Within two minutes of global ischemia or focal stroke, a sudden loss of neuronal and glial membrane potential induces anoxic depolarization (AD) that propagates across gray matter of the cerebral hemispheres at 2-5 mm/min. It induces terminal neuronal damage, forming the initial ischemic core. In healthy gray matter, a milder version termed spreading depression (SD) does not cause tissue damage and generates the migraine aura, often a marching sensory deficit preceding the pain. AD and SD have been well studied in the cerebral and cerebellar cortices, but not the in the brainstem. We induced AD in coronal brain slices of mouse using oxygen/glucose deprived (OGD) saline. SD was induced by briefly elevating the extracellular K+ concentration. AD or SD propagation was imaged as a moving front of elevated light transmittance (LT) in cerebral and cerebellar cortices. Most ventral brainstem areas did not support AD or SD but in the dorsal brainstem some LT mini-fronts were observed in the superficial superior colliculus (SC), edges of inferior colliculus (IC), periaqueductal gray (PAG), tegmental nucleus (TN) and solitary nucleus (SolN). Their AD/SD characteristics were compared to those in the ‘higher’ brain regions. Although time of onset was not different, the present AD and SD propagated more slowly in brainstem gray matter. A non-specific glutamate receptor antagonist kynurenic acid (KYNA) successfully blocked the SD but not AD in PAG, TN and SolN. Two-photon laser scanning microscopy (2-PLSM) of live YFP+ mice brain slices showed that pyramidal neurons in ‘higher’ hippocamal CA1 irreversibly swelled and formed dendritic beads while neurons in the ‘lower’ mesencephalic trigeminal nucleus (Mes) of the midbrain-pons did not significantly swell or display any sign of injury. Finally, dendritic beading was induced in intact mouse neocortex and hippocampus by cardiac perfusion with OGD saline or ice-cold saline. However, dendrites in the brainstem from the same mice showed no obvious beading. Taken together, our study supports the concept that most brainstem regions are comparatively resistant to AD/SD compared to the ‘higher’ regions of cerebral and cerebellar cortices. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2012-10-01 13:42:14.325

Pyramidal neurons (PyNs) in ‘higher’ brain are highly susceptible to acute stroke injury yet ‘lower’ brain regions better survive global ischemia, presumably because of better residual blood flow. Here we show that projection neurons in ‘lower’ brain regions of hypothalamus and brainstem intrinsically resist acute stroke-like injury independent of blood flow in the brain slice. In contrast `higher` projection neurons in neocortex, hippocampus, striatum and thalamus are highly susceptible. In live brain slices from rat deprived of oxygen and glucose (OGD), we imaged anoxic depolarization (AD) as it propagates through these regions. AD, the initial electrophysiological event of stroke, is a depolarizing front that drains residual energy in compromised gray matter. The extent of AD reliably determines ensuing damage in higher brain, but using whole-cell recordings we found that all CNS neurons do not generate a robust AD. Higher neurons generate strong AD and show no functional recovery in contrast to neurons in hypothalamus and brainstem that generate a weak and gradual AD. Most dramatically, lower neurons recover their membrane potential, input resistance and spike amplitude when oxygen and glucose is restored, while higher neurons do not. Following OGD, new recordings could be acquired in all lower (but not higher) brain regions, with some neurons even withstanding multiple OGD exposure. Two-photon laser scanning microscopy confirmed neuroprotection in lower, but not higher gray matter. Specifically pyramidal neurons swell and lose their dendritic spines post-OGD, whereas neurons in hypothalamus and brainstem display no such injury. Exposure to the Na+/K+ ATPase inhibitor ouabain (100 μM), induces depolarization similar to OGD in all cell types tested. Moreover, elevated [K+]o evokes spreading depression (SD), a milder version of AD, in higher brain but not hypothalamus or brainstem so weak AD correlates with the inability to generate SD. In summary, overriding the Na+/K+ pump using OGD, ouabain or elevated [K+]o evokes steep and robust depolarization of higher gray matter. We show that this important regional difference can be largely accounted for by the intrinsic properties of the resident neurons and that Na+/K+ ATPase pump efficiency is a major determining factor generating strong or weak spreading depolarizations. / Thesis (Ph.D, Anatomy & Cell Biology) -- Queen's University, 2012-10-02 17:59:20.589

With their unique photochemical properties, porphyrins have remained a central research topic for decades. Porphyrins can self-assemble into tubular structures at acidic pHs. However, the possibility of the disassembly of the aggregated porphyrin has not been investigated. Furthermore, quantitative understanding of the porphyrin optical activities is complicated by the complex interplay of the photon absorption, scattering, and fluorescence emission that can concurrently occur in porphyrin samples. Using meso- Tetrakis (4-sulfonato phenyl) porphyrin (TPPS) as the model molecule, discussed herein is combined UV-vis extinction, Stokes-shifted fluorescence, and polarized resonance synchronous spectroscopy (PRS2) study of porphyrin assembly and disassembly at acidic solutions. A series of optical constants, including photon absorption, scattering, and fluorescence emission cross-sections as well as its fluorescence and light scattering depolarizations has been quantified. Compared to UV-vis and SSF methods, the PRS2 is significantly more sensitivity in the detection of both concentration- and time-dependent porphyrin aggregation.

MECHANISMS OF DEPOLARIZATION INDUCED DENDRITIC GROWTH OF DROSOPHILA MOTOR NEURONS Cortnie Lauren Cherry The University of Arizona, 2006 Director: Richard B. Levine The study of the cellular mechanisms underlying dendritic growth contributes to our understanding of nervous system development, function and disease. Electrical activity is a fundamental property of neurons, and this property is utilized to influence the mechanisms involved in dendrite formation and maturation. Here we employ the Drosophila transgenic system to quantify dendritic growth of identified motor neurons using both in vitro and in vivo techniques. Two novel techniques are introduced: one a system to visualize and measure dendritic outgrowth in cultured neurons using reporter proteins, and the other using 3D reconstruction to measure the arborization of identified motor neurons in vivo. Both transgenic manipulation of K+ channel function and depolarizing concentrations of K+ in the culture medium result in an acceleration of dendritic outgrowth. Depolarization induced outgrowth is dependent on Plectreurys Toxin (PLTX)-sensitive voltage-gated calcium current and protein synthesis in cultured motor neurons. Depolarization leads to direct induction of fos, a protein that heterodimerizes with jun to make the functional transcription factor, AP-1. Fos, but not jun, is necessary for basal levels of dendritic growth, while both are necessary for depolarization induced outgrowth. Over-expression of AP-1 in control cells is sufficient to cause dendritic outgrowth. The transcription factor Adf-1 is also necessary for basal and depolarization induced growth, but unlike AP-1 is not sufficient to cause outgrowth when over-expressed. Another transcription factor CREB, on the other hand, is not necessary for basal levels of dendritic growth, but is necessary for depolarization induced dendritic growth. Over-expression of CREB, like Adf-1, is not sufficient to cause dendritic outgrowth. These findings present exciting new techniques for the study of the field of dendritic regulation and contribute to our understanding of the cellular mechanisms underlying dendritic growth.

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<p> Peroxynitrite is formed in blood vessels upon reaction of superoxide anion with nitric oxide (NO). It can oxidize proteins and thiols and nitrosylate free or protein bound thiols and tyrosine residues, thereby producing vascular dysfunction. Peroxynitrite therefore, may contribute to hypertension and cardiovascular diseases. We investigated the in vitro effects of commercially available peroxynitrite. De-endothelialized rings from the left descending coronary artery of pig were treated with peroxynitrite for 30 min, washed and then contracted with cyclopiazonic acid (CPA) or by membrane depolarization with KCl. Tissues pre-treated with peroxynitrite showed inhibition of the CPA-induced contraction with an IC50 of ≈100 uM but there was no effect on KCl-induced contraction. Peroxynitrite is stable only at alkaline pH and it may decompose to form superoxide and NO. However, including superoxide dismutase + catalase along with peroxynitrite did not change its effect.</p> <p> Next, we used the same protocol to compare the effects of peroxynitrite and NO generating agents: 3-morpholino sydnonimine (SIN-1), s-nitroso-N-acetylpenicilliamine (SNAP), sodium nitroprusside (SNP) and spermine nonoate. The effectiveness of these agents to inhibit the CPA-induced contraction was SNAP > spermine nonoate ≥ SIN-1 > SNP. SNAP was the most effective in inhibiting the KCl-induced contraction with spermine nononoate being less effective and SIN-1 and SNP not producing any significant inhibition. We further investigated the effect of SNAP. Catalase, superoxide dismutase or CPTIO (a NO scavenger) did not prevent the effects of SNAP on the KCl or the CPA-induced contractions. The guanylate cyclase inhibitor ODQ, partially reversed the effects of only low concentrations of SNAP. Thus, pretreatment with NO generating agents such as SNAP and spermine NONOate appear to be more effective in inhibiting the contraction of the pig coronary artery than with peroxynitrite or the peroxynitrite generating agent SIN-1. Since SIN-1, SNAP, SNP and NONOates produce different amounts of peroxynitrite, nitric oxide and S-nitrosylation products, their effects may be used to delineate the molecular basis of the actions of peroxynitrite and NO on the arterial function.</p> / Thesis / Master of Science (MSc)

Several studies suggest that action potential duration (APD) alternans play an important role in initiation of arrhythmias, while less is known about the alternans of early phases of action potential (AP) and phase relation between the two. Transmembrane potentials recorded from swine and canine ventricles were analyzed to determine the correlation and phase relation between alternans of early and late phases of an AP. In both species, for activation intervals ≤ 400 ms, action potential amplitude (APA) alternans occurred≥ 50% of times when APD alternans occurred and vice versa, both were mostly in phase. Also, alternans of APA and APD were mostly in phase with alternans of maximal rate of depolarization. The correlation between alternans in early and later parts of AP, however, was variable between species; APD10 and APD90 alternans were out of phase 81 % versus 34 % in canines and swines. These observations suggest that ionic mechanisms underlying alternans of depolarization and early repolarization phases may be distinct from those underlying later phases of repolarization. Simulations conducted to see the spatiotemporal effect of phase behavior between these alternans show that out of phase behavior suppresses oscillations in wavelength and minimizes the chances of spatial discordance.

Membrane depolarization is an effective neurotrophic stimulus, with its trophic effect on spiral ganglion neurons (SGNs) even surpassing that of neurotrophins. Thus, SGN cultures are a favorable system to investigate pro-survival signal transduction downstream of depolarization. Depolarization promotes SGN survival by recruiting three distinct kinase pathways: cyclic AMP-dependent protein kinase (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII) and CaMKIV. CaMKIV mediates the pro-survival effect of depolarization by activating CREB in nucleus. However, the mechanisms by which PKA and CaMKII promote survival are still not clear. By targeting constitutively active PKA or a PKA inhibitor (PKI) to the outer mitochondrial membrane (OMM), we showed that PKA activity at the OMM is sufficient to support SGN survival in the absence of other trophic factors and necessary for cAMP-dependent SGN survival. It has been suggested that PKA can promote survival by inactivating pro-apoptotic protein Bad. By cotransfection of SGNs with OMM-PKA and wild-type Bad, we showed that this was the case. We further showed that Ser112 and Ser136 in Bad, but not Ser155, a hypothetical PKA target, were necessary for functional inactivation of Bad by PKA. CaMKII mediates the third depolarization-dependent pro-survival pathway. A specific pro-survival target for CaMKII was identified through a separate investigation of the pro-apoptotic JNK-Jun signaling pathway, which we had identified as active in apoptotic SGNs in vivo. By measuring anti-phosphoJun immunofluorescence, we could quantify JNK-Jun activation in SGNs under different conditions. We showed that JNK inhibition or genetic deletion of JNK3 reduces SGN death after neurotrophic factor withdrawal. Neurotrophins have been shown to suppress JNK activation via their receptor protein tyrosine kinases (PTKs). By expressing constitutively active and dominant negative forms of candidate protein kinases, we identified a novel signaling pathway linking depolarization to JNK: Ca2+ entry - CaMKII - FAK/Pyk2 - PI-3-OH Kinase - Protein Kinase B - inhibition of MLKs (upstream activators of JNK). Thus, depolarization also recruits PTKs - the nonreceptor PTKs FAK and Pyk2 - to suppress JNK activation, implying a conserved PTK-PI3K-PKB pathway for suppression of pro-apoptotic JNK activation by neurotrophic stimuli.

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