Oliver Kann

Elevation of Basal Intracellular Calcium as a Central Element in the Activation of Brain Macrophages (Microglia): Suppression of Receptor-Evoked Calcium Signaling and Control of Release Function

Microglia–brain macrophages are immune-competent cells of the CNS and respond to pathologic events. Using bacterial lipopolysaccharide (LPS) as a tool to activate cultured mouse microglia, we studied alterations in the intracellular calcium concentration ([Ca 2+]i) and in the receptor-evoked generation of transient calcium signals. LPS treatment led to a chronic elevation of basal [Ca 2+]i along with a suppression of evoked calcium signaling, as indicated by reduced [Ca 2+]i transients during stimulation with UTP and complement factor 5a. Presence of the calcium chelator BAPTA prevented the activation-associated changes in [Ca 2+]i and restored much of the signaling efficacy. We also evaluated downstream consequences of a basal [Ca 2+]i lifting during microglial activation and found BAPTA to strongly attenuate the LPS-induced release of nitric oxide (NO) and certain cytokines and chemokines. Furthermore, microglial treatment with ionomycin, an ionophore elevating basal [Ca 2+]i, mimicked the activation-induced calcium signal suppression but failed to induce release activity on its own. Our findings suggest that chronic elevation of basal [Ca 2+]i attenuates receptor-triggered calcium signaling. Moreover, increased [Ca 2+]i is required, but by itself is not sufficient, for release of NO and certain cytokines and chemokines. Elevation of basal [Ca 2+]i could thus prove a central element in the regulation of executive functions in activated microglia.

Activation of mouse microglial cells affects P2 receptor signaling.

Microglial cells are the immunocompetent cells of the CNS, which are known to exist in several activation states. Here we investigated the impact of microglial activation on the P2 receptor-mediated intracellular calcium ([Ca(2+)](i)) signaling by means of fluo-3 based Ca(2+)-imaging. Cultured mouse microglial cells were treated with either astrocyte-conditioned medium to induce a ramified morphology or LPS to shift the cells toward the fully activated stage. The extracellular application of ATP (100 microM) induced a [Ca(2+)](i) elevation in 85% of both untreated and ramified microglial cells, whereas only 50% of the LPS-activated cells responded to the stimulus. To characterise the pharmacological profile of microglial P2 receptors we investigated the effects of various P2 agonists on [Ca(2+)](i) in cultured microglial cells. Untreated and ramified microglial cells demonstrated a very similar sensitivity to the different P2 agonists. In contrast, in LPS-activated microglia, a sharp decrease of responses to P2 agonist stimulation was seen. This indicates that microglial activation influences the capability of microglial cells to generate [Ca(2+)](i) signals upon P2 receptor activation.

Coupling of neuronal activity and mitochondrial metabolism as revealed by nad(p)h fluorescence signals in organotypic hippocampal slice cultures of the rat

During physiological activity neurons may experience localised energy demands which require intracellular signals for stimulation of mitochondrial NADH generation and subsequent delivery of ATP. To elucidate these mechanisms, we applied microfluorimetric monitoring of cytoplasmic (Fluo-3) and mitochondrial (Rhod-2) calcium concentration ([Ca(2+)](c), [Ca(2+)](m)), as well as of mitochondrial oxidative metabolism (NAD(P)H), whilst simultaneously measuring changes in extracellular potassium concentration ([K(+)](o)), as an indicator of neuronal activity in hippocampal slice cultures. Changes in neuronal activity were induced by repetitive stimulation at different frequencies (5, 20, 100 Hz) and intensities. Stimulation parameters were chosen to elicit rises in [K(+)](o) of less than 3 mM which is comparable to physiologically occurring rises in [K(+)](o). The mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) reduced stimulus-induced changes in Rhod-2 fluorescence by 79%, indicating that Rhod-2 signals were primarily of mitochondrial origin. Repetitive stimulation at 20 Hz applied to areas CA1 or CA3 resulted in moderate rises in [K(+)](o) which were associated with stimulus-dependent elevations in [Ca(2+)](c) and [Ca(2+)](m) of up to 15%. The same stimuli also elicited biphasic changes in NAD(P)H fluorescence characterised by an initial decline and a subsequent prolonged elevation of up to 10%. Variation of stimulus parameters revealed close correlations between rises in [K(+)](o), in [Ca(2+)](m) and changes in NAD(P)H fluorescence. To elucidate the role of intracellular Ca(2+) accumulation in induction of NAD(P)H fluorescence signals, the effect of application of Ca(2+)-free solution on these signals evoked by repetitive antidromic stimulation of the alveus during recordings in area CA1 was studied. Lowering extracellular Ca(2+) led to complete blockade of postsynaptic field potential components as well as of rises in [Ca(2+)](c) and [Ca(2+)](m). Amplitudes of NAD(P)H signals were reduced by 59%, though rises in [K(+)](o) were comparable in presence and absence of extracellular Ca(2+). The results suggest i) that mitochondrial oxidative metabolism is fine-tuned to graded physiological activity in neurons and ii) that rapid mitochondrial Ca(2+) signalling represents one of the main determinants for stimulation of oxidative metabolism under physiological conditions.

Microglial Activation by Components of Gram-Positive and -Negative Bacteria: Distinct and Common Routes to the Induction of Ion Channels and Cytokines

Gram-positive Streptococcus pneumoniae is the major pathogen causing lethal meningitis in adults. We used pneumococcal cell walls (PCW) to investigate microglial consequences of a bacterial challenge and to determine the role of serum in the activation process. PCW caused the characteristic induction of an outwardly rectifying K+ channel (IK+(OR)), together with a concomitant suppression of the constitutively expressed inward rectifier K+ current, and evoked the release of tumor necrosis factor-alpha (TNF alpha), interleukin-6 (IL-6), IL-12, KC, macrophage inflammatory protein (MIP) 1alpha and MIP-2. Serum presence strongly facilitated the PCW effects, similarly as observed for lipopolysaccharide (LPS) from gram-negative Escherichia coli. The inflammatory cytokine, interferon-gamma (IFNgamma) induced the same electrophysiological changes, but independent of serum. Recombinant LPS binding protein (LBP) could partially replace serum activity in LPS stimulations. In contrast, neither LBP nor an antibody-mediated blockade of the LPS receptor, CD14 had significant influences on PCW-inducible changes. Cell surface interactions and cofactor involvement in microglial activation by gram-positive bacteria are thus distinct from the mechanisms employed by LPS. Moreover, tyrphostin AG126, a protein kinase inhibitor that prevents activation of the mitogen-activated protein kinase, p42MAPK (ERK2), potently blocked the PCW-stimulated cytokine release while having only a limited effect on LPS-inducible cytokines. In contrast, AG126 did not influence IK+(OR) inductions. This indicates that PCW recruits more than 1 intracellular signaling pathway to trigger the various responses and that different bacterial agents signal through both common and individual routes during microglial activation.

Gamma Oscillations and Spontaneous Network Activity in the Hippocampus Are Highly Sensitive to Decreases in pO2 and Concomitant Changes in Mitochondrial Redox State

Gamma oscillations have been implicated in higher cognitive processes and might critically depend on proper mitochondrial function. Using electrophysiology, oxygen sensor microelectrode, and imaging techniques, we investigated the interactions of neuronal activity, interstitial pO2, and mitochondrial redox state [NAD(P)H and FAD (flavin adenine dinucleotide) fluorescence] in the CA3 subfield of organotypic hippocampal slice cultures. We find that gamma oscillations and spontaneous network activity decrease significantly at pO2 levels that do not affect neuronal population responses as elicited by moderate electrical stimuli. Moreover, pO2 and mitochondrial redox states are tightly coupled, and electrical stimuli reveal transient alterations of redox responses when pO2 decreases within the normoxic range. Finally, evoked redox responses are distinct in somatic and synaptic neuronal compartments and show different sensitivity to changes in pO2. We conclude that the threshold of interstitial pO2 for robust CA3 network activities and required mitochondrial function is clearly above the “critical” value, which causes spreading depression as a result of generalized energy failure. Our study highlights the importance of a functional understanding of mitochondria and their implications on activities of individual neurons and neuronal networks.

Gamma oscillations in the hippocampus require high complex I gene expression and strong functional performance of mitochondria.

Fast neuronal network oscillations in the gamma range (~30-90 Hz) have been implicated in complex brain functions such as sensory processing, memory formation and, perhaps, consciousness, and appear to be exceptionally vulnerable to various pathologies. However, both energy demand and mitochondrial performance underlying gamma oscillations are unknown. We investigated the fundamental relationship between acetylcholine-induced gamma oscillations, mitochondrial gene expression and oxidative metabolism in hippocampal slice preparations of mouse and rat by applying electrophysiology, in situ hybridization, quantitative polymerase chain reaction, oxygen sensor microelectrode (interstitial partial oxygen pressure) and imaging of mitochondrial redox state [nicotinamide adenine dinucleotide (phosphate) and flavin adenine dinucleotide fluorescence]. We show that (i) gamma oscillation power, oxygen consumption and expression of complex I (nicotinamide adenine dinucleotide:ubiquinone oxidoreductase) subunits are higher in hippocampal subfield CA3 than in CA1 and dentate gyrus; (ii) the amount of oxygen consumption of gamma oscillations reaches that of seizure-like events; (iii) gamma oscillations are exquisitely sensitive to pharmacological complex I inhibition; and (iv) gamma oscillations utilize mitochondrial oxidative capacity near limit. These data suggest that gamma oscillations are especially energy demanding and require both high complex I expression and strong functional performance of mitochondria. Our study helps to explain the exceptional vulnerability of complex brain functions in ischaemia as well as in neurodegenerative and psychiatric disorders that are associated with mitochondrial dysfunction.

The protein tyrosine kinase inhibitor AG126 prevents the massive microglial cytokine induction by pneumococcal cell walls

Central nervous system (CNS) infections caused by Streptococcus pneumoniae still have a disastrous outcome. Underlying immunological and CNS cellular events are largely enigmatic. We used pneumococcal cells walls (PCW) to investigate microglial responses as these cells are prominent sensors and effectors during neuropathological changes. PCW stimulation of mouse microglia invitro evoked the release of the cyto‐ and chemokines, TNF‐α, IL‐6, IL‐12, KC, MCP‐1, MIP‐1α, MIP‐2 and RANTES as well as soluble TNF receptor II, a potential TNF‐α antagonist. The release induction followed extremely steep dose‐response relations, and short exposure periods (15 min) were already sufficient to trigger substantial responses. PCW signaling controlling the release depended on both p38 and p42 / p44 (ERK2 / ERK1) MAP kinase activities. The kinase inhibitor, tyrphostin AG126 prevented the PCW‐inducible phosphorylation of p42 / p44MAPK, potently blocked cytokine release and drastically reduced the bioavailable TNF‐α, since it only marginally affected the release of soluble TNF receptors. Moreover, in an in vivo model of pneumococcal meningitis, AG126 significantly attenuated the PCW‐induced leukocyte influx to the cerebrospinal fluid. The findings imply that pneumococcal CNS infection can cause a rapid and massive microglial activation and that ERK / MAPK pathway(s) are potential targets for pharmacological interventions.

ERK activation causes epilepsy by stimulating NMDA receptor activity

The ERK MAPK signalling pathway is a highly conserved kinase cascade linking transmembrane receptors to downstream effector mechanisms. To investigate the function of ERK in neurons, a constitutively active form of MEK1 (caMEK1) was conditionally expressed in the murine brain, which resulted in ERK activation and caused spontaneous epileptic seizures. ERK activation stimulated phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) and augmented NMDA receptor 2B (NR2B) protein levels. Pharmacological inhibition of NR2B function impaired synaptic facilitation in area cornus ammonicus region 3 (CA3) in acute hippocampal slices derived from caMEK1‐expressing mice and abrogated epilepsy in vivo. In addition, expression of caMEK1 caused phosphorylation of the transcription factor, cAMP response element‐binding protein (CREB) and increased transcription of ephrinB2. EphrinB2 overexpression resulted in increased NR2B tyrosine phosphorylation, which was essential for caMEK1‐induced epilepsy in vivo, since conditional inactivation of ephrinB2 greatly reduced seizure frequency in caMEK1 transgenic mice. Therefore, our study identifies a mechanism of epileptogenesis that links MAP kinase to Eph/Ephrin and NMDA receptor signalling.

Mitochondrial Calcium Ion and Membrane Potential Transients Follow the Pattern of Epileptiform Discharges in Hippocampal Slice Cultures

Emerging evidence suggests that mitochondrial dysfunction contributes to the pathophysiology of epilepsy. Recurrent mitochondrial Ca2+ ion load during seizures might act on mitochondrial membrane potential (ΔΨm) and proton motive force. By using electrophysiology and confocal laser-scanning microscopy, we investigated the effects of epileptiform activity, as induced by low-Mg2+ ion perfusion in hippocampal slice cultures, on changes in ΔΨm and in mitochondrial Ca2+ ion concentration ([Ca2+]m). The mitochondrial compartment was identified by monitoring ΔΨm in the soma and dendrites of patched CA3 pyramidal cells using the mitochondria-specific voltage-sensitive dye rhodamine-123 (Rh-123). Interictal activity was accompanied by localized mitochondrial depolarization that was restricted to a few mitochondria in small dendrites. In contrast, robust Rh-123 release into the cytosol was observed during seizure-like events (SLEs), indicating simultaneous depolarization of mitochondria. This was critically dependent on Ca2+ ion uptake and extrusion, because inhibition of the mitochondrial Ca2+ ion uniporter by Ru360 and the mitochondrial Na+/Ca2+ ion exchanger by 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one but not the inhibitor of mitochondrial permeability transition pore, cyclosporin A, decreased the SLE-associated mitochondrial depolarization. The Ca2+ ion dependence of simultaneous mitochondrial depolarization suggested enhanced Ca2+ ion cycling across mitochondrial membranes during epileptiform activity. Indeed, [Ca2+]m fluctuated during interictal activity in single dendrites, and these fluctuations spread over the entire mitochondrial compartment during SLEs, as revealed using mitochondria-specific dyes (rhod-2 and rhod-ff) and spatial frequency-based image analysis. These findings strengthen the hypothesis that epileptic activity results in Ca2+ ion-dependent changes in mitochondrial function that might contribute to the neuronal injury during epilepsy.

Monitoring NAD(P)H autofluorescence to assess mitochondrial metabolic functions in rat hippocampal–entorhinal cortex slices

Changes in neuronal energy metabolism, mitochondrial functions and homeostasis of reactive oxygen species are often supposed to induce alterations in neuronal activity in hippocampal slice models. In order to investigate the NAD(P)H autofluorescence signal in brain slice models, methods to monitor NAD(P)H signal in isolated mitochondria as described by Chance et al. [J. Biol. Chem. 254 (1979) 4764] and dissociated neurons as described by Duchen [Biochem. J. 283 (1992) 41] were adapted to recording conditions required for brain slices. Considering different experimental questions, we established an approach to monitor NAD(P)H autofluorescence signals from hippocampal slices of 400 microm thickness under either submerged or interface conditions. Therefore the procedure described here allows the measurement of NAD(P)H autofluorescence under conditions typically required in electrophysiological experiments. Depolarization of plasma membrane caused by electrical stimulation or application of glutamate (100 microM) resulted in a characteristic initial decrease followed by a long-lasting increase in the NAD(P)H autofluorescence signal. H(2)O(2) (100 microM) evoked a strong NAD(P)H signal decrease indicating direct oxidation to the nonfluorescencend NAD(P)(+). In contrast, the increase in NAD(P)H signal that followed a brief inhibition of mitochondrial respiratory chain complex I using rotenone (1 microM) indicated an accumulation of NAD(P)H. However, in presence of rotenone (1 microM) electrically evoked long-lasting NAD(P)H signal overshoot decreased progressively, due to a negative feedback of accumulated NAD(P)H to the citrate cycle. A comparable reduction in NAD(P)H signal increase were observed during low-Mg(2+) induced epileptiform activity, indicating a relative energy failure. In conclusion, the method presented here allows to monitor NAD(P)H autofluorescence signals to gain insight into the coupling of neuronal activity, energy metabolism and mitochondrial function in brain slice models.