutant mice neocortex (open bars, p = 0.01). The Ca²⁺-independent NOS fraction (gray bars) did not differ between the two mice strains. Data from 3 animals of each strain is displayed as mean±SD.</p

Mutant astroglial NOS activity is unaffected by NOS2 inhibitor.

<p>Example images from slices incubated in the selective NOS2 inhibitor 1400W (3 µM) for at least 30 minutes. At 30 seconds from the beginning of illumination (left panels), neuronal punctate fluorescence (arrow heads) was abundant in slices from either mutant or control mice. At 180 seconds (right panels), astrocytic diffuse fluorescence is abolished in slices from control mice, but not in mutant slices. Images were taken with ND4 filter to slow the response and allow for cells' separation. Scale bar = 20 µm.</p

Diffusely-stained astrocytes are prevalent in confocal images from mutant mice.

<p>(A) Example of confocal images from DAF-2DA incubated neocortical slices. Punctate, putative neuronal staining is dominating in slices from control mice (left), and diffuse fluorescence is dominating in the mutant's slice (right). Scale bar = 25 µm. (B) Diffuse DAF-2DA staining (green) is co-localized with the specific astrocytic marker SR101 (red). Examples from confocal images of a single cell are displayed. Scale bar = 10 µm. (C) Merging image of SR101 and DAF-2DA staining (projection of four 5 µm thick images). Cells which stain with both dyes appear in yellow. Over 95% of the cells displayed co-localization (4 slices, 2 animals). Scale bar = 20 µm.</p

Electrophysiological properties of diffusely fluorescent cells in mutant slices are characteristic of astroglia.

<p>(A) IR/DIC image from a mutant slice displays the recording pipette and the cell's soma. The arrowhead points to the location of the pipette tip. Scale bar = 20 µm. (B) A fluorescent image of the same region as in (A) reveals several diffusely fluorescent cells. The arrowhead position is the same as in (A). (C) A series of current pulses at 0.1 nA increments were delivered through the recording pipette (inset). A plot of the current pulse intensity (I) vs. the voltage deflection (Vm) reveals linear relationship characteristic of astroglia.</p

DAF-2DA fluorescence in mutant mice is faster and stronger.

<p>(A) Slices from mutant and control mice were pre-incubated in DAF-2DA (2 µM) for 10 minutes, and imaged with a fluorescent light source. Example images are displayed at three time points following the start of illumination (marked above). Arrowheads at 30 s point at punctate staining of putative neurons. Note the larger and steadily increasing number of diffusely-fluorescing, putative astroglia in slices from the mutant mouse. Scale bar = 25 µm. (B) Summary diagram of diffuse DAF-2DA fluorescent changes over time. Data are displayed as mean FI±S.E.M from identified single cells (control-26 cells from 4 slices, open circles; mutants–58 cells from 4 slices, closed squares).</p

Mutant mice differed from their controls in stress related aspects of exploratory behavior.

<p>(A) Control mice (n = 16,) were compared to mutant mice (n = 11, open circles) in the Open-Field test. The general motor activity was measured as the total distance traveled during the test. The Freezing was measured as the latency to escape from the center of the field. Rearing and entries to the center of the field episodes were counted. (B) Example parameters from the results of the Hole-Board test are displayed. The motor activity is measured as the total distance traveled during the test. Grooming episodes are counted (control-n = 6, gray closed squares controls; mutants-n = 9, open circles). For all tests, data is displayed as mean±S.E.M. Significant difference between the groups on a specific day are marked: *-p≤0.05; **-p≤0.01; ***-p≤0.001.</p