Constraints of vigilance-dependent noradrenergic signaling to mouse cerebellar Bergmann glia.

Behavioral state plays an important role in determining astroglia Ca2+ signaling. In particular, locomotion-mediated elevated vigilance has been found to trigger norepinephrine-dependent whole cell Ca2+ elevations in astroglia throughout the brain. For cerebellar Bergmann glia it has recently been found that locomotion-induced transient Ca2+ elevations depend on their α1A -adrenergic receptors. With increasing availability and implementation of locomotion as behavioral parameter it becomes important to understand the constraints of noradrenergic signaling to astroglia. Here we evaluated the effect of speed, duration and interval of locomotion on Ca2+ signals in Bergmann glia as well as cerebellar noradrenergic axon terminals. We found almost no dependence on locomotion speed, but following the initial Ca2+ transient prolonged locomotion events revealed a steady-state Ca2+ elevation. Comparison of time course and recovery of transient Bergmann glia and noradrenergic terminal Ca2+ dynamics suggested that noradrenergic terminal Ca2+ activity determines Bergmann glia Ca2+ activation and does not require noradrenergic receptor desensitization to account for attenuation during prolonged locomotion. Further, analyzing the correlation among Ca2+ dynamics within regions within the field of observation we found that coordinated activity among noradrenergic terminals accounts for fluctuations of steady-state Bergmann glia Ca2+ activity. Together, our findings will help to better understand astroglia Ca2+ dynamics during less controlled awake behavior and may guide the identification of behavioral contexts preferably dependent on astroglia Ca2+ signaling.

Noradrenergic terminal short-term potentiation enables modality-selective integration of sensory input and vigilance state.

Recent years have seen compelling demonstrations of the importance of behavioral state on sensory processing and attention. Arousal plays a dominant role in controlling brain-wide neural activity patterns, particularly through modulation by norepinephrine. Noradrenergic brainstem nuclei, including locus coeruleus, can be activated by stimuli of multiple sensory modalities and broadcast modulatory signals via axonal projections throughout the brain. This organization might suggest proportional brain-wide norepinephrine release during states of heightened vigilance. Here, however, we have found that low-intensity, nonarousing visual stimuli enhanced vigilance-dependent noradrenergic signaling locally in visual cortex, revealed using dual-site fiber photometry to monitor noradrenergic Ca2+ responses of astroglia simultaneously in cerebellum and visual cortex and two-photon microscopy to monitor noradrenergic axonal terminal Ca2+ dynamics. Nitric oxide, following N-methyl-d-aspartate receptor activation in neuronal nitric oxide synthase-positive interneurons, mediated transient acceleration of norepinephrine-dependent astroglia Ca2+ activation. These findings reveal a candidate cortical microcircuit for sensory modality-selective modulation of attention.

Potential and Realized Impact of Astroglia Ca2 + Dynamics on Circuit Function and Behavior.

Astroglia display a wide range of spontaneous and behavioral state-dependent Ca2+ dynamics. During heightened vigilance, noradrenergic signaling leads to quasi-synchronous Ca2+ elevations encompassing soma and processes across the brain-wide astroglia network. Distinct from this vigilance-associated global Ca2+ rise are apparently spontaneous fluctuations within spatially restricted microdomains. Over the years, several strategies have been pursued to shed light on the physiological impact of these signals including deletion of endogenous ion channels or receptors and reduction of intracellular Ca2+ through buffering, extrusion or inhibition of release. Some experiments that revealed the most compelling behavioral alterations employed chemogenetic and optogenetic manipulations to modify astroglia Ca2+ signaling. However, there is considerable contrast between these findings and the comparatively modest effects of inhibiting endogenous sources of Ca2+. In this review, we describe the underlying mechanisms of various forms of astroglia Ca2+ signaling as well as the functional consequences of their inhibition. We then discuss how the effects of exogenous astroglia Ca2+ modification combined with our knowledge of physiological mechanisms of astroglia Ca2+ activation could guide further refinement of behavioral paradigms that will help elucidate the natural Ca2+-dependent function of astroglia.

Ethanol abolishes vigilance-dependent astroglia network activation in mice by inhibiting norepinephrine release.

Norepinephrine adjusts sensory processing in cortical networks and gates plasticity enabling adaptive behavior. The actions of norepinephrine are profoundly altered by recreational drugs like ethanol, but the consequences of these changes on distinct targets such as astrocytes, which exhibit norepinephrine-dependent Ca2+ elevations during vigilance, are not well understood. Using in vivo two-photon imaging, we show that locomotion-induced Ca2+ elevations in mouse astroglia are profoundly inhibited by ethanol, an effect that can be reversed by enhancing norepinephrine release. Vigilance-dependent astroglial activation is abolished by deletion of α1A-adrenergic receptor from astroglia, indicating that norepinephrine acts directly on these ubiquitous glial cells. Ethanol reduces vigilance-dependent Ca2+ transients in noradrenergic terminals, but has little effect on astroglial responsiveness to norepinephrine, suggesting that ethanol suppresses their activation by inhibiting norepinephrine release. Since abolition of astroglia Ca2+ activation does not affect motor coordination, global suppression of astroglial networks may contribute to the cognitive effects of alcohol intoxication.

Astroglial FMRP deficiency cell-autonomously up-regulates miR-128 and disrupts developmental astroglial mGluR5 signaling.

The loss of fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS), the most common inherited intellectual disability. How the loss of FMRP alters protein expression and astroglial functions remains essentially unknown. Here we showed that selective loss of astroglial FMRP in vivo up-regulates a brain-enriched miRNA, miR-128-3p, in mouse and human FMRP-deficient astroglia, which suppresses developmental expression of astroglial metabotropic glutamate receptor 5 (mGluR5), a major receptor in mediating developmental astroglia to neuron communication. Selective in vivo inhibition of miR-128-3p in FMRP-deficient astroglia sufficiently rescues decreased mGluR5 function, while astroglial overexpression of miR-128-3p strongly and selectively diminishes developmental astroglial mGluR5 signaling. Subsequent transcriptome and proteome profiling further suggests that FMRP commonly and preferentially regulates protein expression through posttranscriptional, but not transcriptional, mechanisms in astroglia. Overall, our study defines an FMRP-dependent cell-autonomous miR pathway that selectively alters developmental astroglial mGluR5 signaling, unveiling astroglial molecular mechanisms involved in FXS pathogenesis.

Reorganization of Destabilized Nodes of Ranvier in ßIV Spectrin Mutants Uncovers Critical Timelines for Nodal Restoration and Prevention of Motor Paresis.

Disorganization of nodes of Ranvier is associated with motor and sensory dysfunctions. Mechanisms that allow nodal recovery during pathological processes remain poorly understood. A highly enriched nodal cytoskeletal protein ßIV spectrin anchors and stabilizes the nodal complex to actin cytoskeleton. Loss of murine ßIV spectrin allows the initial nodal organization, but causes gradual nodal destabilization. Mutations in human ßIV spectrin cause auditory neuropathy and impairment in motor coordination. Similar phenotypes are caused by nodal disruption due to demyelination. Here we report on the precise timelines of nodal disorganization and reorganization by following disassembly and reassembly of key nodal proteins in ßIV spectrin mice of both sexes before and after ßIV spectrin re-expression at specifically chosen developmental time points. We show that the timeline of nodal restoration has different outcomes in the PNS and CNS with respect to nodal reassembly and functional restoration. In the PNS, restoration of nodes occurs within 1 month regardless of the time of ßIV spectrin re-expression. In contrast, the CNS nodal reorganization and functional restoration occurs within a critical time window; after that, nodal reorganization diminishes, leading to less efficient motor recovery. We demonstrate that timely restoration of nodes can improve both the functional properties and the ultrastructure of myelinated fibers affected by long-term nodal disorganization. Our studies, which indicate a critical timeline for nodal restoration together with overall motor performance and prolonged life span, further support the idea that nodal restoration is more beneficial if initiated before any axonal damage, which is critically relevant to demyelinating disorders.SIGNIFICANCE STATEMENT Nodes of Ranvier are integral to efficient and rapid signal transmission along myelinated fibers. Various demyelinating disorders are characterized by destabilization of the nodal molecular complex, accompanied by severe reduction in nerve conduction and the onset of motor and sensory dysfunctions. This study is the first to report in vivo reassembly of destabilized nodes with sequential improvement in overall motor performance. Our study reveals that nodal restoration is achievable before any axonal damage, and that long-term nodal destabilization causes irreversible axonal structural changes that prevent functional restoration. Our studies provide significant insights into timely restoration of nodal domains as a potential therapeutic approach in treatment of demyelinating disorders.

Comparison of GCaMP3 and GCaMP6f for studying astrocyte Ca2+ dynamics in the awake mouse brain.

In recent years it has become increasingly clear that astrocytes play a much more active role in neural processes than the traditional view of them as supporting cells suggests. Although not electrically excitable, astrocytes exhibit diverse Ca2+ dynamics across spatial and temporal scales, more or less dependent on the animal's behavioral state. Ca2+ dynamics range from global elevations lasting multiple seconds encompassing the soma up to the finest processes, to short elevations restricted to so-called microdomains within fine processes. Investigations of astrocyte Ca2+ dynamics have particularly benefitted from the development of Genetically-Encoded Calcium Indicators (GECIs). GECI expression can be achieved non-invasively in a cell type-specific manner and it can be genetically targeted to subcellular domains. The GCaMP family, a group of GECIs derived from the green fluorescent protein, has experienced some of the fastest advancements during the past decade. As a consequence we are now facing the challenge of needing to compare published data obtained with different versions of GECIs. With the intention to provide some guidance, here we compared Ca2+ dynamics across scales in awake transgenic mice expressing either the well-established GCaMP3, or the increasingly popular GCaMP6f, specifically in astrocytes. We found that locomotion-induced global Ca2+ elevations in cortical astrocytes displayed only minor kinetic differences and their apparent dynamic ranges for Ca2+ sensing were not different. In contrast, Ca2+ waves in processes and microdomain Ca2+ transients were much more readily detectable with GCaMP6f. Our findings suggest that behavioral state-dependent global astrocyte Ca2+ responses can be studied with either GCaMP3 or GCaMP6f whereas the latter is more appropriate for studies of spatially restricted weak and fast Ca2+ dynamics.

Norepinephrine controls astroglial responsiveness to local circuit activity.

Astrocytes perform crucial supportive functions, including neurotransmitter clearance, ion buffering, and metabolite delivery. They can also influence blood flow and neuronal activity by releasing gliotransmitters in response to intracellular Ca(2+) transients. However, little is known about how astrocytes are engaged during different behaviors in vivo. Here we demonstrate that norepinephrine primes astrocytes to detect changes in cortical network activity. We show in mice that locomotion triggers simultaneous activation of astrocyte networks in multiple brain regions. This global stimulation of astrocytes was inhibited by alpha-adrenoceptor antagonists and abolished by depletion of norepinephrine from the brain. Although astrocytes in visual cortex of awake mice were rarely engaged when neurons were activated by light stimulation alone, pairing norepinephrine release with light stimulation markedly enhanced astrocyte Ca(2+) signaling. Our findings indicate that norepinephrine shifts the gain of astrocyte networks according to behavioral state, enabling astrocytes to respond to local changes in neuronal activity.

Reduction of motion artifacts during in vivo two-photon imaging of brain through heartbeat triggered scanning.

Two-photon imaging of fluorescence in brain enables analysis of the structure and dynamic activity of neurons and glial cells in living animals. However, vital functions such as beating of the heart cause pulsations in brain tissue, leading to image distortion and loss of resolution. We find that synchronizing imaging scans to the cardiac cycle reduces motion artifacts, significantly improving the resolution of cellular structures. By interlacing multiple heartbeat triggered imaging scans, it was possible to image large brain volumes with negligible distortion. This approach can be readily incorporated into conventional microscopes to achieve substantial reductions in motion artifacts during two-photon imaging.

Zones of enhanced glutamate release from climbing fibers in the mammalian cerebellum.

Purkinje cells in the mammalian cerebellum are remarkably homogeneous in shape and orientation, yet they exhibit regional differences in gene expression. Purkinje cells that express high levels of zebrin II (aldolase C) and the glutamate transporter EAAT4 cluster in parasagittal zones that receive input from distinct groups of climbing fibers (CFs); however, the physiological properties of CFs that target these molecularly distinct Purkinje cells have not been determined. Here we report that CFs that innervate Purkinje cells in zebrin II-immunoreactive (Z(+)) zones release more glutamate per action potential than CFs in Z(-) zones. CF terminals in Z(+) zones had larger pools of release-ready vesicles, exhibited enhanced multivesicular release, and produced larger synaptic glutamate transients. As a result, CF-mediated EPSCs in Purkinje cells decayed more slowly in Z(+) zones, which triggered longer-duration complex spikes containing a greater number of spikelets. The differences in the duration of CF EPSCs between Z(+) and Z(-) zones persisted in EAAT4 knock-out mice, indicating that EAAT4 is not required for maintaining this aspect of CF function. These results indicate that the organization of the cerebellum into discrete longitudinal zones is defined not only by molecular phenotype of Purkinje cells within zones, but also by the physiological properties of CFs that project to these distinct regions. The enhanced release of glutamate from CFs in Z(+) zones may alter the threshold for synaptic plasticity and prolong inhibition of cerebellar output neurons in deep cerebellar nuclei.

Candidate amino acids involved in H+ gating of acid-sensing ion channel 1a.

Acid-sensing ion channels are ligand-gated cation channels, gated by extracellular H(+). H(+) is the simplest ligand possible, and whereas for larger ligands that gate ion channels complex binding sites in the three-dimensional structure of the proteins have to be assumed, H(+) could in principle gate a channel by titration of a single amino acid. Experimental evidence suggests a more complex situation, however. For example, it has been shown that extracellular Ca(2+) ions compete with H(+); probably Ca(2+) ions bound to the extracellular loop of ASICs stabilize the closed state of the channel and have to be displaced before the channel can open. In such a scheme, amino acids contributing to Ca(2+) binding would also be candidates contributing to H(+) gating. In this study we systematically screened more than 40 conserved, charged amino acids in the extracellular region of ASIC1a for a possible contribution to H(+) gating. We identified four amino acids where substitution strongly affects H(+) gating: Glu(63), His(72)/His(73), and Asp(78). These amino acids are highly conserved among H(+)-sensitive ASICs and are candidates for the "H(+) sensor" of ASICs.

Isolation of the ocular surface to treat dysfunctional tear syndrome associated with computer use.

Dysfunctional tear syndrome (DTS) associated with computer use is characterized by mild irritation, itching, redness, and intermittent tearing after extended staring. It frequently involves foreign body or sandy sensation, blurring of vision, and fatigue, worsening especially at the end of the day. We undertook a study to determine the effectiveness of periocular isolation using microenvironment glasses (MEGS) alone and in combination with artificial tears in alleviating the symptoms and signs of dry eye related to computer use. At the same time, we evaluated the relative ability of a battery of clinical tests for dry eye to distinguish dry eyes from normal eyes in heavy computer users. Forty adult subjects who used computers 3 hours or more per day were divided into dry eye sufferers and controls based on their scores on the Ocular Surface Disease Index (OSDI). Baseline scores were recorded and ocular surface assessments were made. On four subsequent visits, the subjects played a computer game for 30 minutes in a controlled environment, during which one of four treatment conditions were applied, in random order, to each subject: 1) no treatment, 2) artificial tears, 3) MEGS, and 4) artificial tears combined with MEGS. Immediately after each session, subjects were tested on: a subjective comfort questionnaire, tear breakup time (TBUT), fluorescein staining, lissamine green staining, and conjunctival injection. In this study, a significant correlation was found between cumulative lifetime computer use and ocular surface disorder, as measured by the standardized OSDI index. The experimental and control subjects were significantly different (P<0.05) in the meibomian gland assessment and TBUT; they were consistently different in fluorescein and lissamine green staining, but with P>0.05. Isolation of the ocular surface alone produced significant improvements in comfort scores and TBUT and a consistent trend of improvement in fluorescein staining and lissamine green staining. Isolation plus tears produced a significant improvement in lissamine green staining. The subjective comfort inventory and the TBUT test were most effective in distinguishing between the treatments used. Computer users with ocular surface complaints should have a detailed ocular surface examination and, if symptomatic, they can be effectively treated with isolation of the ocular surface, artificial tears therapy, and effective environmental manipulations.

Synaptic communication between neurons and NG2+ cells.

Chemical synaptic transmission provides the basis for much of the rapid signaling that occurs within neuronal networks. However, recent studies have provided compelling evidence that synapses are not used exclusively for communication between neurons. Physiological and anatomical studies indicate that a distinct class of glia known as NG2(+) cells also forms direct synaptic junctions with both glutamatergic and GABAergic neurons. Glutamatergic signaling can influence intracellular Ca(2+) levels in NG2(+) cells by activating Ca(2+) permeable AMPA receptors, and these inputs can be potentiated through high frequency stimulation. Although the significance of this highly differentiated form of communication remains to be established, these neuro-glia synapses might enable neurons to influence rapidly the behavior of this ubiquitous class of glial progenitors.

Strong modulation by RFamide neuropeptides of the ASIC1b/3 heteromer in competition with extracellular calcium.

Acid-sensing ion channels are excitatory receptors for extracellular H+. Since the extracellular H+ concentration can significantly increase during an inflammation, one of the proposed functions for ASICs is peripheral perception of pain. The ASIC1b and ASIC3 subunits are specifically expressed in sensory ganglia neurons and are candidate sensors of peripheral acidosis. However, the function of these ASIC subunits is limited by their steady-state desensitization during a small but persistent increase of the H+ concentration and by their desensitization after stronger H+ stimuli. Here we show that ASIC1b and ASIC3 form a heteromeric channel that, at steady-state, desensitizes at more acidic values than either homomeric ASIC1b or homomeric ASIC3 alone. Moreover, we show that RFamide neuropeptides, putative modulators of ASIC activity during inflammation, drastically slow down the desensitization of the ASIC1b/3 heteromer with an apparent dissociation constant of approximately 24microM. The apparent affinity for RFamide-induced effects was about 3-fold higher at low extracellular calcium concentrations. Our results suggest that the ASIC1b/3 heteromer is a possible target for RFamide neuropeptides in the peripheral nervous system.

Xenopus TRPN1 (NOMPC) localizes to microtubule-based cilia in epithelial cells, including inner-ear hair cells.

In vertebrates, the senses of hearing and balance depend on hair cells, which transduce sounds with their hair bundles, containing actin-based stereocilia and microtubule-based kinocilia. A longstanding question in auditory science is the identity of the mechanically sensitive transduction channel of hair cells, thought to be localized at the tips of their stereocilia. Experiments in zebrafish implicated the transient receptor potential (TRP) channel NOMPC (drTRPN1) in this role; TRPN1 is absent from the genomes of higher vertebrates, however, and has not been localized in hair cells. Another candidate for the transduction channel, TRPA1, apparently is required for transduction in mammalian and nonmammalian vertebrates. This discrepancy raises the question of the relative contribution of TRPN1 and TRPA1 to transduction in nonmammalian vertebrates. To address this question, we cloned the TRPN1 ortholog from the amphibian Xenopus laevis, generated an antibody against the protein, and determined the protein's cellular and subcellular localization. We found that TRPN1 is prominently located in lateral-line hair cells, auditory hair cells, and ciliated epidermal cells of developing Xenopus embryos. In ciliated epidermal cells TRPN1 staining was enriched at the tips and bases of the cilia. In saccular hair cells, TRPN1 was located prominently in the kinocilial bulb, a component of the mechanosensory hair bundles. Moreover, we observed redistribution of TRPN1 upon treatment of hair cells with calcium chelators, which disrupts the transduction apparatus. This result suggests that although TRPN1 is unlikely to be the transduction channel of stereocilia, it plays an essential role, functionally related to transduction, in the kinocilium.

Identification of the Ca2+ blocking site of acid-sensing ion channel (ASIC) 1: implications for channel gating.

Acid-sensing ion channels ASIC1a and ASIC1b are ligand-gated ion channels that are activated by H+ in the physiological range of pH. The apparent affinity for H+ of ASIC1a and 1b is modulated by extracellular Ca2+ through a competition between Ca2+ and H+. Here we show that, in addition to modulating the apparent H+ affinity, Ca2+ blocks ASIC1a in the open state (IC50 approximately 3.9 mM at pH 5.5), whereas ASIC1b is blocked with reduced affinity (IC50 > 10 mM at pH 4.7). Moreover, we report the identification of the site that mediates this open channel block by Ca2+. ASICs have two transmembrane domains. The second transmembrane domain M2 has been shown to form the ion pore of the related epithelial Na+ channel. Conserved topology and high homology in M2 suggests that M2 forms the ion pore also of ASICs. Combined substitution of an aspartate and a glutamate residue at the beginning of M2 completely abolished block by Ca2+ of ASIC1a, showing that these two amino acids (E425 and D432) are crucial for Ca2+ block. It has previously been suggested that relief of Ca2+ block opens ASIC3 channels. However, substitutions of E425 or D432 individually or in combination did not open channels constitutively and did not abolish gating by H+ and modulation of H+ affinity by Ca2+. These results show that channel block by Ca2+ and H+ gating are not intrinsically linked.

A family of acid-sensing ion channels from the zebrafish: widespread expression in the central nervous system suggests a conserved role in neuronal communication.

Acid-sensing ion channels (ASICs) are excitatory receptors for extracellular H(+). Proposed functions include synaptic transmission, peripheral perception of pain, and mechanosensation. Despite the physiological importance of these functions, the precise role of ASICs has not yet been established. In order to increase our understanding of the physiological role and basic structure-function relationships of ASICs, we report here the cloning of six new ASICs from the zebrafish (zASICs). zASICs possess the basic functional properties of mammalian ASICs: activation by extracellular H(+), Na(+) selectivity, and block by micromolar concentrations of amiloride. The zasic genes are broadly expressed in the central nervous system, whereas expression in the peripheral nervous system is scarce. This pattern suggests a predominant role for zASICs in neuronal communication. Our results suggest a conserved function for receptors of extracellular H(+) in the central nervous system of vertebrates.

Alternative splicing and interaction with di- and polyvalent cations control the dynamic range of acid-sensing ion channel 1 (ASIC1).

Homomeric acid-sensing ion channel 1 (ASIC1) can be activated by extracellular H(+) in the physiological pH range and may, therefore, contribute to neurotransmission and peripheral pain perception. ASIC1a and ASIC1b are alternative splice products of the ASIC1 gene. Here we show that both splice variants show steady-state inactivation when exposed to slightly decreased pH, limiting their operational range. Compared with ASIC1a, steady-state inactivation and pH activation of ASIC1b are shifted to more acidic values by 0.25 and 0.7 pH units, respectively, extending the dynamic range of ASIC1. Shifts of inactivation and activation are intimately linked; only two amino acids in the ectodomain, which are exchanged by alternative splicing, control both properties. Moreover, we show that extracellular, divalent cations like Ca(2+) and Mg(2+) as well as the polyvalent cation spermine shift the steady-state inactivation of ASIC1a and ASIC1b to more acidic values. This leads to a potentiation of the channel response and is due to a stabilization of the resting state. Our results indicate that ASIC1b is an effective sensor of transient H(+) signals during slight acidosis and that, in addition to alternative splicing, interaction with di- and polyvalent cations extends the dynamic range of ASIC H(+) sensors.

The P2X(7) receptor from Xenopus laevis: formation of a large pore in Xenopus oocytes.

The purinergic P2X(7) receptor is an ATP-receptor channel predominantly expressed in immune cells. P2X(7) has been cloned from human, rat and mouse. Here we report cloning of the Xenopus laevis P2X(7) receptor (xP2X(7)). xP2X(7) is only about 50% identical to the mammalian homologues, shows a broad tissue expression pattern, and has the electrophysiological characteristics typical of a P2X(7) receptor: low agonist affinity (EC(50) about 2.6 mM) and a non-desensitizing current. Moreover, expression of xP2X(7) in Xenopus oocytes is sufficient to induce the formation of a large pore, which is permeable to large cations such as NMDG(+). Identification of a non-mammalian P2X(7) receptor may help to identify functionally important parts of the protein.