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FRIMEDBIO-Fri prosj.st. med.,helse,biol

Dynamics, mechanisms, and modulation of subthreshold electrical signalling in cortical neurons in vivo

Awarded: NOK 6.6 mill.

Electrical activity in neurons (nerve cells) and circuits of the cerebral cortex is the basis for behavior and conscious experiences.. How do neuronal mechanisms determine the activity of cortical cercuits? How do these change the state of the brain, e.g. from awake, conscious state to unconscious sleep and general anesthesia? We know that such changes are caused by chemical signal substances that modulate (change) ion channels (molecules conducting ionic currents through the cell membrane) that are opened by small changes in the membrane potential, "sub-threshold" for nerve impulse generation, thus altering neuronal properties. We and others have previously studied in isolated brain slices how signal substances such as noradrenaline and acetyl choline affect the ion channels that regulate rhythms (oscillations) and impulse patterns. In this project, we will also study mechanisms in the intact brain (in vivo) in wake, sleep, and anesthesia, at several levels and with different methods, from intracellular measurements (patch clamp) to brain waves (EEG) and mathematical models. So far in this project, we have : (1) Discovered limitations in a commonly used method for measuring electrical activity in cortical cells in vivo, and a new method for correcting these; (2) Established intracellular measurements («whole cell patch clamp») the intact mouse brain (in vivo), e.g. of what happens during electrical rhythms in cortex under sleep, wake and anesthesia («up- & down-states», UDS; «high-conductance state», HCS, caused synaptic bombardment by the network etc.), slow after-hyperpolarizations («sAHP») etc. (3) Established, with Swiss and other groups, the use of large mathematical models of brain networks for testing exactly how "sub-threshold" ionic currents affect HCS, UDS etc. (4) Established methods for activating brain cells by light (optogenetic, with Channel Rhodopsin2; (5) Discovered that a type of ion channels («D-channels»), that are opened "sub-threshold", change strongly during brain development. (6) Discovered 2 other types of ion channels that are opened "sub-threshold" («M-channels») and by calcium («SK-channels») have complementary functions in some cortical neurons (DG granule cells). (6) Discovered that M-channels also exist in ME cortex stellate cells and are regulated by acetyl choline. This was surprising, since leading researchers in USA could not find M-channels there, and interesting since these stellate cells are «grid cells», discovered by E. and MB Moser (Nobel prize 2015), and we showed before that M-channels regulate brain rhythms (theta) that may cause the grid pattern. (7) Discovered, surprisingly, that M-channels have different functions in the same type of pyramidal cells in different parts of the cortex: they are much more active in dorsal than ventral parts of the brain. Since these channels dampen epileptic seizures, this may explain why epilepsy is more common in ventral parts of this cortex. (8) Discovered, that the «sAHP-channels», which also act "sub-threshold», they are much more active in dorsal parts of the brain, but in a different cell type (DG granule cells). Thus is surprising, suggesting a new principle for regulating cortical activity. Since the «sAHP-channels» are modulated by noradrenaline, acetyl choline, etc., which regulate wake, sleep, and anesthesia, this is of interest for control of consciousness. (9) We found that the sAHP is not caused by «IK? ion channels as claimed by a leading group in USA. (10) We and others have previously found a high density of M-channels in nerve fibres (axsons) in the cortex, but it has been unknown what they do there and how they are modulated. We think that we have now found the answer, by mathematical models and measuring directly from the axons; and that the axonal M-channels are important for changes from sleep to wake. (11) Discovered, by experiments and mathematical models i that the sAHP-channels can regulate cortical rhythms in sleep and wake, by dampening the rhythms that are important for wake, memory, and navigation. (12) Discovered how Kv2-kanaler regulate firing patterns in MEC-stellate celles (grid cells); (13) Established methods for accurate brain wave recording (hdEEG) in sleep and wake, in vivo, and magnetic stimulation (TMS) of the cortex, for measuring changes in brain state and connectivity (complexity, integration osv.). EEG and TMS are non-invasive methods that can be used in both animals and humans. Based on these results, we will compare changes in brain state in wake, sleep and anesthesia in animals and humans, and in models, to understand mechanisms at different levels from cells to circuits. This is the basis also for new collaborations with groups in Europe and USA, and a new EU project (Human Brain Project, HBP/SP3), started April 1st 2016, coordinated by JFS. The NRC project is also basis for 2 PhD theses in 2016.

A fundamental question is how neuronal mechanisms determine electrical signalling in cortical circuits, and how they are modulated in different behavioural states. Although only two ionic currents (fast Na+ and K+) are needed to generate action potential s, cortical neurons have many additional channel types operating in the subthreshold voltage domain. Although far smaller and slower han the fast spike currents, these subthreshold currents (or conductances) are apparently, according to brain slice data, highly important for synaptic integration, thus determining whether, when, and how often spikes are generated. However, in vivo intracellular recordings from cortical neurons in anaesthetized animals indicate that they are in a high-conductance state due to synaptic bombardment by the network. This would be expected to largely shunt the effects of the weaker intrinsic subthreshold currents, suggesting that the latter have little impact in vivo. This apparent paradox can only be resolved by directly exami ning the effects of intrinsic currents by intracellular recording in vivo. Because these properties may be altered by anaesthesia and behavioral state, these questions must be addressed by intracellular recording in unanestethized animals. Recent progres s now allows studies by whole-cell patch clamp recordings from awake, behaving mice, using blind patching or visually guided shadow-patching in head-restrained mice running on a spherical treadmill. We will establish these highly potent methods and use th em to study the subthreshold membrane potential dynamics of neocortical layer 5 and 2/3 pyramidal cells in visual and somatosensory cortex. Thus, we will address the high-conductance state paradox and other fundamental questions regarding the impact of su bthreshold ionic conductances involved in intrinsic subthreshold oscillations, resonance, after-hyperpolarizations, after-depolarizations, and other aspects of subthreshold dynamics in the intact cortex.

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FRIMEDBIO-Fri prosj.st. med.,helse,biol

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