Fluoride Induces a Volume Reduction in CA1 Hippocampal Slices Via MAP Kinase Pathway Through Volume Regulated Anion Channels

Regulation of cell volume is an important aspect of cellular homeostasis during neural activity. This volume regulation is thought to be mediated by activation of specific transporters, aquaporin, and volume regulated anion channels (VRAC). In cultured astrocytes, it was reported that swelling-induced mitogen-activated protein (MAP) kinase activation is required to open VRAC, which are thought to be important in regulatory volume decrease and in the response of CNS to trauma and excitotoxicity. It has been also described that sodium fluoride (NaF), a recognized G-protein activator and protein phosphatase inhibitor, leads to a significant MAP kinase activation in endothelial cells. However, NaF's effect in volume regulation in the brain is not known yet. Here, we investigated the mechanism of NaF-induced volume change in rat and mouse hippocampal slices using intrinsic optical signal (IOS) recording, in which we measured relative changes in intracellular and extracellular volume as changes in light transmittance through brain slices. We found that NaF (1~5 mM) application induced a reduction in light transmittance (decreased volume) in CA1 hippocampus, which was completely reversed by MAP kinase inhibitor U0126 (10 µM). We also observed that NaF-induced volume reduction was blocked by anion channel blockers, suggesting that NaF-induced volume reduction could be mediated by VRAC. Overall, our results propose a novel molecular mechanism of NaF-induced volume reduction via MAP kinase signaling pathway by activation of VRAC

Imaging Single Photons and Intrinsic Optical Signals for Studies of Vesicular and Non-Vesicular ATP Release from Axons

The temporal and spatial dynamics of neurotransmitter release are fundamental to understanding activity-dependent signaling between axons and other cells, including neurons, glia, and vascular cells. A microscopic imaging technique is described that enables studying release of the neurotransmitter ATP from axons in response to action potentials. The method combines imaging single-photons, intrinsic optical signal imaging, and high magnification time-lapse microcopy to enable investigations of action potential-induced ATP release together with cell morphology and activity-dependent axon swelling. ATP released from axons catalyzes a chemiluminescent reaction between luciferin and luciferase that generates single photons that can be imaged individually. In addition to vesicular release, ATP release through membrane channels activated by axon swelling was monitored simultaneously with intrinsic optical signals. Repeated emissions of photons were observed from localized 15 μm regions of axons, with a frequency distribution that differed from a normal distribution and from the frequency of emissions outside these localized regions

Neuronal and astroglial correlates underlying spatiotemporal Intrinsic Optical Signal in the rat hippocampal slice

Widely used for mapping afferent activated brain areas in vivo, the label-free intrinsic optical signal (IOS) is mainly ascribed to blood volume changes subsequent to glial glutamate uptake. By contrast, IOS imaged in vitro is generally attributed to neuronal and glial cell swelling, however the relative contribution of different cell types and molecular players remained largely unknown. We characterized IOS to Schaffer collateral stimulation in the rat hippocampal slice using a 464-element photodiode-array device that enables IOS monitoring at 0.6 ms time-resolution in combination with simultaneous field potential recordings. We used brief half-maximal stimuli by applying a medium intensity 50 Volt-stimulus train within 50 ms (20 Hz). IOS was primarily observed in the str. pyramidale and proximal region of the str. radiatum of the hippocampus. It was eliminated by tetrodotoxin blockade of voltage-gated Na+ channels and was significantly enhanced by suppressing inhibitory signaling with gamma-aminobutyric acid(A) receptor antagonist picrotoxin. We found that IOS was predominantly initiated by postsynaptic Glu receptor activation and progressed by the activation of astroglial Glu transporters and Mg2+-independent astroglial N-methyl-D-aspartate receptors. Under control conditions, role for neuronal K+/Cl- cotransporter KCC2, but not for glial Na+/K+/Cl- cotransporter NKCC1 was observed. Slight enhancement and inhibition of IOS through non-specific Cl- and volume-regulated anion channels, respectively, were also depicted. High-frequency IOS imaging, evoked by brief afferent stimulation in brain slices provide a new paradigm for studying mechanisms underlying IOS genesis. Major players disclosed this way imply that spatiotemporal IOS reflects glutamatergic neuronal activation and astroglial response, as observed within the hippocampus. Our model may help to better interpret in vivo IOS and support diagnosis in the future

The Mechanism of Downregulated Interstitial Fluid Drainage Following Neuronal Excitation.

The drainage of brain interstitial fluid (ISF) has been observed to slow down following neuronal excitation, although the mechanism underlying this phenomenon is yet to be elucidated. In searching for the changes in the brain extracellular space (ECS) induced by electrical pain stimuli in the rat thalamus, significantly decreased effective diffusion coefficient (DECS) and volume fraction (α) of the brain ECS were shown, accompanied by the slowdown of ISF drainage. The morphological basis for structural changes in the brain ECS was local spatial deformation of astrocyte foot processes following neuronal excitation. We further studied aquaporin-4 gene (APQ4) knockout rats in which the changes of the brain ECS structure were reversed and found that the slowed DECS and ISF drainage persisted, confirming that the down-regulation of ISF drainage following neuronal excitation was mainly attributable to the release of neurotransmitters rather than to structural changes of the brain ECS. Meanwhile, the dynamic changes in the DECS were synchronized with the release and elimination processes of neurotransmitters following neuronal excitation. In conclusion, the downregulation of ISF drainage following neuronal excitation was found to be caused by the restricted diffusion in the brain ECS, and DECS mapping may be used to track the neuronal activity in the deep brain

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

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

Causal relationship between local field potential and intrinsic optical signal in epileptiform activity in vitro

The directed causal relationship were examined between the local field potential (LFP) and the intrinsic optical signal (IOS) during induced epileptiform activity in in vitro cortical slices by the convergent cross-mapping causality analysis method. Two components of the IOS signal have been distinguished: a faster, activity dependent component (IOSh) which changes its sign between transmitted and reflected measurement, thus it is related to the reflectance or the scattering of the tissue and a slower component (IOSl), which is negative in both cases, thus it is resulted by the increase of the absorption of the tissue. We have found a strong, unidirectional, delayed causal effect from LFP to IOSh with 0.5- 1s delay, without signs of feedback from the IOSh to the LFP, while the correlation was small and the peaks of the cross correlation function did not reflect the actual causal dependency. Based on these observations, a model has been set up to describe the dependency of the IOSh on the LFP power and IOSh was reconstructed, based on the LFP signal. This study demonstrates that causality analysis can lead to better understanding the physiological interactions, even in case of two data series with drastically different time scales

Current Techniques for Investigating the Brain Extracellular Space

The brain extracellular space (ECS) is a continuous reticular compartment that lies between the cells of the brain. It is vast in extent relative to its resident cells, yet, at the same time the nano- to micrometer dimensions of its channels and reservoirs are commonly finer than the smallest cellular structures. Our conventional view of this compartment as largely static and of secondary importance for brain function is rapidly changing, and its active dynamic roles in signaling and metabolite clearance have come to the fore. It is further emerging that ECS microarchitecture is highly heterogeneous and dynamic and that ECS geometry and diffusional properties directly modulate local diffusional transport, down to the nanoscale around individual synapses. The ECS can therefore be considered an extremely complex and diverse compartment, where numerous physiological events are unfolding in parallel on spatial and temporal scales that span orders of magnitude, from milliseconds to hours, and from nanometers to centimeters. To further understand the physiological roles of the ECS and identify new ones, researchers can choose from a wide array of experimental techniques, which differ greatly in their applicability to a given sample and the type of data they produce. Here, we aim to provide a basic introduction to the available experimental techniques that have been applied to address the brain ECS, highlighting their main characteristics. We include current gold-standard techniques, as well as emerging cutting-edge modalities based on recent super-resolution microscopy. It is clear that each technique comes with unique strengths and limitations and that no single experimental method can unravel the unknown physiological roles of the brain ECS on its own.This work was supported by the grants from the Spanish Ministry for Research and Innovation SAF2017-83776-R and RYC-2014-15994 to JT, IJCI-2017-32114 to FS, University of the Basque Country grant GIU18/094 to OP and JT, and a Basque Government grant PIBA 2019-65 to JT

Dynamics of Synaptic Transmission in the CNS: Contribution of Neuron-glia Interactions

The last 30 years have seen a growing appreciation of the importance for CNS functioning of the internal state of neural tissues, which is exquisitely reflective of the immediate-to-long-term history of the preceding neural activity experienced by those tissues. This dissertation comprises three research projects that together address different, but related aspects of dynamics of the state of neural tissues, with a focus on the roles played by astroglia and GABAergic synaptic transmission. The first two projects study the relationship between stimulus-evoked glial and neuronal activities within local networks of the dorsal horn of the spinal cord and sensitivity of GABAergic actions to the state of local glia and prior sensory stimulation, whereas the third project investigates the sensitivity of GABAergic actions to prior sensory stimulation in the neocortex. Project 1: Origins of Optical Intrinsic Signal and its significance. In rat spinal cord slice, repetitive electrical stimulation of the dorsal root at an intensity that activates C-fibers evokes a slow-to-develop and prolonged (30-50 s) change in light transmittance (OISDR) in the superficial part of the ipsilateral dorsal horn (DHs). Inhibition of astrocyte metabolism by bath-applied fluoroacetate and glutamine (FAc+Gln), or interference with glial and neuronal K+ transport by 4-aminopyridine (4-AP) lead to dissociation of the OISDR and the postsynaptic DHs response to a single-pulse dorsal root stimulus (P-PSPDR). The OISDR decreases under FAc+Gln, whereas the P-PSPDR remains unaltered; under 4-AP, the P-PSPDR increases, but the OISDR decreases. In contrast, both the OISDR and P-PSPDR increase when K+o is elevated. These observations indicate that the OISDR mainly reflects cell volume and light scattering changes associated with DHs astrocyte uptake of K+ and glutamate (GLU). In slices from subjects that received an intracutaneous injection of formalin 3-5 days earlier, both the OISDR and the response of the DHs to local application of K+ or GLU are profoundly reduced, and the normally exquisite sensitivity of the DHs to elevated K+o is decreased. Considered collectively, the observations raise the possibility that impaired regulation of DHs K+o and GLUo may contribute to initiation and maintenance of the CNS pain circuit and sensorimotor abnormalities that develop following intracutaneous formalin injection. Project 2: Effects of alteration of glia on neuronal plasticity. Transient (20min) exposure of the spinal cord slice to fluorocitrate (FC; a reversible inhibitor of glial energy production via the TCA cycle) is shown to be accompanied by a protracted decrease of the superficial dorsal horn (DHs) optical response to repetitive electrical stimulation of the ipsilateral dorsal root, and by a similarly protracted increase in the postsynaptic response of the DHs to single-pulse stimulation of the attached dorsal root (LTPFC). It also is shown that LTPFC does not occur in the presence of d-aminophosphopentanoic acid (APV), becomes progressively smaller as [K+]o in the perfusion solution is decreased from 3.0 mM (normal) to 0.0 mM, and is reduced or eliminated by bath application of 1 mM bicuculline. Somal whole-cell patch recordings were carried out to evaluate the effects of FC on the response of DHs neurons to puffer-applied GABA. The observations reveal that transient exposure of the slice to FC is reliably accompanied by a prolonged (>1 hr) depolarizing shift of the equilibrium potential for the DHs neuron transmembrane ionic currents evoked by GABA (average EGABApreFC: -75 mV ; EGABApostFC: -50 mV). Considered collectively, the findings demonstrate that LTPFC involves (1) elevation of [K+]o in the DHs, (2) NMDA receptor activation, and (3) conversion of the effect of GABA on DHs neurons from inhibition to excitation. It is proposed that a transient impairment of astrocyte energy production via the TCA cycle can trigger the cascade of dorsal horn mechanisms that underlies hyperalgesia and persistent pain. Project 3: Contribution of GABA to cerebral cortical dynamics. Imaging of the optical intrinsic signal (OIS), evoked in the rat sensorimotor cortical slice by 1s-long 20Hz electrical stimulation applied to locus at the layer VI/white matter junction, was used to delineate a column-shaped cortical region responding to a local thalamocortical input drive, and whole-cell patch clamp recordings were obtained from layer II-III pyramidal neurons residing in that region. Puffs of pressure-ejected GABA were released from a micropipette in a close vicinity of the recorded neuron's soma before and also immediately after conditioning electrical stimulation. Prior to conditioning stimulation, GABA puffs hyperpolarized the recorded neurons, whereas for ~15s subsequent to conditioning stimulation GABA puffs depolarized the same neurons. Two-photon Cl- imaging in cortical slices taken from CLM1 Clomeleon mice revealed that conditioning stimulation transiently elevates [Cl-]i in the stimulated cortical column; this increase is blocked by SR95531 (gabazine), a selective GABAA receptor antagonist. Next, two-photon Ca2+ imaging revealed that isoguvacine (GABAA receptor agonist) increases Ca2+ influx into neurons in the stimulated cortical column. Finally, OIS imaging in the presence of GABA antagonist bicuculline suggests that the depolarizing action of GABA is confined to the center of the stimulated cortical region, while at its margins GABA remains hyperpolarizing. Taken together, these findings suggest that synaptically released GABA can be either inhibitory or excitatory, depending on the activity state of the local network. Such activity dependence of GABA action can be expected to funnel stimulus-evoked activity in a cortical area into the central, most strongly driven cortical columns