Micro-Electrode-Array recordings : a tool to study calcium signaling pathways involved in neuronal network plasticity and late phase long-term potentiation

The molecular mechanisms of many biological signaling pathways are highly conserved in evolution and are, often, regulated by second messengers. One of the most important second messengers is calcium, Ca2+. The huge diversity of calcium-regulated signaling pathways spans from fertilization of the oocyte over the activation of the immune system right up to the activity-regulated transmitter release in neurons. Furthermore, it is known that in cultured neurons, increases in nuclear calcium concentrations are of vital importance for CREB-mediated gene transcription. Those genes control, among other phenomena, neuronal survival and certain forms of learning and memory. The hippocampal formation is essential for spatial memory and associative learning, and has been extensively characterized in terms of its circuitry and molecular mechanisms which underlie plasticity. The potentiation of Schaffer collateral – CA1 synapses by the activation of NMDAR-mediated postsynaptic signaling cascades is known as long-term potentiation (LTP) and serves as a model for learning and memory in the mammalian central nervous system. The induction of LTP requires postsynaptic calcium influx, the activation of calcium-dependent kinases and relative signaling cascades. These processes are now well-understood but less is known about the mechanisms which make LTP persistent for hours and days. The objective of this study was to establish procedures, by means of Micro-Electrode-Arrays (MEAs), which allow studying especially the signaling pathways involved in the CREB-regulated transcription-dependent late phase of LTP (L-LTP). In one study I cultured primary hippocampal neurons for two weeks on MEAs and analyzed spontaneous activity of these networks by extracellular recordings. The spontaneous activity pattern strongly influences neuronal network information processing and thus modulation of neuronal network activity, e.g. upon external stimulations, is likely to be a basic feature of processes involved in learning and memory. In this part I could show that the specific inhibition of nuclear calcium signaling (and the associated inhibition of CREB-mediated gene transcription) alters the periodic activity pattern of developing neuronal networks. Moreover, I demonstrated that one target gene of the nuclear calcium signaling pathway, vegf-d, is involved in keeping neuronal networks fire. Interestingly, so far VEGF-D has been mainly known as a growth factor important for angiogenesis and lymphatogenesis. In another study I tried to transfer the results of my work on neuronal networks to acute slice preparations, a system closer to the in vivo condition. Acute hippocampal slices enabled detailed studies concerning the initiation of LTP but less is known about the mechanisms that make LTP persist for extended time periods. This is partly due to the difficulty of maintaining stable recordings over several hours from acute slice preparations. MEAs offer stable extracellular field recordings from many points on a brain slice. I could show that on MEAs LTP induced by high-frequency stimulation lasted four hours and longer, and was NMDA receptor- as well as translation-sensitive. To investigate whether the expression of late phase LTP is nuclear calcium-sensitive and which genes exactly are necessary for the maintenance phase of LTP I established virus-mediated gene transfer into the hippocampus of adult rats to generate genetically modified animals. In summary, I established two novel methods suitable to investigate especially the signaling pathways important for the maintenance phase of LTP in hippocampal neurons. Both methods can be used to screen for candidate genes involved in L-LTP

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 nucleusspecific calcium signal perturbation strategy in vivo complemented by gene profiling, bioinformatics and functional analyses, we discovered a pain-associated, nuclear calciumregulated gene program in spinal excitatory neurons. This includes C1q, a novel 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

Nuclear Calcium Sensors Reveal that Repetition of Trains of Synaptic Stimuli Boosts Nuclear Calcium Signaling in CA1 Pyramidal Neurons

Nuclear calcium is a key signal in the dialogue between synapse and nucleus that controls the genomic responses required for persistent adaptations, including memory and acquired neuroprotection. The amplitude and duration of nuclear calcium transients specify activity-induced transcriptional changes. However, the precise relationship between synaptic input and nuclear calcium output is unknown. Here, we used stereotaxic delivery to the rat brain of recombinant adeno-associated viruses encoding nuclear-targeted calcium sensors to assess nuclear calcium transients in CA1 pyramidal neurons after stimulation of the Schaffer collaterals. We show that in acute hippocampal slices, a burst of synaptic activity elicits a nuclear calcium signal with a regenerative component at above-threshold stimulation intensities. Using classical stimulation paradigms (i.e., high-frequency stimulation (HFS) and θ burst stimulation (TBS)) to induce early LTP (E-LTP) and transcription-dependent late LTP (L-LTP), we found that the magnitude of nuclear calcium signals and the number of action potentials activated by synaptic stimulation trains are greatly amplified by their repetition. Nuclear calcium signals and action potential generation were reduced by blockade of either NMDA receptors or L-type voltage-gated calcium channels, but not by procedures that lead to internal calcium store depletion or by blockade of metabotropic glutamate receptors. These findings identify a repetition-induced switch in nuclear calcium signaling that correlates with the transition from E-LTP to L-LTP, and may explain why the transcription-dependent phase of L-LTP is not induced by a single HFS or TBS but requires repeated trains of activity. Recombinant, nuclear-targeted indicators may prove useful for further analysis of nuclear calcium signaling in vivo