Interferon-γ enhances neocortical synaptic inhibition by promoting membrane association and phosphorylation of GABAA receptors in a protein kinase C-dependent manner.

Interferon-γ (IFN-γ), an important mediator of the antiviral immune response, can also act as a neuromodulator. CNS IFN-γ levels rise acutely in response to infection and therapeutically applied IFN-γ provokes CNS related side effects. Moreover, IFN-γ plays a key role in neurophysiological processes and a variety of chronic neurological and neuropsychiatric conditions. To close the gap between basic research, behavioral implications and clinical applicability, knowledge of the mechanism behind IFN-γ related changes in brain function is crucial. Here, we studied the underlying mechanism of acutely augmented neocortical inhibition by IFN-γ (1.000 IU ml-1) in layer 5 pyramidal neurons of male Wistar rats. We demonstrate postsynaptic mediation of IFN-γ augmented inhibition by pressure application of GABA and analysis of paired pulse ratios. IFN-γ increases membrane presence of GABAAR γ2, as quantified by cell surface biotinylation and functional synaptic GABAAR number, as determined by peak-scaled non-stationary noise analysis. The increase in functional receptor number was comparable to the increase in underlying miniature inhibitory postsynaptic current (mIPSC) amplitudes. Blockage of putative intracellular mediators, namely phosphoinositide 3-kinase and protein kinase C (PKC) by Wortmannin and Calphostin C, respectively, revealed PKC-dependency of the pro-inhibitory IFN-γ effect. This was corroborated by increased serine phosphorylation of P-serine PKC motifs on GABAAR γ2 upon IFN-γ application. GABAAR single channel conductance, intracellular chloride levels and GABAAR driving force are unlikely to contribute to the effect, as shown by single channel recordings and chloride imaging. The effect of IFN-γ on mIPSC amplitudes was similar in female and male rats, suggesting a gender-independent mechanism of action. Collectively, these results indicate a novel mechanism for the regulation of inhibition by IFN-γ, which could impact on neocortical function and therewith behavior.

Photosynthetic hydrogen production: Novel protocols, promising engineering approaches and application of semi-synthetic hydrogenases.

Photosynthetic production of molecular hydrogen (H2 ) by cyanobacteria and green algae is a potential source of renewable energy. These organisms are capable of water biophotolysis by taking advantage of photosynthetic apparatus that links water oxidation at Photosystem II and reduction of protons to H2 downstream of Photosystem I. Although the process has a theoretical potential to displace fossil fuels, photosynthetic H2 production in its current state is not yet efficient enough for industrial applications due to a number of physiological, biochemical, and engineering barriers. This article presents a short overview of the metabolic pathways and enzymes involved in H2 photoproduction in cyanobacteria and green algae and our present understanding of the mechanisms of this process. We also summarize recent advances in engineering photosynthetic cell factories capable of overcoming the major barriers to efficient and sustainable H2 production.

Peri-Infarct Hot-Zones Have Higher Susceptibility to Optogenetic Functional Activation-Induced Spreading Depolarizations.

BACKGROUND AND PURPOSE: Spreading depolarizations (SDs) are recurrent and ostensibly spontaneous depolarization waves that may contribute to infarct progression after stroke. Somatosensory activation of the metastable peri-infarct tissue triggers peri-infarct SDs at a high rate. METHODS: We directly measured the functional activation threshold to trigger SDs in peri-infarct hot zones using optogenetic stimulation after distal middle cerebral artery occlusion in Thy1-ChR2-YFP mice. RESULTS: Optogenetic activation of peri-infarct tissue triggered SDs at a strikingly high rate (64%) compared with contralateral homotopic cortex (8%; P=0.004). Laser speckle perfusion imaging identified a residual blood flow of 31±2% of baseline marking the metastable tissue with a propensity to develop SDs. CONCLUSIONS: Our data reveal a spatially distinct increase in SD susceptibility in peri-infarct tissue where physiological levels of functional activation are capable of triggering SDs. Given the potentially deleterious effects of peri-infarct SDs, the effect of sensory overstimulation in hyperacute stroke should be examined more carefully.

Secondary Bleeding During Acute Experimental Intracerebral Hemorrhage.

Background and Purpose- Mechanisms contributing to acute hematoma growth in intracerebral hemorrhage are not well understood. Neuropathological studies suggest that the initial hematoma may create mass effect that can tear vessels in the vicinity by shearing, causing further bleeding and hematoma growth. Methods- To test this in mice, we simulated initial intracerebral hemorrhage by intrastriatal injection of a liquid polymer that coagulates upon contact with tissue and measured the presence and volume of bleeding secondary to the mass effect using Hemoglobin ELISA 15 minutes after injection. Results- Secondary hemorrhage occurred in a volume-dependent (4, 7.5, or 15 µL of polymer) and rate-dependent (0.05, 0.5, or 5 µL/s) manner. Anticoagulation (warfarin or dabigatran) exacerbated the secondary hemorrhage volume. In a second model of hematoma expansion, we confirmed that intrastriatal whole blood injection (15 µL, 0.5 µL/s) also caused secondary bleeding, using acute Evans blue extravasation as a surrogate. Anticoagulation once again exacerbated secondary hemorrhage after intrastriatal whole blood injection. Secondary hemorrhage directly and significantly correlated with arterial blood pressures in both nonanticoagulated and anticoagulated mice, when modulated by phenylephrine or labetalol. Conclusions- Our study provides the first proof of concept for secondary vessel rupture and bleeding as a potential mechanism for intracerebral hematoma growth.

Synthesis of Arabinoxylan Oligosaccharides by Preactivation-Based Iterative Glycosylations.

A concise synthetic strategy has been developed for assembling densely substituted arabinoxylan oligosaccharides, which are valuable substrates for characterizing hemicellulose-degrading enzymes. The xylan backbone has been prepared by an iterative preactivation-based glycosylation approach with phenyl thioglycosides. The preactivation has been performed with in situ generated p-nitrobenzenesulfenyl triflate prior to addition of the acceptor. The glycosylation temperature was shown to have an important impact on the yield of the coupling. The arabinose substituents have been introduced in one high-yielding glycosylation with an N-phenyl trifluoroacetimidate donor. The strategy has been successfully employed for the synthesis of three heptasaccharides in seven steps and overall yields of 24-36% from the corresponding monosaccharide building blocks.

A concept of dual-responsive prodrugs based on oligomerization-controlled reactivity of ester groups: an improvement of cancer cells versus neutrophils selectivity of camptothecin.

Many known chemotherapeutic anticancer agents exhibit neutropenia as a dose-limiting side effect. In this paper we suggest a prodrug concept solving this problem for camptothecin (HO-cpt). The prodrug is programmed according to Boolean "AND" logic. In the absence of H2O2 (trigger T1), e.g. in the majority of normal cells, it exists as an inactive oligomer. In cancer cells and in primed neutrophils (high H2O2), the oligomer is disrupted forming intermediate (inactive) lipophilic cationic species. These are accumulated in mitochondria (Mit) of cancer cells, where they are activated by hydrolysis at mitochondrial pH 8 (trigger T2) with formation of camptothecin. In contrast, the intermediates remain stable in neutrophils lacking Mit and therefore a source of T2. In this paper we demonstrated a proof-of-concept. Our prodrug exhibits antitumor activity both in vitro and in vivo, but is not toxic to normal cell and neutrophils in contrast to known single trigger prodrugs and the parent drug HO-cpt.

Neurovascular coupling during optogenetic functional activation: Local and remote stimulus-response characteristics, and uncoupling by spreading depression.

Neurovascular coupling is a fundamental response that links activity to perfusion. Traditional paradigms of neurovascular coupling utilize somatosensory stimulation to activate the primary sensory cortex through subcortical relays. Therefore, examination of neurovascular coupling in disease models can be confounded if the disease process affects these multisynaptic pathways. Optogenetic stimulation is an alternative to directly activate neurons, bypassing the subcortical relays. We employed minimally invasive optogenetic cortical activation through intact skull in Thy1-channelrhodopsin-2 transgenic mice, examined the blood flow changes using laser speckle imaging, and related these to evoked electrophysiological activity. Our data show that optogenetic activation of barrel cortex triggers intensity- and frequency-dependent hyperemia both locally within the barrel cortex (>50% CBF increase), and remotely within the ipsilateral motor cortex (>30% CBF increase). Intriguingly, activation of the barrel cortex causes a small (∼10%) but reproducible hypoperfusion within the contralateral barrel cortex, electrophysiologically linked to transhemispheric inhibition. Cortical spreading depression, known to cause neurovascular uncoupling, diminishes optogenetic hyperemia by more than 50% for up to an hour despite rapid recovery of evoked electrophysiological activity, recapitulating a unique feature of physiological neurovascular coupling. Altogether, these data establish a minimally invasive paradigm to investigate neurovascular coupling for longitudinal characterization of cerebrovascular pathologies.

Non-invasively triggered spreading depolarizations induce a rapid pro-inflammatory response in cerebral cortex.

Cortical spreading depolarization (CSD) induces pro-inflammatory gene expression in brain tissue. However, previous studies assessing the relationship between CSD and inflammation have used invasive methods that directly trigger inflammation. To eliminate the injury confounder, we induced CSDs non-invasively through intact skull using optogenetics in Thy1-channelrhodopsin-2 transgenic mice. We corroborated our findings by minimally invasive KCl-induced CSDs through thinned skull. Six CSDs induced over 1 h dramatically increased cortical interleukin-1ß (IL-1ß), chemokine (C-C motif) ligand 2 (CCL2), and tumor necrosis factor-α (TNF-α) mRNA expression peaking around 1, 2 and 4 h, respectively. Interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1) were only modestly elevated. A single CSD also increased IL-1ß, CCL2, and TNF-α, and revealed an ultra-early IL-1ß response within 10 min. The response was blunted in IL-1 receptor-1 knockout mice, implicating IL-1ß as an upstream mediator, and suppressed by dexamethasone, but not ibuprofen. CSD did not alter systemic inflammatory indices. In summary, this is the first report of pro-inflammatory gene expression after non-invasively induced CSDs. Altogether, our data provide novel insights into the role of CSD-induced neuroinflammation in migraine headache pathogenesis and have implications for the inflammatory processes in acute brain injury where numerous CSDs occur for days.

Real-time non-invasive in vivo visible light detection of cortical spreading depolarizations in mice.

BACKGROUND: Cortical spreading depolarization (CSD) is a phenomenon classically associated with migraine aura. CSDs have also been implicated in secondary injury following ischemic stroke, intracerebral hemorrhage, subarachnoid hemorrhage, and traumatic brain injury; however, most investigations involving these disease processes do not account for the occurrence of CSDs. A major barrier to detection of CSDs in experimental models is that currently validated methods are invasive and require specialized equipment and a high level of expertise to implement. NEW METHOD: We present a low-cost, easy-to-implement approach to the detection of CSDs in the mouse through full-thickness intact skull. Our method uses the optical intrinsic signal from white light illumination (OIS-WL) and allows for real-time in vivo detection of CSDs using readily available USB cameras. RESULTS: OIS-WL detected 100% of CSDs that were seen with simultaneous electrode recording (69 CSDs in 28 mice), laser Doppler flowmetry (82 CSDs in 10 mice), laser speckle flowmetry (68 CSDs in 25 mice), or combined electrode recording plus laser speckle flowmetry (29 CSDs in 20 mice). OIS-WL detected 1 additional CSD that was missed by laser Doppler flowmetry. COMPARISON WITH EXISTING METHODS: OIS-WL is less invasive than electrophysiological recordings and easier to implement than laser speckle flowmetry. Moreover, it provides excellent spatial and temporal resolution for dynamic imaging of CSDs in the setting of brain injury. CONCLUSIONS: Detection of CSDs with an inexpensive USB camera and white light source provides a reliable method for the in vivo and non-invasive detection of CSDs through unaltered mouse skull.