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

Enhanced stress reactivity in nitric oxide synthase type 2 mutant mice : findings in support of astrocytic nitrosative modulation of behavior

Alterations of nitric oxide (NO) metabolism in the brain have been associated with modifications of stress-related behavior in animal models. It has been generally assumed that these behavioral changes are due to the neuronal nitrosative activity. On the other hand, glial NO production has been demonstrated mainly as a slow reaction to brain insults through the activity of an inducible nitric oxide synthase (NOS) isoform (NOS2). Recently we uncovered increased NOS activity in astrocytes of mice with a NOS2 mutation. Interestingly, these mice revealed a behavioral phenotype suggestive of increased susceptibility to stress. In the present study we investigated the responses of these mutants to stress by exposing them to predator scent. Seven days later, mutant mice exhibited significantly higher anxiety-like behavior in the elevated-plus maze, increased acoustic startle responses, and higher plasma corticosterone levels compared with their controls. Systemic administration of a NOS inhibitor prior to the stress exposure reversed these stress-related effects without affecting controls' behavior. These findings are in agreement with previous studies showing an association between increased NO levels and enhanced anxiety-like responses. In addition, mutant mice performed better in the Morris water maze prior to stress exposure, but the two animal groups performed alike in an object-recognition test. Taken together, our results suggest the involvement of astrocytic-derived NO in modulating behavior

Biphasic cuirass ventilation is better than bag-valve mask ventilation for resuscitation following organophosphate poisoning

Objective: Exposure to organophosphates (OP) may lead to a life threatening cholinergic crisis with death attributed to a rapidly progressive respiratory failure. In a toxicological mass casualty event involving organophosphate exposure, many of the victims may depend on immediate short-term ventilation to overcome the respiratory distress which may exhaust life supporting resources. In addition, the mandatory use of personal protective gear by first responders emphasizes the need for a noninvasive, easy-to-operate ventilation device. Our objective was to assess the efficacy of MRTX, a Biphasic Cuirass Ventilation device, in comparison with standard bag-valve mask ventilation following acute organophosphate poisoning. Methods: Pigs were exposed to paraoxon poisoning (1.4 LD50), and treated 8 min later with atropine (0.05 mg/kg). The control group received no further support (n = 9), the two experimental groups received ventilation support initiated 15 min post exposure and lasted for 25 min: one group was ventilated with the commonly used bag-valve mask (Mask group, n = 7) and the other was ventilated with the Biphasic Cuirass Ventilation device (Cuirass group, n = 7). Clinical signs and physiological parameters were monitored during the first hour, and mortality up to 24 h post exposure was recorded. Results: No mortality was observed in the Cuirass group following OP poisoning, while mortality in the Control and in the Mask groups was high (67% and 71%, respectively). Mouth excretions of the cuirass-ventilated animals were frothy white as in deep suctioning, as opposed to the clear saliva-like appearance of secretions in the other two groups. No further group differences were recorded. Conclusions: The noninvasive, easy-to-operate Biphasic Cuirass Ventilation device was effective in reducing OP-induced mortality and might be advantageous in an organophosphate mass casualty event. This finding should be validated in further investigations

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

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

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

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 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