Potassium Dependent Regulation of Astrocyte Water Permeability Is Mediated by cAMP Signaling

Astrocytes express potassium and water channels to support dynamic regulation of potassium homeostasis. Potassium kinetics can be modulated by aquaporin-4 (AQP4), the essential water channel for astrocyte water permeability regulation. We investigated whether extracellular potassium ([K+]o) can regulate astrocyte water permeability and the mechanisms of such an effect. Studies were performed on rat primary astrocytes and a rat astrocyte cell line transfected with AQP4. We found that 10mM [K+]o caused an immediate, more than 40%, increase in astrocyte water permeability which was sustained in 5min. The water channel AQP4 was a target for this regulation. Potassium induced a significant increase in intracellular cAMP as measured with a FRET based method and with enzyme immunoassay. We found that protein kinase A (PKA) could phosphorylate AQP4 in vitro. Further elevation of [K+]o to 35mM induced a global intracellular calcium response and a transient water permeability increase that was abolished in 5min. When inwardly rectifying potassium (Kir)-channels were blocked, 10mM [K+]o also induced a calcium increase and the water permeability increase no longer persisted. In conclusion, we find that elevation of extracellular potassium regulates AQP4 and astrocyte water permeability via intracellular signaling involving cAMP. A prolonged increase of astrocyte water permeability is Kir-channel dependent and this response can be impeded by intracellular calcium signaling. Our results support the concept of coupling between AQP4 and potassium handling in astrocytes

Disentangling astroglial physiology with a realistic cell model in silico

Electrically non-excitable astroglia take up neurotransmitters, buffer extracellular K+ and generate Ca2+ signals that release molecular regulators of neural circuitry. The underlying machinery remains enigmatic, mainly because the sponge-like astrocyte morphology has been difficult to access experimentally or explore theoretically. Here, we systematically incorporate multi-scale, tri-dimensional astroglial architecture into a realistic multi-compartmental cell model, which we constrain by empirical tests and integrate into the NEURON computational biophysical environment. This approach is implemented as a flexible astrocyte-model builder ASTRO. As a proof-of-concept, we explore an in silico astrocyte to evaluate basic cell physiology features inaccessible experimentally. Our simulations suggest that currents generated by glutamate transporters or K+ channels have negligible distant effects on membrane voltage and that individual astrocytes can successfully handle extracellular K+ hotspots. We show how intracellular Ca2+ buffers affect Ca2+ waves and why the classical Ca2+ sparks-and-puffs mechanism is theoretically compatible with common readouts of astroglial Ca2+ imaging

Low Dose Isoflurane Exerts Opposing Effects on Neuronal Network Excitability in Neocortex and Hippocampus

The anesthetic excitement phase occurring during induction of anesthesia with volatile anesthetics is a well-known phenomenon in clinical practice. However, the physiological mechanisms underlying anesthetic-induced excitation are still unclear. Here we provide evidence from in vitro experiments performed on rat brain slices that the general anesthetic isoflurane at a concentration of about 0.1 mM can enhance neuronal network excitability in the hippocampus, while simultaneously reducing it in the neocortex. In contrast, isoflurane tissue concentrations above 0.3 mM expectedly caused a pronounced reduction in both brain regions. Neuronal network excitability was assessed by combining simultaneous multisite stimulation via a multielectrode array with recording intrinsic optical signals as a measure of neuronal population activity

Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain

Imaging techniques based on optical contrast analysis can be used to visualize dynamic and functional properties of the nervous system via optical signals resulting from changes in blood volume, oxygen consumption and cellular swelling associated with brain physiology and pathology. Here we report in vivo noninvasive transdermal and transcranial imaging of the structure and function of rat brains by means of laser-induced photoacoustic tomography (PAT). The advantage of PAT over pure optical imaging is that it retains intrinsic optical contrast characteristics while taking advantage of the diffraction-limited high spatial resolution of ultrasound. We accurately mapped rat brain structures, with and without lesions, and functional cerebral hemodynamic changes in cortical blood vessels around the whisker-barrel cortex in response to whisker stimulation. We also imaged hyperoxia- and hypoxia-induced cerebral hemodynamic changes. This neuroimaging modality holds promise for applications in neurophysiology, neuropathology and neurotherapy

Loop Diuretics Have Anxiolytic Effects in Rat Models of Conditioned Anxiety

A number of antiepileptic medications that modulate GABAA mediated synaptic transmission are anxiolytic. The loop diuretics furosemide (Lasix) and bumetanide (Bumex) are thought to have antiepileptic properties. These drugs also modulate GABAA mediated signalling through their antagonism of cation-chloride cotransporters. Given that loop diuretics may act as antiepileptic drugs that modulate GABAergic signalling, we sought to investigate whether they also mediate anxiolytic effects. Here we report the first investigation of the anxiolytic effects of these drugs in rat models of anxiety. Furosemide and bumetanide were tested in adult rats for their anxiolytic effects using four standard anxiety models: 1) contextual fear conditioning; 2) fear-potentiated startle; 3) elevated plus maze, and 4) open-field test. Furosemide and bumetanide significantly reduced conditioned anxiety in the contextual fear-conditioning and fear-potentiated startle models. At the tested doses, neither compound had significant anxiolytic effects on unconditioned anxiety in the elevated plus maze and open-field test models. These observations suggest that loop diuretics elicit significant anxiolytic effects in rat models of conditioned anxiety. Since loop diuretics are antagonists of the NKCC1 and KCC2 cotransporters, these results implicate the cation-chloride cotransport system as possible molecular mechanism involved in anxiety, and as novel pharmacological target for the development of anxiolytics. In view of these findings, and since furosemide and bumetanide are safe and well tolerated drugs, the clinical potential of loop diuretics for treating some types of anxiety disorders deserves further investigation

Functional Diffusion Tensor Imaging: Measuring Task-Related Fractional Anisotropy Changes in the Human Brain along White Matter Tracts

Functional neural networks in the human brain can be studied from correlations between activated gray matter regions measured with fMRI. However, while providing important information on gray matter activation, no information is gathered on the co-activity along white matter tracts in neural networks.We report on a functional diffusion tensor imaging (fDTI) method that measures task-related changes in fractional anisotropy (FA) along white matter tracts. We hypothesize that these fractional anisotropy changes relate to morphological changes of glial cells induced by axonal activity although the exact physiological underpinnings of the measured FA changes remain to be elucidated. As expected, these changes are very small as compared to the physiological noise and a reliable detection of the signal change would require a large number of measurements. However, a substantial increase in signal-to-noise ratio was achieved by pooling the signal over the complete fiber tract. Adopting such a tract-based statistics enabled us to measure the signal within a practically feasible time period. Activation in the sensory thalamocortical tract and optic radiation in eight healthy human subjects was found during tactile and visual stimulation, respectively.The results of our experiments indicate that these FA changes may serve as a functional contrast mechanism for white matter. This noninvasive fDTI method may provide a new approach to study functional neural networks in the human brain

Stomatin Inhibits Pannexin-1-Mediated Whole-Cell Currents by Interacting with Its Carboxyl Terminal

The pannexin-1 (Panx1) channel (often referred to as the Panx1 hemichannel) is a large-conductance channel in the plasma membrane of many mammalian cells. While opening of the channel is potentially detrimental to the cell, little is known about how it is regulated under physiological conditions. Here we show that stomatin inhibited Panx1 channel activity. In transfected HEK-293 cells, stomatin reduced Panx1-mediated whole-cell currents without altering either the total or membrane surface Panx1 protein expression. Stomatin coimmunoprecipitated with full-length Panx1 as well as a Panx1 fragment containing the fourth membrane-spanning domain and the cytosolic carboxyl terminal. The inhibitory effect of stomatin on Panx1-mediated whole-cell currents was abolished by truncating Panx1 at a site in the cytosolic carboxyl terminal. In primary culture of mouse astrocytes, inhibition of endogenous stomatin expression by small interfering RNA enhanced Panx1-mediated outward whole-cell currents. These observations suggest that stomatin may play important roles in astrocytes and other cells by interacting with Panx1 carboxyl terminal to limit channel opening

Dibucaine Mitigates Spreading Depolarization in Human Neocortical Slices and Prevents Acute Dendritic Injury in the Ischemic Rodent Neocortex

Spreading depolarizations that occur in patients with malignant stroke, subarachnoid/intracranial hemorrhage, and traumatic brain injury are known to facilitate neuronal damage in metabolically compromised brain tissue. The dramatic failure of brain ion homeostasis caused by propagating spreading depolarizations results in neuronal and astroglial swelling. In essence, swelling is the initial response and a sign of the acute neuronal injury that follows if energy deprivation is maintained. Choosing spreading depolarizations as a target for therapeutic intervention, we have used human brain slices and in vivo real-time two-photon laser scanning microscopy in the mouse neocortex to study potentially useful therapeutics against spreading depolarization-induced injury.We have shown that anoxic or terminal depolarization, a spreading depolarization wave ignited in the ischemic core where neurons cannot repolarize, can be evoked in human slices from pediatric brains during simulated ischemia induced by oxygen/glucose deprivation or by exposure to ouabain. Changes in light transmittance (LT) tracked terminal depolarization in time and space. Though spreading depolarizations are notoriously difficult to block, terminal depolarization onset was delayed by dibucaine, a local amide anesthetic and sodium channel blocker. Remarkably, the occurrence of ouabain-induced terminal depolarization was delayed at a concentration of 1 µM that preserves synaptic function. Moreover, in vivo two-photon imaging in the penumbra revealed that, though spreading depolarizations did still occur, spreading depolarization-induced dendritic injury was inhibited by dibucaine administered intravenously at 2.5 mg/kg in a mouse stroke model.Dibucaine mitigated the effects of spreading depolarization at a concentration that could be well-tolerated therapeutically. Hence, dibucaine is a promising candidate to protect the brain from ischemic injury with an approach that does not rely on the complete abolishment of spreading depolarizations

Astrocytic Mechanisms Explaining Neural-Activity-Induced Shrinkage of Extraneuronal Space

Neuronal stimulation causes ∼30% shrinkage of the extracellular space (ECS) between neurons and surrounding astrocytes in grey and white matter under experimental conditions. Despite its possible implications for a proper understanding of basic aspects of potassium clearance and astrocyte function, the phenomenon remains unexplained. Here we present a dynamic model that accounts for current experimental data related to the shrinkage phenomenon in wild-type as well as in gene knockout individuals. We find that neuronal release of potassium and uptake of sodium during stimulation, astrocyte uptake of potassium, sodium, and chloride in passive channels, action of the Na/K/ATPase pump, and osmotically driven transport of water through the astrocyte membrane together seem sufficient for generating ECS shrinkage as such. However, when taking into account ECS and astrocyte ion concentrations observed in connection with neuronal stimulation, the actions of the Na+/K+/Cl− (NKCC1) and the Na+/HCO3− (NBC) cotransporters appear to be critical determinants for achieving observed quantitative levels of ECS shrinkage. Considering the current state of knowledge, the model framework appears sufficiently detailed and constrained to guide future key experiments and pave the way for more comprehensive astroglia–neuron interaction models for normal as well as pathophysiological situations