Anna M. Hagenston

Mitochondrial calcium uniporter Mcu controls excitotoxicity and is transcriptionally repressed by neuroprotective nuclear calcium signals

The recent identification of the mitochondrial Ca2+ uniporter gene (Mcu/Ccdc109a) has enabled us to address its role, and that of mitochondrial Ca2+ uptake, in neuronal excitotoxicity. Here we show that exogenously expressed Mcu is mitochondrially localized and increases mitochondrial Ca2+ levels following NMDA receptor activation, leading to increased mitochondrial membrane depolarization and excitotoxic cell death. Knockdown of endogenous Mcu expression reduces NMDA-induced increases in mitochondrial Ca2+, resulting in lower levels of mitochondrial depolarization and resistance to excitotoxicity. Mcu is subject to dynamic regulation as part of an activity-dependent adaptive mechanism that limits mitochondrial Ca2+ overload when cytoplasmic Ca2+ levels are high. Specifically, synaptic activity transcriptionally represses Mcu, via a mechanism involving the nuclear Ca2+ and CaM kinase-mediated induction of Npas4, resulting in the inhibition of NMDA receptor-induced mitochondrial Ca2+ uptake and preventing excitotoxic death. This establishes Mcu and the pathways regulating its expression as important determinants of excitotoxicity, which may represent therapeutic targets for excitotoxic disorders.

Nuclear Calcium Signaling in Spinal Neurons Drives a Genomic Program Required for Persistent Inflammatory Pain

Persistent pain induced by noxious stimuli is characterized by the transition from normosensitivity to hypersensitivity. Underlying mechanisms are not well understood, although gene expression is considered important. Here, we show that persistent nociceptive-like activity triggers calcium transients in neuronal nuclei within the superficial spinal dorsal horn, and that nuclear calcium is necessary for the development of long-term inflammatory hypersensitivity. Using a nucleus-specific calcium signal perturbation strategy in vivo complemented by gene profiling, bioinformatics, and functional analyses, we discovered a pain-associated, nuclear calcium-regulated gene program in spinal excitatory neurons. This includes C1q, a modulator of synaptic spine morphogenesis, which we found to contribute to activity-dependent spine remodelling on spinal neurons in a manner functionally associated with inflammatory hypersensitivity. Thus, nuclear calcium integrates synapse-to-nucleus communication following noxious stimulation and controls a spinal genomic response that mediates the transition between acute and long-term nociceptive sensitization by modulating functional and structural plasticity.

Calcium Signaling in Synapse-to-Nucleus Communication

Changes in the intracellular concentration of calcium ions in neurons are involved in neurite growth, development, and remodeling, regulation of neuronal excitability, increases and decreases in the strength of synaptic connections, and the activation of survival and programmed cell death pathways. An important aspect of the signals that trigger these processes is that they are frequently initiated in the form of glutamatergic neurotransmission within dendritic trees, while their completion involves specific changes in the patterns of genes expressed within neuronal nuclei. Accordingly, two prominent aims of research concerned with calcium signaling in neurons are determination of the mechanisms governing information conveyance between synapse and nucleus, and discovery of the rules dictating translation of specific patterns of inputs into appropriate and specific transcriptional responses. In this article, we present an overview of the avenues by which glutamatergic excitation of dendrites may be communicated to the neuronal nucleus and the primary calcium-dependent signaling pathways by which synaptic activity can invoke changes in neuronal gene expression programs.

Inositol-1,4,5-trisphosphate receptor-mediated Ca2+ waves in pyramidal neuron dendrites propagate through hot spots and cold spots

We studied inositol‐1,4,5‐trisphosphate (IP3) receptor‐dependent intracellular Ca2+ waves in CA1 hippocampal and layer V medial prefrontal cortical pyramidal neurons using whole‐cell patch‐clamp recordings and Ca2+ fluorescence imaging. We observed that Ca2+ waves propagate in a saltatory manner through dendritic regions where increases in the intracellular concentration of Ca2+ ([Ca2+]i) were large and fast (‘hot spots’) separated by regions where increases in [Ca2+]i were comparatively small and slow (‘cold spots’). We also observed that Ca2+ waves typically initiate in hot spots and terminate in cold spots, and that most hot spots, but few cold spots, are located at dendritic branch points. Using immunohistochemistry, we found that IP3 receptors (IP3Rs) are distributed in clusters along pyramidal neuron dendrites and that the distribution of inter‐cluster distances is nearly identical to the distribution of inter‐hot spot distances. These findings support the hypothesis that the dendritic locations of Ca2+ wave hot spots in general, and branch points in particular, are specially equipped for regenerative IP3R‐dependent internal Ca2+ release. Functionally, the observation that IP3R‐dependent [Ca2+]i rises are greater at branch points raises the possibility that this novel Ca2+ signal may be important for the regulation of Ca2+‐dependent processes in these locations. Futhermore, the observation that Ca2+ waves tend to fail between hot spots raises the possibility that influences on Ca2+ wave propagation may determine the degree of functional association between distinct Ca2+‐sensitive dendritic domains.

Reliable optical detection of coherent neuronal activity in fast oscillating networks in vitro.

Cognitive and behavioral functions depend on the activation of stable neuronal assemblies, i.e. distributed groups of co-active neurons within neuronal networks. It is therefore crucial to monitor distributed patterns of activity in real time with single-neuron resolution. Microelectrode recordings allow detection of coincidence between discharges of identified units at high temporal resolution, but are not able to reveal the full spatial pattern of activity in multi-cellular assemblies. Therefore, observation of such distributed sets of neurons is a stronghold of optical techniques, but the required resolution, sensitivity, and speed are still challenging current technology. Here, we report a new approach for monitoring neuronal assemblies, using memory-related network oscillations in rodent hippocampal circuits as a model. The cytosolic calcium-sensitive fluorescent protein GCaMP3.NES was expressed using recombinant adeno-associated viral (rAAV)-mediated gene transfer in CA3 pyramidal neurons of cultured mouse hippocampal slices. After 14-21 days in culture, field potential recordings revealed spontaneous occurrence of sharp wave-ripple network events during which a fraction of local neurons is coherently activated. Using a custom-built epi-fluorescence microscope we could monitor a field of view of 410 μm × 410 μm with single-neuron optical resolution (20× objective, 0.4 NA). We developed a highly sensitive and specific wavelet-based method of cell identification allowing simultaneous observation of more than 150 neurons at frame rates of up to 60 Hz. Our recording configuration and image analysis provide a tool to investigate cognition-related activity patterns in the hippocampus and other circuits.

Neuronal calcium signaling in chronic pain

Acute physiological pain, the unpleasant sensory response to a noxious stimulus, is essential for animals and humans to avoid potential injury. Pathological pain that persists after the original insult or injury has subsided, however, not only results in individual suffering but also imposes a significant cost on society. Improving treatments for long-lasting pathological pain requires a comprehensive understanding of the biological mechanisms underlying pain perception and the development of pain chronicity. In this review, we aim to highlight some of the major findings related to the involvement of neuronal calcium signaling in the processes that mediate chronic pain.

Nuclear Calcium Buffering Capacity Shapes Neuronal Architecture.

Background: Calcium-binding proteins regulate calcium dynamics and downstream signaling events. Results: Increasing the calcium buffering capacity of the nucleus alters the expression of genes that regulate neuronal architecture. Conclusion: The nuclear calcium buffering capacity is an important determinant of neuronal morphology. Significance: Nuclear calcium buffers represent a new target for modulating gene expression and neuronal structure. Calcium-binding proteins (CaBPs) such as parvalbumin are part of the cellular calcium buffering system that determines intracellular calcium diffusion and influences the spatiotemporal dynamics of calcium signals. In neurons, CaBPs are primarily localized to the cytosol and function, for example, in nerve terminals in short-term synaptic plasticity. However, CaBPs are also expressed in the cell nucleus, suggesting that they modulate nuclear calcium signals, which are key regulators of neuronal gene expression. Here we show that the calcium buffering capacity of the cell nucleus in mouse hippocampal neurons regulates neuronal architecture by modulating the expression levels of VEGFD and the complement factor C1q-c, two nuclear calcium-regulated genes that control dendrite geometry and spine density, respectively. Increasing the levels of nuclear calcium buffers by means of expression of a nuclearly targeted form of parvalbumin fused to mCherry (PV.NLS-mC) led to a reduction in VEGFD expression and, as a result, to a decrease in total dendritic length and complexity. In contrast, mRNA levels of the synapse pruning factor C1q-c were increased in neurons expressing PV.NLS-mC, causing a reduction in the density and size of dendritic spines. Our results establish a close link between nuclear calcium buffering capacity and the transcription of genes that determine neuronal structure. They suggest that the development of cognitive deficits observed in neurological conditions associated with CaBP deregulation may reflect the loss of necessary structural features of dendrites and spines.

Cerebral Glucose Metabolism and Sedation in Brain-injured Patients: A Microdialysis Study.

Background: Disturbed brain metabolism is a signature of primary damage and/or precipitates secondary injury processes after severe brain injury. Sedatives and analgesics target electrophysiological functioning and are as such well-known modulators of brain energy metabolism. Still unclear, however, is how sedatives impact glucose metabolism and whether they differentially influence brain metabolism in normally active, healthy brain and critically impaired, injured brain. We therefore examined and compared the effects of anesthetic drugs under both critical (<1 mmol/L) and noncritical (>1 mmol/L) extracellular brain glucose levels. Methods: We performed an explorative, retrospective analysis of anesthetic drug administration and brain glucose concentrations, obtained by bedside microdialysis, in 19 brain-injured patients. Result: Our investigations revealed an inverse linear correlation between brain glucose and both the concentration of extracellular glutamate (Pearson r=−0.58, P=0.01) and the lactate/glucose ratio (Pearson r=−0.55, P=0.01). For noncritical brain glucose levels, we observed a positive linear correlation between midazolam dose and brain glucose (P<0.05). For critical brain glucose levels, extracellular brain glucose was unaffected by any type of sedative. Conclusions: These findings suggest that the use of anesthetic drugs may be of limited value in attempts to influence brain glucose metabolism in injured brain tissue.

Reversible occlusion (on-off) valves in shunted tumor patients.

The first commercially produced adjustable valve for shunted hydrocephalus patients was introduced by H. Portnoy and R. Schulte in 1973. This valve is still in use and known as reversible occlusion or on–off valve. The reversible occlusion valve is mainly used in conjunction with an existing shunt in patients receiving intraventricular cytostatic therapy. The valve has a simple mechanical lock that is closed by external pressure application with a single finger. The study method is a retrospective clinical series of 15 patients undergoing a total of 16 valve implantations between 2003 and 2010 was carried out, and the valve was tested in vitro. We report a high incidence of accidental occlusions leading to a loss of consciousness in five patients (33.3%). We furthermore demonstrate in vitro that accidental occlusions can occur. The reversible occlusion valve is needed in shunted tumor patients receiving intrathecal administration of cytostatica. The mechanism works as long as no external pressure compresses the valve. However, head positions pose significant risks for unintentional occlusions. We stress the importance of: (1) a position near the midline avoiding the retroauricular or occipital regions, (2) a handling training for nurses and doctors, (3) instruction of patients and relatives, and (4) removal of the device after intrathecal cytostatic treatment.

Intracellular Calcium Waves Transmit Synaptic Information to the Nucleus in Hippocampal Pyramidal Neurons

Ca2+ waves provide a spatially and temporally unique intracellular signal that carries information from one region of the neuron to another. Despite the computational potential of such a mechanism, relatively little is known about the consequences of Ca2+ waves on neuronal function. In this chapter we review the basic properties of internal Ca2+ release and Ca2+ waves in hippocampal CA1 pyramidal neurons and how synaptically elicited Ca2+ waves influence the transcription factor CREB in an age-dependent manner.