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Ion channels in glial cells. Brain Res Brain Res Rev
Abstract
Functional and molecular analysis of glial voltage- and ligand-gated ion channels underwent tremendous boost over the last 15 years. The traditional image of the glial cell as a passive, structural element of the nervous system was transformed into the concept of a plastic cell, capable of expressing a large variety of ion channels and neurotransmitter receptors. These molecules might enable glial cells to sense neuronal activity and to integrate it within glial networks, e.g., by means of spreading calcium waves. In this review we shall give a comprehensive summary of the main functional properties of ion channels and ionotropic receptors expressed by macroglial cells, i.e., by astrocytes, oligodendrocytes and Schwann cells. In particular we will discuss in detail glial sodium, potassium and anion channels, as well as glutamate, GABA and ATP activated ionotropic receptors. A majority of available data was obtained from primary cell culture, these results have been compared with corresponding studies that used acute tissue slices or freshly isolated cells. In view of these data, an active glial participation in information processing seems increasingly likely and a physiological role for some of the glial channels and receptors is gradually emerging.
... So far, there has been no model addressing the unique features of astrocyte. Based on our previous model [4] and the literature reported data [5][6][7][8], we have implemented the relatively comprehensive simulations on Ca 2+ oscillations in astrocytes mediated by A Methods on the simulation In the model astrocyte (see Figure 1), different types of voltage-gated calcium channels (VGCCs) [5] form the Ca 2+ influx J VGCC from the extracellular space (ECS) to the intracellular space (ICS). The electrophysiological properties of these VGCCs were described by the Hodgkin-Huxley (HH) equations. ...
... So far, there has been no model addressing the unique features of astrocyte. Based on our previous model [4] and the literature reported data [5][6][7][8], we have implemented the relatively comprehensive simulations on Ca 2+ oscillations in astrocytes mediated by A Methods on the simulation In the model astrocyte (see Figure 1), different types of voltage-gated calcium channels (VGCCs) [5] form the Ca 2+ influx J VGCC from the extracellular space (ECS) to the intracellular space (ICS). The electrophysiological properties of these VGCCs were described by the Hodgkin-Huxley (HH) equations. ...
... A can activate the L-type channels and increase the concentration of intracellular Ca 2+ . In turn, the block of L-type VGCC can protect cells from the detrimental effects of AAstrocytes exhibit ionic excitability [5] mediated by Aβ. This work indicates that preventing the effects from Acan prevent the development of Alzheimer's disease. ...
Disruptions of astrocyte Ca ²⁺ signaling is important in Alzheimer’s disease (AD) with the unclear mechanism of amyloid beta peptide (Aβ). We have modified our previous computational model of spontaneous Ca ²⁺ oscillations in astrocytes to investigate the effects of Aβ on intracellular Ca ²⁺ dynamics. The simulation results have shown consistence with the previous experiments. Aβ can increase the resting concentration of intracellular Ca ²⁺ and change the regime of Ca ²⁺ oscillations by activating L-type voltage-gated calcium channels and the metabolic glutamate receptors, or by increasing ryanodine receptors sensitivity and Ca ²⁺ leakage, respectively. This work have provided a toolkit to study the influence of Aβ on intracellular Ca ²⁺ dynamics in AD. It is helpful for understanding the toxic role of Aβ during the progression of AD.
Statement of Significance
Alzheimer’s disease (AD) is the most common neurodegenerative disease with the unclear mechanism of amyloid beta peptide (Aβ). This work have implemented a computational model to address the Ca ²⁺ dynamics of astrocyte mediated by Aβ with the four different pathways: voltage-gated calcium channels, metabotropic glutamate receptors 5, ryanodine receptor channels and membrane leak. The Ca ²⁺ oscillations and bifurcation diagram indicate that astrocytes exhibit ionic excitability mediated by Aβ and become the potential targets of Aβ neurotoxicity. We expect this shared computational model would advance the understanding of AD.
... Astrocytes support grey matter micro-architecture and compose almost half of the CNS's volume. They are endowed with ion channels and transport pathways and actively participate in the regulation of the neuronal environment and activity [48]. They assist in several brain functions, from providing trophic support for neurons to controlling extracellular ion and neurotransmitter concentrations and from assisting synapse formation, function, and pruning to preserving the blood-brain barrier [49]. ...
... Astrocytes regulate their own activities via Ca 2+ and Na + signals and affect neuronal excitability [48]. Glial cells communicate with their surroundings by increasing intracellular Ca 2+ concentrations and by propagating the signal as spontaneously occurring Ca 2+ waves or in response to a variety of stimuli. ...
Ever since its presence was reported in the brain, the nature and role of hydrogen sulfide (H2S) in the Central Nervous System (CNS) have changed. Consequently, H2S has been elected as the third gas transmitter, along with carbon monoxide and nitric oxide, and a number of studies have focused on its neuromodulatory and protectant functions in physiological conditions. The research on H2S has highlighted its many facets in the periphery and in the CNS, and its role as a double-faced compound, switching from protective to toxic depending on its concentration. In this review, we will focus on the bell-shaped nature of H2S as an angiogenic factor and as a molecule released by glial cells (mainly astrocytes) and non-neuronal cells acting on the surrounding environment (paracrine) or on the releasing cells themselves (autocrine). Finally, we will discuss its role in Amyotrophic Lateral Sclerosis, a paradigm of a neurodegenerative disease.
... [24,25] Glutamate Glutamate regulates proliferation, migration, and differentiation of OPCs and remyelination after damage. [26,27] AMPA/KAR c-fos, c-jun, and jun-B modulation. [28] AMPA/KAR OPC proliferation and differentiation blocking. ...
... Rat and mouse sciatic nerve studies have shown that dehydrogenase glutamic acid decarboxylase (GAD67) and glutamine synthetase are expressed in in vitro SCs and in nerve slices [34,53,54]. In vitro studies have also shown that, through the expression of excitatory acid transporter-1, SCs take up glutamate from the extracellular environment, and after metabolization they synthesise new neuromediators for release as autocrine and paracrine signalling on neighbouring SCs [27,55]. ...
The cross talk between neurons and glial cells during development, adulthood, and disease, has been extensively documented. Among the molecules mediating these interactions, neurotransmitters play a relevant role both in myelinating and non-myelinating glial cells, thus resulting as additional candidates regulating the development and physiology of the glial cells. In this review, we summarise the contribution of the main neurotransmitter receptors in the regulation of the morphogenetic events of glial cells, with particular attention paid to the role of acetylcholine receptors in Schwann cell physiology. In particular, the M2 muscarinic receptor influences Schwann cell phenotype and the α7 nicotinic receptor is emerging as influential in the modulation of peripheral nerve regeneration and inflammation. This new evidence significantly improves our knowledge of Schwann cell development and function and may contribute to identifying interesting new targets to support the activity of these cells in pathological conditions.
... GBM originates from the brain's supporting cells, and these cells express a myriad of ion channels, including sodium, potassium, and anion channels [6]. Genomic analysis of mutations present in GBM has shown the presence of mutations in the genes encoding these ion channels in 90% of the glioblastoma samples examined [7]. ...
... Measures of association for patients taking digoxin and glioblastoma developmentTables 5,6 show the baseline characteristics and measures of association for our patients taking amiodarone. A total of 543,288 patients were taking amiodarone; of this number, 51 patients (0.009%) developed a glioblastoma. ...
Background
Ion channels play a role in the development and progression of glioblastoma multiforme. This study investigates the association between the risk of developing glioblastoma multiforme in patients taking these medications.
Methods
A retrospective propensity score-matched analysis was performed using the TriNetX multinational electronic health record database for patients taking verapamil, digoxin, amiodarone, or diltiazem versus those not taking these medications. The outcome of interest was the incidence of glioblastoma multiforme.
Results
Verapamil users had an OR of 0.494 (p < 0.0001) of developing glioblastoma versus verapamil non-users. Patients on digoxin had an OR of 0.793 (p = 0.2393), patients on amiodarone had an OR of 0.600 (p = 0.0035), patients on diltiazem had an OR of 0.584 (p < 0.0001), and patients on verapamil, digoxin, amiodarone, or diltiazem had an OR of 0.641 (p < 0.0001) of developing glioblastoma versus patients not taking these medications.
Conclusion
In patients taking the ion channel blockers diltiazem, amiodarone, or verapamil, the odds of developing glioblastoma multiforme were lower than in patients not taking these medications.
... Microscopy in combination with electrophysiology forms a powerful system for studying physiology of live cells (Scanziani & Häusser, 2009) and, crucially, with a gentle touch, such that cellular processes continue unhindered. Especially in neuroscience the application of a microscopy/electrophysiology system is indispensable for defining the electrical activity of single cells and neuronal networks in both, in vitro and in vivo conditions (Margrie et al., 2002;Russo & Taverna, 2014;Verkhratsky & Steinhäuser, 2000;Walz & Bekar, 2001) in order to generate a comprehensive understanding of the nervous system function in health and in disease. In the patch-clamp electrophysiological technique, a pipette brought into the firm contact with the plasma membrane of the single cell forms the giga-ohm seal and allows recording of ion currents through the cell membrane (Hamill et al., 1981;Sakmann & Neher, 1984). ...
... Furthermore, we found no difference in the membrane current responses between control and SOD1 G93A microglia. Electrophysiological studies of glial cells in culture are beneficial as they allow assessment of cellular properties that are cell autonomous and experimenter can isolate one factor at a time to study its cause-and-effect relationship (Verkhratsky, 2006;Verkhratsky & Steinhäuser, 2000;Walz & Bekar, 2001). Custom approach therefore is valuable for initial and effective inspecting of the glial membrane properties. ...
We describe an approach for studying the physiology of single live cells using the conceptionally novel upright microscope/patch‐clamp configuration. Electrophysiology experiments typically require a microscope with the fixed stage position and the motion control of the microscope objective. Here, we demonstrate that a microscope with a z ‐axis movable stage and a fixed objective can also be efficiently used in combination with the patch‐clamp technique. We define a set of underlying principles governing the operation of this microscope/patch‐clamp configuration and demonstrate its performance in practice using cultured astrocytes, microglia, and oligodendrocytes. Experimental results show that our custom configuration provides stable recordings, has a high success rate of the whole‐cell patch‐clamp trials, can be effectively applied to study cellular physiology of glial cells, and provides comparable performance and usability to the commercially available systems. Our system can be easily replicated or adapted to suit the needs of the research groups and can be cost‐effective in reducing the investments in purchasing additional equipment. We provide step‐by‐step instructions on implementing an upright microscope with z ‐axis movable stage as a routine workhorse for patch‐clamping.
Research Highlights
Approach for efficient patch‐clamping using an upright microscope with a z ‐axis movable stage.
Routine study of live cell physiology.
Custom microscope/patch‐clamp configuration that is cost‐effective and overcomes equipment limitations.
... At the molecular level, this powerful tool is driven by an ensemble current emergent from tiny single-channel ionic currents (single channel conductance is merely $ 1 pS (Lin et al., 2009)), and can introduce massive perturbations to even large nervous systems manifesting as large changes in spikes and LFPs (Kim et al., 2017;Oliva et al., 2018;Fernandez-Ruiz et al., 2019;Jun and Cardin, 2020;McKenzie et al., 2021). The power of such ion channels in altering network physiology begs attention to the study of endogenous ion channels that express on neuronal and glial (Verkhratsky and Steinhauser, 2000) membranes in shaping LFPs, apart from their established roles in altering neural activity and behavior. ...
... Furthermore, astrocytes themselves express several voltage-gated ion channels and receptors, and can release calcium and neurotransmitter molecules (Verkhratsky and Steinhauser, 2000;Halassa and Haydon, 2010;Araque et al., 2014;Bazargani and Attwell, 2016;Ashhad and Narayanan, 2019). Through this gliotransmission, it has also been observed that astrocytes can induce large, long-lasting and slow excitatory potentials (SEPs) or dendritic plateau potentials in CA1 pyramidal neurons (Ashhad and Narayanan, 2016). ...
Neurons and glial cells are endowed with membranes that express a rich repertoire of ion channels, transporters, and receptors. The constant flux of ions across the neuronal and glial membranes results in voltage fluctuations that can be recorded from the extracellular matrix. The high frequency components of this voltage signal contain information about the spiking activity, reflecting the output from the neurons surrounding the recording location. The low frequency components of the signal, referred to as the local field potential (LFP), have been traditionally thought to provide information about the synaptic inputs that impinge on the large dendritic trees of various neurons. In this review, we discuss recent computational and experimental studies pointing to a critical role of several active dendritic mechanisms that can influence the genesis and the location-dependent spectro-temporal dynamics of LFPs, spanning different brain regions. We strongly emphasize the need to account for the several fast and slow dendritic events and associated active mechanisms –– including gradients in their expression profiles, inter- and intra-cellular spatio-temporal interactions spanning neurons and glia, heterogeneities and degeneracy across scales, neuromodulatory influences, and activity-dependent plasticity — towards gaining important insights about the origins of LFP under different behavioral states in health and disease. We provide simple but essential guidelines on how to model LFPs taking into account these dendritic mechanisms, with detailed methodology on how to account for various heterogeneities and electrophysiological properties of neurons and synapses while studying LFPs.
... Astroglial cells even in the normal brain contain a comparatively large cellular iron pool that may play a role in neurotransmitter homeostasis [90]. Astrocytic processes form parts of the blood brain barrier [4,25] and regulate the uptake and distribution of metal ions within the brain [24,41,67,89,90], which makes them the first parenchymal cell type that will get in contact with iron after having crossed the blood brain barrier [41]. ...
... Astroglial cells even in the normal brain contain a comparatively large cellular iron pool that may play a role in neurotransmitter homeostasis [90]. Astrocytic processes form parts of the blood brain barrier [4,25] and regulate the uptake and distribution of metal ions within the brain [24,41,67,89,90], which makes them the first parenchymal cell type that will get in contact with iron after having crossed the blood brain barrier [41]. Due to their large intracellular iron pool together with their close contacts to various other brain cell types [3,35,39,67,89], they play a key role in processes of iron distribution within the neuropil [68]. ...
Iron is essential for neurons and glial cells, playing key roles in neurotransmitter synthesis, energy production and myelination. In contrast, high concentrations of free iron can be detrimental and contribute to neurodegeneration, through promotion of oxidative stress. Particularly in Parkinson’s disease (PD) changes in iron concentrations in the substantia nigra (SN) was suggested to play a key role in degeneration of dopaminergic neurons in nigrosome 1. However, the cellular iron pathways and the mechanisms of the pathogenic role of iron in PD are not well understood, mainly due to the lack of quantitative analytical techniques for iron quantification with subcellular resolution. Here, we quantified cellular iron concentrations and subcellular iron distributions in dopaminergic neurons and different types of glial cells in the SN both in brains of PD patients and in non-neurodegenerative control brains (Co). To this end, we combined spatially resolved quantitative element mapping using m icro p article i nduced X -ray e mission (µPIXE) with nickel-enhanced immunocytochemical detection of cell type-specific antigens allowing to allocate element-related signals to specific cell types. Distinct patterns of iron accumulation were observed across different cell populations. In the control (Co) SNc, oligodendroglial and astroglial cells hold the highest cellular iron concentration whereas in PD, the iron concentration was increased in most cell types in the substantia nigra except for astroglial cells and ferritin-positive oligodendroglial cells. While iron levels in astroglial cells remain unchanged, ferritin in oligodendroglial cells seems to be depleted by almost half in PD. The highest cellular iron levels in neurons were located in the cytoplasm, which might increase the source of non-chelated Fe ³⁺ , implicating a critical increase in the labile iron pool. Indeed, neuromelanin is characterised by a significantly higher loading of iron including most probable the occupancy of low-affinity iron binding sites. Quantitative trace element analysis is essential to characterise iron in oxidative processes in PD. The quantification of iron provides deeper insights into changes of cellular iron levels in PD and may contribute to the research in iron-chelating disease-modifying drugs.
... In particular, GABA A (i.e., α2, α3, β1, β2 and β3 subunits) and GABA B (i.e., GABA B1 and GABA B2 ) receptors have been identified in peripheral nerves and Schwann cells [15,20,55]. Furthermore, the rat sural nerve expresses AMPA subunits, NMDA receptor 1 subunits, GluR 5, 6 and 7 kainate subunits [56,57], and Schwann cells of mammalian peripheral vestibular system express GluR 2, 3 and 4 [57,58]. Finally, PROG and its metabolites may also bind the membrane progesterone receptors (mPRs) [59,60]. ...
... In particular, GABA A (i.e., α2, α3, β1, β2 and β3 subunits) and GABA B (i.e., GABA B1 and GABA B2 ) receptors have been identified in peripheral nerves and Schwann cells [15,20,55]. Furthermore, the rat sural nerve expresses AMPA subunits, NMDA receptor 1 subunits, GluR 5, 6 and 7 kainate subunits [56,57], and Schwann cells of mammalian peripheral vestibular system express GluR 2, 3 and 4 [57,58]. Finally, PROG and its metabolites may also bind the membrane progesterone receptors (mPRs) [59,60]. ...
Peripheral neuropathy (PN) refers to many conditions involving damage to the peripheral nervous system (PNS). Usually, PN causes weakness, numbness and pain and is the result of traumatic injuries, infections, metabolic problems, inherited causes, or exposure to chemicals. Despite the high prevalence of PN, available treatments are still unsatisfactory. Neuroactive steroids (i.e., steroid hormones synthesized by peripheral glands as well as steroids directly synthesized in the nervous system) represent important physiological regulators of PNS functionality. Data obtained so far and here discussed, indeed show that in several experimental models of PN the levels of neuroactive steroids are affected by the pathology and that treatment with these molecules is able to exert protective effects on several PN features, including neuropathic pain. Of note, the observations that neuroactive steroid levels are sexually dimorphic not only in physiological status but also in PN, associated with the finding that PN show sex dimorphic manifestations, may suggest the possibility of a sex specific therapy based on neuroactive steroids.
... of membrane potentials around astrocyte resting membrane potentials, i.e., from Ϫ108 mV to Ϫ66 mV. Within this voltage range, activation of voltage-gated outward and voltageindependent inwardly rectifying K ϩ channels should have marginal contribution to the observed passive conductance (Verkhratsky and Steinhäuser, 2000;Zhou and Kimelberg, 2000;Olsen and Sontheimer, 2005;Xie et al., 2007); thus, the passive conductance could be more accurately defined as a resting K ϩ conductance. ...
... The presence of other rectifying K ϩ channels by astrocytes has also been supported by several immunocytochemical studies and cannot be excluded by whole-cell current measurements for the reason just mentioned (Verkhratsky and Steinhäuser, 2000;Olsen and Sontheimer, 2005). Also, outward rectifying K ϩ current components were also seen in 30% excised patch recordings from astrocytic somata, where voltage clamping over a wide range of membrane potential can be achieved . ...
Expression of a linear current-voltage (I-V) relationship (passive) K ϩ membrane conductance is a hallmark of mature hippocampal astrocytes. However, the molecular identifications of the K ϩ channels underlying this passive conductance remain unknown. We provide the following evidence supporting significant contribution of the two-pore domain K ϩ channel (K 2P) isoforms, TWIK-1 and TREK-1, to this conductance. First, both passive astrocytes and the cloned rat TWIK-1 and TREK-1 channels expressed in CHO cells conduct significant amounts of Cs ϩ currents, but vary in their relative P Cs /P K permeability, 0.43, 0.10, and 0.05, respectively. Second, quinine, which potently inhibited TWIK-1 (IC 50 ϭ 85 M) and TREK-1 (IC 50 ϭ 41 M) currents, also inhibited astrocytic passive conductance by 58% at a concentration of 200 M. Third, a moderate sensitivity of passive conductance to low extracellular pH (6.0) supports a combined expression of acid-insensitive TREK-1, and to a lesser extent, acid-sensitive TWIK-1. Fourth, the astrocyte passive conductance showed low sensitivity to extracellular Ba 2ϩ , and extracellular Ba 2ϩ blocked TWIK-1 channels at an IC 50 of 960 M and had no effect on TREK-1 channels. Finally, an immunocytochemical study showed colocalization of TWIK-1 and TREK-1 proteins with the astrocytic markers GLAST and GFAP in rat hippocampal stratum radiatum. In contrast, another K 2P isoform TASK-1 was mainly colocalized with the neuronal marker NeuN in hippocampal pyramidal neurons and was expressed at a much lower level in astrocytes. These results support TWIK-1 and TREK-1 as being the major components of the long-sought K ϩ channels underlying the passive conductance of mature hippocampal astrocytes.
... In the case of glia, it is possible that even biphasic charge injection can cause charge imbalances along the membranes of cells. This in turn may trigger activation of glial ion channels, voltage-gated or otherwise, in an effort to restore perturbed membrane potentials back to their resting values (Nowak et al., 1987;Verkhratsky and Steinhäuser, 2000). Doing so may also cause cells to become damaged through changes in tonicity (Turner and Sontheimer, 2014). ...
Neural interfacing devices interact with the central nervous system to alleviate functional deficits arising from disease or injury. This often entails the use of invasive microelectrode implants that elicit inflammatory responses from glial cells and leads to loss of device function. Previous work focused on improving implant biocompatibility by modifying electrode composition; here, we investigated the direct effects of electrical stimulation on glial cells at the electrode interface. A high-throughput in vitro system that assesses primary glial cell response to biphasic stimulation waveforms at 0 mA, 0.15 mA, and 1.5 mA was developed and optimized. Primary mixed glial cell cultures were generated from heterozygous CX3CR-1+/EGFP mice, electrically stimulated for 4 h/day over 3 days using 75 μm platinum-iridium microelectrodes, and biomarker immunofluorescence was measured. Electrodes were then imaged on a scanning electron microscope to assess sustained electrode damage. Fluorescence and electron microscopy analyses suggest varying degrees of localized responses for each biomarker assayed (Hoescht, EGFP, GFAP, and IL-1β), a result that expands on comparable in vivo models. This system allows for the comparison of a breadth of electrical stimulation parameters, and opens another avenue through which neural interfacing device developers can improve biocompatibility and longevity of electrodes in tissue.
... With processes that extend into the synaptic space, astrocytes are in close proximity to neuronal synapses [19] making them ideally located to respond to environmental cues from neighboring neurons [20]. In fact, they express most neurotransmitter receptors [21][22][23][24] and are capable of responding to synaptic neurotransmitter release [25][26][27][28], including glutamate [29,30], ACh [25,31,32], ...
Astrocytes release numerous factors known to contribute to the process of synaptogenesis, yet knowledge about the signals that control their release is limited. We hypothesized that neuron-derived signals stimulate astrocytes, which respond to neurons through the modulation of astrocyte-released synaptogenic factors. Here we investigate the effect of cholinergic stimulation of astrocytes on synaptogenesis in co-cultured neurons. Using a culture system where primary rat astrocytes and primary rat neurons are first grown separately allowed us to independently manipulate astrocyte cholinergic signaling. Subsequent co-culture of pre-stimulated astrocytes with naïve neurons enabled us to assess how prior stimulation of astrocyte acetylcholine receptors uniquely modulates neuronal synapse formation. Pre-treatment of astrocytes with the acetylcholine receptor agonist carbachol increased the expression of synaptic proteins, the number of pre- and postsynaptic puncta, and the number of functional synapses in hippocampal neurons after 24 h in co-culture. Astrocyte secretion of the synaptogenic protein thrombospondin-1 increased after cholinergic stimulation and inhibition of the receptor for thrombospondins prevented the increase in neuronal synaptic structures. Thus, we identified a novel mechanism of neuron-astrocyte-neuron communication, where neuronal release of acetylcholine stimulates astrocytes to release synaptogenic proteins leading to increased synaptogenesis in neurons. This study provides new insights into the role of neurotransmitter receptors in developing astrocytes and into our understanding of the modulation of astrocyte-induced synaptogenesis.
... Computational studies suggest that any elevations in astrocytic glutamate concentrations can retain the glutamate in the synaptic cleft for longer periods and thus lead to an increased magnitude of slow inward currents (SICs), potentially resulting in hyperexcitability (Li et al., 2016a;Flanagan et al., 2018). Therefore, the activity of glutamate transporters in astrocytes is strictly regulated by transmembrane Na + concentrations, that indirectly contribute to shaping synaptic transmission (Verkhratsky and Steinhäuser, 2000;Lalo et al., 2011). Particularly, Bergmann glial (BG) GLAST is indispensable for the excitatory synaptic wiring and wrapping of Purkinje cells in the cerebellar cortex (Miyazaki et al., 2017) and its reduced expression causes increased Purkinje cell firing, hyperactivity, and subsequent loss of Purkinje cells contributing to myotonic dystrophy and spinocerebellar ataxia type 1 (SCA1) (Cvetanovic, 2015;Sicot et al., 2017). ...
Neuronal signalling is a key element in neuronal communication and is essential for the proper functioning of the CNS. Astrocytes, the most prominent glia in the brain play a key role in modulating neuronal signalling at the molecular, synaptic, cellular, and network levels. Over the past few decades, our knowledge about astrocytes and their functioning has evolved from considering them as merely a brain glue that provides structural support to neurons, to key communication elements. Astrocytes can regulate the activity of neurons by controlling the concentrations of ions and neurotransmitters in the extracellular milieu, as well as releasing chemicals and gliotransmitters that modulate neuronal activity. The aim of this review is to summarise the main processes through which astrocytes are modulating brain function. We will systematically distinguish between direct and indirect pathways in which astrocytes affect neuronal signalling at all levels. Lastly, we will summarize pathological conditions that arise once these signalling pathways are impaired focusing on neurodegeneration.
... With processes that extend into the synaptic space, astrocytes are in close proximity to neuronal synapses [19] making them ideally located to respond to environmental cues from neighboring neurons [20]. In fact, they express most neurotransmitter receptors [21][22][23][24] and are capable of responding to synaptic neurotransmitter release [25][26][27][28], including glutamate [29,30], ACh [25,31,32], γ-aminobutyric acid (GABA) [33], adenosine triphosphate (ATP) [34], and endocannabinoids [35][36][37]. ...
Astrocytes release numerous factors known to contribute to the process of synaptogenesis, yet knowledge about the signals that control their release is limited. We hypothesized that neuron-derived signals stimulate astrocytes, which respond by signaling back to neurons through the modulation of astrocyte-released synaptogenic factors. Here we investigate the effect of cholinergic stimulation of astrocytes on synaptogenesis in co-cultured neurons. Using a culture system where primary rat astrocytes and primary rat neurons are first grown separately allowed us to independently manipulate astrocyte cholinergic signaling. Subsequent co-culture of pre-stimulated astrocytes with naïve neurons enabled us to assess how prior stimulation of astrocyte acetylcholine receptors uniquely modulates neuronal synapse formation. Pre-treatment of astrocytes with the acetylcholine receptor agonist carbachol increased the expression of synaptic proteins, the number of pre- and postsynaptic puncta, and the number of functional synapses in hippocampal neurons after 24 hours in co-culture. Astrocyte secretion of the synaptogenic protein thrombospondin-1 increased after cholinergic stimulation and the inhibition of the target receptor for thrombospondins prevented the observed increase in neuronal synaptic structures. Thus, we identified a novel mechanism of neuron-astrocyte-neuron communication, i.e. , neuronal release of acetylcholine stimulates astrocytes to release synaptogenic proteins leading to increased synaptogenesis in neurons. This study provides new insights into the role of neurotransmitter receptors in developing astrocytes and into our understanding of the modulation of astrocyte-induced synaptogenesis.
... In astrocytes, the probability of Kir4.1 opening at V rest is high. This contributes to their negative resting membrane potential near the reversal K + potential (Verkhratsky & Steinhäuser, 2000) and to a large portion of K + permeability, which, in turn, is required for extracellular K + clearance. These findings are reminiscent of previous reports in retinal Muller cells, spinal oligodendrocytes, and complex glia in CA1 stratum radiatum, in which a lack of Kir4.1 caused full loss of inward currents and depolarization of the V rest (Djukic et al., 2007;Kofuji et al., 2000;Neusch et al., 2001). ...
Neuron‐restrictive silencer factor/repressor element 1 (RE1)‐silencing transcription factor (NRSF/REST) is a transcriptional repressor of a large cluster of neural genes containing RE1 motifs in their promoter region. NRSF/REST is ubiquitously expressed in non‐neuronal cells, including astrocytes, while it is down‐regulated during neuronal differentiation. While neuronal NRSF/REST homeostatically regulates intrinsic excitability and synaptic transmission, the role of the high NRSF/REST expression levels in the homeostatic functions of astrocytes is poorly understood. Here, we investigated the functional consequences of NRSF/REST deletion in primary cortical astrocytes derived from NRSF/REST conditional knockout mice (KO). We found that NRSF/REST KO astrocyte displayed a markedly reduced activity of inward rectifying K⁺ channels subtype 4.1 (Kir4.1) underlying spatial K⁺ buffering that was associated with a decreased expression and activity of the glutamate transporter‐1 (GLT‐1) responsible for glutamate uptake by astrocytes. The effects of the impaired astrocyte homeostatic functions on neuronal activity were investigated by co‐culturing wild‐type hippocampal neurons with NRSF/REST KO astrocytes. Interestingly, neurons experienced increased neuronal excitability at high firing rates associated with decrease after hyperpolarization and increased amplitude of excitatory postsynaptic currents. The data indicate that astrocytic NRSF/REST directly participates in neural circuit homeostasis by regulating intrinsic excitability and excitatory transmission and that dysfunctions of NRSF/REST expression in astrocytes may contribute to the pathogenesis of neurological disorders. image
... Channels involved in the transfer of sodium (Na +) , K + , and Ca 2+ ions are one of the pathways frequently damaged in GBM, because ion channels are expressed in glial cells in various ways [52,53]. Ion channels can favor GBM invasiveness, by altering ion and water transport via the cell membrane, thus, resting membrane potential, and facilitating cell shape and volume' changes [54]. ...
Simple Summary
This paper summarizes the crosstalk between tumor/non-tumor cells and other elements of the glioblastoma (GB) microenvironment. In tumor pathology, glial cells result in the highest number of cancers, and GB is considered the most lethal tumor of the central nervous system (CNS). The tumor microenvironment (TME) is a complex peritumoral hallo composed of tumor cells and several non-tumor cells (e.g., nervous cells, stem cells, fibroblasts, vascular and immune cells), which might be a key factor for the ineffective treatment since the microenvironment modulates the biologic status of the tumor with the increase in its evasion capacity. A deeper understanding of cell–cell interactions in the TME and with the tumor cells could be the basis for a more efficient therapy.
Abstract
The central nervous system (CNS) represents a complex network of different cells, such as neurons, glial cells, and blood vessels. In tumor pathology, glial cells result in the highest number of cancers, and glioblastoma (GB) is considered the most lethal tumor in this region. The development of GB leads to the infiltration of healthy tissue through the interaction between all the elements of the brain network. This results in a GB microenvironment, a complex peritumoral hallo composed of tumor cells and several non-tumor cells (e.g., nervous cells, stem cells, fibroblasts, vascular and immune cells), which might be the principal factor for the ineffective treatment due to the fact that the microenvironment modulates the biologic status of the tumor with the increase in its evasion capacity. Crosstalk between glioma cells and the brain microenvironment finally inhibits the beneficial action of molecular pathways, favoring the development and invasion of the tumor and its increasing resistance to treatment. A deeper understanding of cell–cell interactions in the tumor microenvironment (TME) and with the tumor cells could be the basis for a more efficient therapy.
... -Canaux ioniques Les canaux ioniques présents à la membrane astrocytaire peuvent être perméables aux ions K + , Na + et Ca 2+ (Alexej Verkhratsky & Steinhäuser, 2000). Le plus abondant des canaux ioniques astrocytaires est le canal potassique K ir 4.1, particulièrement exprimé dans les PAPs et dans les pieds astrocytaires (Olsen, 2012). ...
Les astrocytes sont les cellules gliales les plus abondantes du système nerveux central et sont impliquées dans la plupart des fonctions physiologiques. Grâce à leur morphologie complexe et plastique, ils contactent à la fois des vaisseaux sanguins, des synapses et d’autres astrocytes. Les astrocytes expriment un fort taux de connexines (Cx), protéines formant des jonctions communicantes. Il a été montré au sein de l’équipe que les changements de morphologie et de couverture synaptique des astrocytes, sous-tendus par les modulations d’expression de la Cx30, altéraient la transmission synaptique excitatrice de l’hippocampe. Par ailleurs, il a été montré dans le noyau accumbens (NAc), zone du striatum ventral impliquée dans le circuit de la récompense, qu’un défaut de transmission synaptique participait à un comportement de recherche de drogue, dans le développement d’une addiction à la cocaïne. Ainsi, au cours de mes travaux de thèse, je me suis intéressée à la modulation de la morphologie astrocytaire par la Cx30 dans le NAc, et son implication dans la neurotransmission et les comportements induits par la cocaïne. Ces travaux ont permis de mettre en avant un nouveau déterminant de la morphologie astrocytaire du NAc, la Cx30, son rôle dans la sensibilisation à la cocaïne et son intérêt comme potentielle cible thérapeutique. Dans un second temps, je me suis intéressée au rôle de la Cx30 dans diverses conditions pathologiques, ainsi qu’au rôle du calcium dans la morphologie astrocytaire en physiologie. J’ai pu identifier de nouvelles voies de régulation de la morphologie astrocytaire, en conditions physio- et pathologiques.
... In addition to inward rectifying channels, astrocytes express voltage-dependent K + channels, which include K v 1.5 (KCNA5), K v 1.4 (KCNA4) and K v 11.1/ERG1 (Bordey and Sontheimer 2000;Verkhratsky and Steinhauser 2000;Edwards et al. 2002) and K v 1, K v 3 and K v 4 mediating fast A-type K + currents (Bekar et al. 2005). Finally, cortical astrocytes express SK (small conductance K Ca 2.3/KCNN3) and IK (intermediate conductance K Ca 3.1/KCNN4) Ca 2+ -dependent K + channels in their somata (Armstrong et al. 2005;Longden et al. 2011) and large-conductance (225 pS) BK (big conductance) channels in their endfeet (Filosa et al. 2006). ...
Astroglia are a diverse group of cells in the central nervous system. They are of the ectodermal, neuroepithelial origin and vary in morphology and function, yet, they can be collectively defined as cells having principle function to maintain homeostasis of the central nervous system at all levels of organisation, including homeostasis of ions, pH and neurotransmitters; supplying neurones with metabolic substrates; supporting oligodendrocytes and axons; regulating synaptogenesis, neurogenesis, and formation and maintenance of the blood-brain barrier; contributing to operation of the glymphatic system; and regulation of systemic homeostasis being central chemosensors for oxygen, CO2 and Na⁺. Their basic physiological features show a lack of electrical excitability (inapt to produce action potentials), but display instead a rather active excitability based on variations in cytosolic concentrations of Ca²⁺ and Na⁺. It is expression of neurotransmitter receptors, pumps and transporters at their plasmalemma, along with transports on the endoplasmic reticulum and mitochondria that exquisitely regulate the cytosolic levels of these ions, the fluctuation of which underlies most, if not all, astroglial homeostatic functions.
... Fully functional astrocytes in vivo are endowed with a variety of ion channels and transporters some of which have unequivocally been shown to have housekeeping homeostatic roles [61,83]. Astrocytes express a large background potassium (K + ) conductance carried mainly by the weakly inward-rectifier K + channel 4.1 (Kir4.1); ...
The capacity of astrocytes to adapt their biochemical and functional features upon physiological and pathological stimuli is a fundamental property at the basis of their ability to regulate the homeostasis of the central nervous system (CNS). It is well known that in primary cultured astrocytes, the expression of plasma membrane ion channels and transporters involved in homeostatic tasks does not closely reflect the pattern observed in vivo. The individuation of culture conditions that promotes the expression of the ion channel array found in vivo is crucial when aiming at investigating the mechanisms underlying their dynamics upon various physiological and pathological stimuli. A chemically defined medium containing growth factors and hormones (G5) was previously shown to induce the growth, differentiation, and maturation of primary cultured astrocytes. Here we report that under these culture conditions, rat cortical astrocytes undergo robust morphological changes acquiring a multi-branched phenotype that develop gradually during the 2-week period of culturing. The shape changes were paralleled by variations in passive membrane properties and background conductance owing to the differential temporal development of inwardly rectifying chloride (Cl−) and potassium (K+) currents. Confocal and immunoblot analyses showed that morphologically differentiated astrocytes displayed a robust increase in the expression of the inward rectifier Cl− and K+ channels ClC-2 and Kir4.1, respectively, which are relevant ion channels in vivo. Finally, they exhibited a large diminution of the intermediate filaments glial fibrillary acidic protein (GFAP) and vimentin which are upregulated in reactive astrocytes in vivo. Taken together the data indicate that long-term culturing of cortical astrocytes in this chemical-defined medium promotes a quiescent functional phenotype. This culture model could aid to address the regulation of ion channel expression involved in CNS homeostasis in response to physiological and pathological challenges.
... Astrocytes express the genes of iGluRs, albeit at lower levels than neurons. All four AMPA receptor subunits (GluA1-GluA4) have been detected in astrocytes [1,103], although with some regional differences in expression [104,105]. For example, GluA1 and GluA4 are the most common subunits in cortical astrocytes and potentially localize to astrocyte processes [104]. ...
Astrocytes are complex glial cells that play many essential roles in the brain, including the fine-tuning of synaptic activity and blood flow. These roles are linked to fluctuations in intracellular Ca2+ within astrocytes. Recent advances in imaging techniques have identified localized Ca2+ transients within the fine processes of the astrocytic structure, which we term microdomain Ca2+ events. These Ca2+ transients are very diverse and occur under different conditions, including in the presence or absence of surrounding circuit activity. This complexity suggests that different signalling mechanisms mediate microdomain events which may then encode specific astrocyte functions from the modulation of synapses up to brain circuits and behaviour. Several recent studies have shown that a subset of astrocyte microdomain Ca2+ events occur rapidly following local neuronal circuit activity. In this review, we consider the physiological relevance of microdomain astrocyte Ca2+ signalling within brain circuits and outline possible pathways of extracellular Ca2+ influx through ionotropic receptors and other Ca2+ ion channels, which may contribute to astrocyte microdomain events with potentially fast dynamics.
... Coordination in the neuronal network plays a crucial role in epileptic electrical activities, mainly through GJ channels that are the backbone for intercellular electrical coupling in the central nervous system (Hamidi et al., 2014). Astrocytes express many ion channels and transmitter receptors (Verkhratsky and Steinhäuser, 2000) and are functionally conjugated to other astrocytes and to oligodendrocytes by GJs (Kettenmann and Ransom, 1988;Butt and Ransom, 1989;Robinson et al., 1993;von Blankenfeld et al., 1993;Venance et al., 1995;Pastor et al., 1998). The intercellular permeability between astrocytes facilitates the diffusion of metabolites throughout the CNS tissues (Valiunas et al., 2005). ...
Epilepsy affects approximately 50 million people worldwide, with 60% of adult epilepsies presenting an onset of focal origin. The most common focal epilepsy is temporal lobe epilepsy (TLE). The role of astrocytes in the presentation and development of TLE has been increasingly studied and discussed within the literature. The most common histopathological diagnosis of TLE is hippocampal sclerosis. Hippocampal sclerosis is characterized by neuronal cell loss within the Cornu ammonis and reactive astrogliosis. In some cases, mossy fiber sprouting may be observed. Mossy fiber sprouting has been controversial in its contribution to epileptogenesis in TLE patients, and the mechanisms surrounding the phenomenon have yet to be elucidated. Several studies have reported that mossy fiber sprouting has an almost certain co-existence with reactive astrogliosis within the hippocampus under epileptic conditions. Astrocytes are known to play an important role in the survival and axonal outgrowth of central and peripheral nervous system neurons, pointing to a potential role of astrocytes in TLE and associated cellular alterations. Herein, we review the recent developments surrounding the role of astrocytes in the pathogenic process of TLE and mossy fiber sprouting, with a focus on proposed signaling pathways and cellular mechanisms, histological observations, and clinical correlations in human patients.
... Astrocytes have historically been thought to serve a primarily structural role by supporting surrounding neurons (1). Over the last 40 years, however, a growing body of evidence has suggested that astrocytes serve important roles in normal brain function, and are critical for axonal growth, energy metabolism, neurotransmitter homeostasis and water/electrolyte balance (2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Moreover, abnormal astrocyte function has been postulated to contribute to the pathogenesis of a wide range of neurological and psychiatric disorders (12)(13)(14)(15)(16)(17). ...
The enzyme glutamine synthetase (GS), also referred to as glutamate ammonia ligase, is abundant in astrocytes and catalyzes the conversion of ammonia and glutamate to glutamine. Deficiency or dysfunction of astrocytic GS in discrete brain regions have been associated with several types of epilepsy, including medically-intractable mesial temporal lobe epilepsy (MTLE), neocortical epilepsies, and glioblastoma-associated epilepsy. Moreover, experimental inhibition or deletion of GS in the entorhinal-hippocampal territory of laboratory animals causes an MTLE-like syndrome characterized by spontaneous, recurrent hippocampal-onset seizures, loss of hippocampal neurons, and in some cases comorbid depressive-like features. The goal of this review is to summarize and discuss the possible roles of astroglial GS in the pathogenesis of epilepsy.
... Following the injury, due to the combination of various damages discussed earlier, the concentration of extracellular glutamate significantly increases, causing the depolarization of neurons [21]. Glutamate binds to NMDA, AMPA and kainite receptors causing an influx of calcium in neurons, glia, oligodendrocytes, endothelial cells and astrocytes [38,[52][53][54]. Due to excess intracellular Ca 2+ , astrocytes release glutamate which is not up-taken due to lipid peroxidation leading to further increase in extracellular glutamate concentration [38]. ...
Despite substantial development in medical treatment strategies scientists are struggling to find a cure against spinal cord injury (SCI) which causes long term disability and paralysis. The prime rationale behind it is the enlargement of primary lesion due to an initial trauma to the spinal cord which spreads to the neighbouring spinal tissues It begins from the time of traumatic event happened and extends to hours and even days. It further causes series of biological and functional alterations such as inflammation, excitotoxicity and ischemia, and promotes secondary lesion to the cord which worsens the life of individuals affected by SCI. Oxidative DNA damage is a stern consequence of oxidative stress linked with secondary injury causes oxidative base alterations and strand breaks, which provokes cell death in neurons. It is implausible to stop primary damage however it is credible to halt the secondary lesion and improve the quality of the patient’s life to some extent. Therefore it is crucial to understand the hidden perspectives of cell and molecular biology affecting the pathophysiology of SCI. Thus the focus of the review is to connect the missing links and shed light on the oxidative DNA damages and the functional repair mechanisms, as a consequence of the injury in neurons. The review will also probe the significance of neuroprotective strategies in the present scenario.
• HIGHLIGHTS
• Spinal cord injury, a pernicious condition, causes excitotoxicity and ischemia, ultimately leading to cell death.
• Oxidative DNA damage is a consequence of oxidative stress linked with secondary injury, provoking cell death in neurons.
• Base excision repair (BER) is one of the major repair pathways that plays a crucial role in repairing oxidative DNA damages.
• Neuroprotective therapies curbing SCI and boosting BER include the usage of pharmacological drugs and other approaches.
... Astrocytes have been well documented to express different ion channels and transporters to maintain extracellular homeostasis. For example, glutamate transporters and GABA transporters take up the neurotransmitters glutamate and GABA, respectively [7,8]. In addition, astrocytes express different types of neurotransmitter receptors including ionotropic receptors and G protein-coupled receptors (GPCRs) [9][10][11][12][13]. ...
Astrocytes are the major glial cells in the central nervous system, but unlike neurons, they do not produce action potentials. For many years, astrocytes were considered supporting cells in the central nervous system (CNS). Technological advances over the last two decades are changing the face of glial research. Accumulating data from recent investigations show that astrocytes display transient calcium spikes and regulate synaptic transmission by releasing transmitters called gliotransmitters. Many new powerful technologies are used to interfere with astrocytic activity, in order to obtain a better understanding of the roles of astrocytes in the brain. Among these technologies, chemogenetics has recently been used frequently. In this review, we will summarize new functions of astrocytes in the brain that have been revealed using this cutting-edge technique. Moreover, we will discuss the possibilities and challenges of manipulating astrocytic activity using this technology.
... Potassium voltage-gated channels (Kv) is a big gene family with at least 40 genes divided into 12 subfamilies (KV1-Kv12) (Gutman et al., 2003). Among them, a large number has brain specific expression, primarily in neurons with a subset expressed in glial cells (Verkhratsky and Steinhäuser, 2000;Vacher et al., 2008). In presented case Potassium voltage-gated channel subfamily A member 1 and 6 genes (KCNA1, HGNC ID:6218; KCNA6, HGNC ID:6225) are deleted. ...
... Experiments in vitro, in cell cultures, demonstrated that astrocytes are potentially capable of expressing all types of receptors to neurotransmitters (both ionotropic and metabotropic, Fig. 2) and neurohormones [61][62][63][64][65]. At the same time, expression of these receptors in the tissue in situ and in vivo is restricted and controlled by a local neurochemical environment, so that astrocytes specifically express receptors congruent with the neurotransmitters released in their vicinity [6,17]. ...
Astroglia are neural cells, heterogeneous in form and function, which act as supportive elements of the central nervous system; astrocytes contribute to all aspects of neural functions in health and disease. Through their highly ramified processes, astrocytes form close physical contacts with synapses and blood vessels, and are integrated into functional syncytia by gap junctions. Astrocytes interact among themselves and with other cells types (e.g., neurons, microglia, blood vessel cells) by an elaborate repertoire of chemical messengers and receptors; astrocytes also influence neural plasticity and synaptic transmission through maintaining homeostasis of neurotransmitters, K+ buffering, synaptic isolation and control over synaptogenesis and synaptic elimination. Satellite glial cells (SGCs) are the most abundant glial cells in sensory ganglia, and are believed to play major roles in sensory functions, but so far research into SGCs attracted relatively little attention. In this review we compare SGCs to astrocytes with the purpose of using the vast knowledge on astrocytes to explore new aspects of SGCs. We survey the main properties of these two cells types and highlight similarities and differences between them. We conclude that despite the much greater diversity in morphology and signaling mechanisms of astrocytes, there are some parallels between them and SGCs. Both types serve as boundary cells, separating different compartments in the nervous system, but much more needs to be learned on this aspect of SGCs. Astrocytes and SGCs employ chemical messengers and calcium waves for intercellular signaling, but their significance is still poorly understood for both cell types. Both types undergo major changes under pathological conditions, which have a protective function, but an also contribute to disease, and chronic pain in particular. The knowledge obtained on astrocytes is likely to benefit future research on SGCs.
... They have numerous fine processes that extent from primary and secondary processes, which give them a "bushy" appearance (Bushong EA,Martone ME,Jones YZ and Ellisman MH, 2002;Halassa MM et al., 2007). Astrocytes are endowed with a diversity of G protein-coupled receptors (GPCRs), including metabotropic glutamate receptors, P2Y receptors, cannabinoid receptors as well as norepinephrine receptors, through which they can sense neuronal activity (Araque A et al., 2014;Chevaleyre V et al., 2006;Khakh BS and McCarthy KD, 2015;Porter JT and McCarthy KD, 1997;Verkhratsky A and Steinhauser C, 2000). Activation of these GPCRs very often impacts on calcium signaling that allows astrocytes integrating different neuronal inputs (Perea G and Araque A, 2005). ...
Astrocytes perform various supporting functions, including ion buffering, metabolic supplying and neurotransmitter clearance. They can also sense neuronal activity owing to the presence of specific receptors for neurotransmitters. In turn, astrocytes can regulate synaptic activity through the release of gliotransmitters. Evidence has shown that astrocytes are very sensitive to the locus coeruleus (LC) afferents. However, little is known about how LC neuromodulatory norepinephrine (NE) modulates synaptic transmission through astrocytic activity. In mouse dentate gyrus (DG), we demonstrated an increase in the frequency of miniature excitatory postsynaptic currents (mEPSC) in response to NE, which required the release of glutamate from astrocytes. The rise in glutamate release probability is likely due to the activation of presynaptic GluN2B-containing NMDA receptors. Moreover, we showed that the activation of NE signaling in DG is necessary for the formation of contextual learning memory. Thus, NE signaling activation during fear conditioning training contributed to enduring changes in the frequency of mEPSC in DG. Our results strongly support the physiological neuromodulatory role of NE signaling, which is derived from activation of astrocytes.
... Ca 2+ is a highly versatile intracellular signaling molecule that regulates many different cellular processes, like cellular metabolism, proliferation and invasion [23,24]. Hence concentration of intracellular Ca 2+ in the cytosol needs to be permanently constant [25]. We observed that the treatment with CAPE and Dasatinib strongly decreases the expression of Ca 2+ which would influence a multitude of cellular reactions due to diversity of Ca 2+ binding proteins via Ca 2+ signaling [26,27]. ...
Gliomas are often recognized as highly heterogeneous cancerous phenotype. They are perpetually recurrent, obstinately resistant to treatment and hence almost incurable. Drug development studies to date have revealed only modest effect in attenuating growth of these tumors. The present study was aimed at elucidating the potential of targeting glioma through a novel combination of drugs in comparison to single agent. Here, we show that the combined administration of Caffeic acid phenethyl ester [CAPE] and Dasatinib exerts a strong antitumor action on C6 glioma cells. Combinational treatment inhibits proliferation, induces apoptosis, modulates astrocytic phenotype and decreases cell density. Results suggest that combinational therapy inhibits migration and invasiveness, decreases cell survival fraction and hence clonogenic property of C6 cells. The Nitric oxide [NO] levels were significantly reduced by combination treatment at all time points and effect was persistent over the time in comparison to single drug treatment. Atomic Absorption Spectroscopy [AAS] analysis of intracellular and extracellular calcium revealed that the treatment with CAPE and Dasatinib strongly modulates the calcium [Ca 2+ ] levels. Herein, we demonstrate that treatment of C6 glioma cells with CAPE and Dasatinib significantly decrease the activity of catalase [CAT]. The results in totality suggest that the combinational therapy remarkably reduces the proliferation of glioma cells possibly through different mechanisms, targeting multiple pathways involved in tumor growth, proliferation and development implicating the relevance of using these drugs in combination therapy for effective treatment of glioma. In vitro results suggest that CAPE and Dasatinib cotreatment could be therapeutically exploited for the management of gliomas.
... No reuse allowed without permission. Oligodendrocyte lineage cells express glutamate receptors including Kainate receptor (Verkhratsky and Steinhauser, 2000) and respond to Kainate stimulation ( Figure 4F). Therefore, we also measured the Kainate response in DsRed+ cells. ...
Rat embryonic stem (ES) cells offer the potential for sophisticated genome engineering in this valuable biomedical model species. However, germline transmission has been rare following conventional homologous recombination and clonal selection. Here we used the CRISPR/Cas9 system to target genomic mutations and insertions. We first evaluated utility for directed mutagenesis and recovered clones with biallelic deletions in Lef1. Mutant cells exhibited reduced sensitivity to glycogen synthase kinase 3 inhibition during self-renewal. We then generated a non-disruptive knock-in of DsRed at the Sox10 locus. Two clones produced germline chimaeras. Comparative expression of DsRed and Sox10 validated the fidelity of the reporter. To illustrate utility, oligodendrocyte lineage cells were visualised by live imaging of DsRed in neonatal brain slices and subjected to patch clamp recording. Overall these results show that CRISPR/Cas9 gene editing technology in germline competent rat ES cells is enabling for in vitro studies and for generating genetically modified rats.
... With respect to GABAergic signaling, astrocytes display internal Ca 2+ increases following GABA A receptor-mediated depolarization through voltage-gated Ca 2+ channels (VGCCs; Nilsson et al., 1993;Verkhratsky and Steinhäuser, 2000;Meier et al., 2008;Parpura et al., 2011;Verkhratsky et al., 2012). However, given the low membrane input resistance of mature astrocytes, the contribution of GABA A receptors to Ca 2+ responses in vivo remains controversial. ...
Astroglial networks constitute a non-neuronal communication system in the brain and are acknowledged modulators of synaptic plasticity. A sophisticated set of transmitter receptors in combination with distinct secretion mechanisms enables astrocytes to sense and modulate synaptic transmission. This integrative function evolved around intracellular Ca2+ signals, by and large considered as the main indicator of astrocyte activity. Regular brain physiology meticulously relies on the constant reciprocity of excitation and inhibition (E/I). Astrocytes are metabolically, physically, and functionally associated to the E/I convergence. Metabolically, astrocytes provide glutamine, the precursor of both major neurotransmitters governing E/I in the central nervous system (CNS): glutamate and γ-aminobutyric acid (GABA). Perisynaptic astroglial processes are structurally and functionally associated with the respective circuits throughout the CNS. Astonishingly, in astrocytes, glutamatergic as well as GABAergic inputs elicit similar rises in intracellular Ca2+ that in turn can trigger the release of glutamate and GABA as well. Paradoxically, as gliotransmitters, these two molecules can thus strengthen, weaken or even reverse the input signal. Therefore, the net impact on neuronal network function is often convoluted and cannot be simply predicted by the nature of the stimulus itself. In this review, we highlight the ambiguity of astrocytes on discriminating and affecting synaptic activity in physiological and pathological state. Indeed, aberrant astroglial Ca2+ signaling is a key aspect of pathological conditions exhibiting compromised network excitability, such as epilepsy. Here, we gather recent evidence on the complexity of astroglial Ca2+ signals in health and disease, challenging the traditional, neuro-centric concept of segregating E/I, in favor of a non-binary, mutually dependent perspective on glutamatergic and GABAergic transmission.
... The disturbance of the blood-brain barrier (BBB) by Ca +2 , K + , Na + and Cl − channels facilitates the ionic translocation into the brain tissue (Morrone et al., 2016;Rapôso et al., 2012;Verkhratsky and Steinhäuser 2000). These voltage and ligand-gated ion channels actively modulate the overall levels of neuronal activity in the glial cellular network. ...
There has been a significantly rising discussion on how neuronal plasticity communicates with the glioma growth and invasion. This literature review aims to determine which neurotransmitters, ion channels and signaling pathways are involved in this context, how information is transferred from synaptic sites to the glioma cells and how glioma cells apply established mechanics of synaptic plasticity for their own increment. This work is a compilation of some outstanding findings related to the influence of the glutamate, calcium, potassium, chloride and sodium channels and other important brain plasticity molecules over the glioma progression. These topics also include the relevant molecular signaling data which could prove to be helpful for an effective clinical management of brain tumors in the future.
... Astroglial physiology is defined by highly hyperpolarised (− 80 to − 85 mV) resting membrane potential, by inability to generate action potentials, by high activity of plasmalemmal transporters underlying astroglial homoeostatic responses and by intracellular ionic excitability [204]. Astroglial membrane potential lies close to the equilibrium potential for K + ions; astroglial membrane behaves as an almost ideal K + electrode because of high density of several types of K + channels (inward and delayed rectifying, voltage-dependent and voltage independent [135,178,208]), which ensure the stability of membrane potential even upon substantial depolarising stimuli. Gap junction connectivity of astrocytes also contributes to the isopotentiality of astroglial syncytia [105] and astrocyte input resistance [1,153]. ...
Astroglia represent a class of heterogeneous, in form and function, cells known as astrocytes, which provide for homoeostasis and defence of the central nervous system (CNS). Ageing is associated with morphological and functional remodelling of astrocytes with a prevalence of morphological atrophy and loss of function. In particular, ageing is associated with (i) decrease in astroglial synaptic coverage, (ii) deficits in glutamate and potassium clearance, (iii) reduced astroglial synthesis of synaptogenic factors such as cholesterol, (iv) decrease in aquaporin 4 channels in astroglial endfeet with subsequent decline in the glymphatic clearance, (v) decrease in astroglial metabolic support through the lactate shuttle, (vi) dwindling adult neurogenesis resulting from diminished proliferative capacity of radial stem astrocytes, (vii) decline in the astroglial-vascular coupling and deficient blood-brain barrier and (viii) decrease in astroglial ability to mount reactive astrogliosis. Decrease in reactive capabilities of astroglia are associated with rise of age-dependent neurodegenerative diseases. Astroglial morphology and function can be influenced and improved by lifestyle interventions such as intellectual engagement, social interactions, physical exercise, caloric restriction and healthy diet. These modifications of lifestyle are paramount for cognitive longevity.
... Indeed, astrocytes differ between various regions of gray matter, or within a single brain region Tsai et al., 2012;Clavreul et al., 2019;Batiuk et al., 2020). Astrocytes express gap junction proteins (Belliveau and Naus, 1994), neurotransmitter receptors, transporters (Zhou and Kimelberg, 2001;Matthias et al., 2003) and ion channels (Verkhratsky and Steinhauser, 2000). These specific molecular characteristics allow astrocytes to fulfill a range of homeostatic functions. ...
Maintenance of cerebral blood vessel integrity and regulation of cerebral blood flow ensure proper brain function. The adult human brain represents only a small portion of the body mass, yet about a quarter of the cardiac output is dedicated to energy consumption by brain cells at rest. Due to a low capacity to store energy, brain health is heavily reliant on a steady supply of oxygen and nutrients from the bloodstream, and is thus particularly vulnerable to stroke. Stroke is a leading cause of disability and mortality worldwide. By transiently or permanently limiting tissue perfusion, stroke alters vascular integrity and function, compromising brain homeostasis and leading to widespread consequences from early-onset motor deficits to long-term cognitive decline. While numerous lines of investigation have been undertaken to develop new pharmacological therapies for stroke, only few advances have been made and most clinical trials have failed. Overall, our understanding of the acute and chronic vascular responses to stroke is insufficient, yet a better comprehension of cerebrovascular remodeling following stroke is an essential prerequisite for developing novel therapeutic options. In this review, we present a comprehensive update on post-stroke cerebrovascular remodeling, an important and growing field in neuroscience, by discussing cellular and molecular mechanisms involved, sex differences, limitations of preclinical research design and future directions.
... Astrocytes from different brain regions express a multitude of ion channels and receptors (Bordey and Sontheimer, 2000;Verkhratsky and Steinhäuser, 2000). Expression of astroglial receptors, transporters and channels undergo substantial and multi-directional developmental changes. ...
It is widely recognised that astrocytes are able to shape synaptic transmission by restricting glutamate transients to the synaptic cleft. In this thesis, I demonstrate that during synaptic transmission K+ efflux through postsynaptic NMDA receptors depolarises the astrocytic membrane and thus slows down glial glutamate uptake. This effect involves the rectifying K+ channels (Kir4.1), predominantly located at perisynaptic astrocytic processes (PAPs). Genetic upregulation of this channel subtype in astrocytes does not affect glutamate transporters efficiency but curtails increase in presynaptic glutamate release probability during extracellular K+ rises. Thus, activity-dependent accumulation of extracellular K+ can boost glutamate release from the presynaptic site while decreasing astroglial glutamate uptake. Both factors occasion increased extrasynaptic glutamate escape and therefore inter-synaptic crosstalk in the hippocampus.
... However, the surviving neurons show strong intrinsic regenerative potential. Increased glial responses influence gray matter plasticity by regulating ion diffusion in pre-and postsynaptic elements, increasing antioxidant activity [14][15][16][17][18]. Excitatory glutamatergic inputs are pruned from the motoneuron cell body, contributing to an inhibitory state over the alpha motoneurons [10,19,20], thereby suppressing action potentials to favor cell repair [21]. ...
Background:
Ventral root avulsion (VRA) is an experimental approach in which there is an abrupt separation of the motor roots from the surface of the spinal cord. As a result, most of the axotomized motoneurons degenerate by the second week after injury, and the significant loss of synapses and increased glial reaction triggers a chronic inflammatory state. Pharmacological treatment associated with root reimplantation is thought to overcome the degenerative effects of VRA. Therefore, treatment with dimethyl fumarate (DMF), a drug with neuroprotective and immunomodulatory effects, in combination with a heterologous fibrin sealant/biopolymer (FS), a biological glue, may improve the regenerative response.
Methods:
Adult female Lewis rats were subjected to VRA of L4-L6 roots followed by reimplantation and daily treatment with DMF for four weeks. Survival times were evaluated 1, 4 or 12 weeks after surgery. Neuronal survival assessed by Nissl staining, glial reactivity (anti-GFAP for astrocytes and anti-Iba-1 for microglia) and synapse preservation (anti-VGLUT1 for glutamatergic inputs and anti-GAD65 for GABAergic inputs) evaluated by immunofluorescence, gene expression (pro- and anti-inflammatory molecules) and motor function recovery were measured.
Results:
Treatment with DMF at a dose of 15 mg/kg was found to be neuroprotective and immunomodulatory because it preserved motoneurons and synapses and decreased astrogliosis and microglial reactions, as well as downregulated the expression of pro-inflammatory gene transcripts.
Conclusion:
The pharmacological benefit was further enhanced when associated with root reimplantation with FS, in which animals recovered at least 50% of motor function, showing the efficacy of employing multiple regenerative approaches following spinal cord root injury.
... Classical astrocytes express a variety of different K + channels, that is inwardly rectifying (Kir), two-pore domain (K 2 P) and voltage-activated (K V ) channels (for review see e.g. Verkhratsky & Nedergaard, 2018;Verkhratsky & Steinhauser, 2000). Among those, Kir4.1 is of particular interest as it is the predominant K + channel in fully developed astrocytes, massively influencing their physiology and behaviour. ...
In the rodent forebrain, the majority of astrocytes are generated during the early postnatal phase. Following differentiation, astrocytes undergo maturation which accompanies the development of the neuronal network. Neonate astrocytes exhibit a distinct morphology and domain size which differs to their mature counterparts. Moreover, many of the plasma membrane proteins prototypical for fully developed astrocytes are only expressed at low levels at neonatal stages. These include connexins and Kir4.1, which define the low membrane resistance and highly negative membrane potential of mature astrocytes. Newborn astrocytes moreover express only low amounts of GLT‐1, a glutamate transporter critical later in development. Furthermore, they show specific differences in the properties and spatio‐temporal pattern of intracellular calcium signals, resulting from differences in their repertoire of receptors and signaling pathways. Therefore, roles fulfilled by mature astrocytes, including ion and transmitter homeostasis, are underdeveloped in the young brain. Similarly, astrocytic ion signaling in response to neuronal activity, a process central to neuron‐glia interaction, differs between the neonate and mature brain. This review describes the unique functional properties of astrocytes in the first weeks after birth and compares them to later stages of development. We conclude that with an immature neuronal network and wider extracellular space, astrocytic support might not be as demanding and critical compared to the mature brain. The delayed differentiation and maturation of astrocytes in the first postnatal weeks might thus reflect a reduced need for active, energy‐consuming regulation of the extracellular space and a less tight control of glial feedback onto synaptic transmission.
... The resident microglia are part of the larger neuroglial cellular system, which includes non-neuronal cells of the nervous system, including astrocytes, oligodendrocytes, and pericytes. Those cells not only provide structural support to the brain parenchyma, but also respond to injury, regulate the ionic and chemical composition of the extracellular milieu, form the myelin insulation of the brain wiring, guide neuronal migration during development, and exchange metabolites with neurons (Verkhratsky and Steinhäuser, 2000). ...
Most neurological disorders seemingly have heterogenous pathogenesis, with overlapping contribution of neuronal, immune and vascular mechanisms of brain injury. The perivascular space in the brain represents a crossroad where those mechanisms interact, as well as a key anatomical component of the recently discovered glymphatic pathway, which is considered to play a crucial role in the clearance of brain waste linked to neurodegenerative diseases. The pathological interplay between neuronal, immune and vascular factors can create an environment that promotes self-perpetration of mechanisms of brain injury across different neurological diseases, including those that are primarily thought of as neurodegenerative, neuroinflammatory or cerebrovascular. Changes of the perivascular space can be monitored in humans in vivo using magnetic resonance imaging (MRI). In the context of glymphatic clearance, MRI-visible enlarged perivascular spaces (EPVS) are considered to reflect glymphatic stasis secondary to the perivascular accumulation of brain debris, although they may also represent an adaptive mechanism of the glymphatic system to clear them. EPVS are also established correlates of dementia and cerebral small vessel disease (SVD) and are considered to reflect brain inflammatory activity. In this review, we describe the “perivascular unit” as a key anatomical and functional substrate for the interaction between neuronal, immune and vascular mechanisms of brain injury, which are shared across different neurological diseases. We will describe the main anatomical, physiological and pathological features of the perivascular unit, highlight potential substrates for the interplay between different noxae and summarize MRI studies of EPVS in cerebrovascular, neuroinflammatory and neurodegenerative disorders.
... We have also found augmented Cav1.2 activity in cortical astrocytes treated with the endotoxin lipopolysaccharide LPS and in a model of astrocyte mechanical injury (Cheli et al., 2016a). Along these lines, in vitro and in vivo evidence suggest that reactive astrocytes upregulate voltage-gated Na 1 channels and downregulate delayed rectifier K 1 currents (Verkhratsky and Steinhäuser, 2000;Pappalardo et al., 2014). Thus, the increase in Na 1 channels density in combination with a decrease in delayed rectifier K 1 currents could make these cells excitable. ...
To determine whether Cav1.2 voltage-gated Ca ²⁺ channels contribute to astrocyte activation, we generated an inducible conditional knock-out mouse in which the Cav1.2 α subunit was deleted in GFAP-positive astrocytes. This astrocytic Cav1.2 knock-out mouse was tested in the cuprizone model of myelin injury and repair which causes astrocyte and microglia activation in the absence of a lymphocytic response. Deletion of Cav1.2 channels in GFAP-positive astrocytes during cuprizone-induced demyelination leads to a significant reduction in the degree of astrocyte and microglia activation and proliferation in mice of either sex. Concomitantly, the production of proinflammatory factors such as TNFα, IL1β and TGFβ1 was significantly decreased in the corpus callosum and cortex of Cav1.2 knock-out mice through demyelination. Furthermore, this mild inflammatory environment promotes oligodendrocyte progenitor cells maturation and myelin regeneration across the remyelination phase of the cuprizone model. Similar results were found in animals treated with nimodipine, a Cav1.2 Ca ²⁺ channel inhibitor with high affinity to the CNS. Mice of either sex injected with nimodipine during the demyelination stage of the cuprizone treatment displayed a reduced number of reactive astrocytes and showed a faster and more efficient brain remyelination. Together, these results indicate that Cav1.2 Ca ²⁺ channels play a crucial role in the induction and proliferation of reactive astrocytes during demyelination; and that attenuation of astrocytic voltage-gated Ca ²⁺ influx may be an effective therapy to reduce brain inflammation and promote myelin recovery in demyelinating diseases.
SIGNIFICANCE STATEMENT Reducing voltage-gated Ca ²⁺ influx in astrocytes during brain demyelination significantly attenuates brain inflammation and astrocyte reactivity. Furthermore, these changes promote myelin restoration and oligodendrocyte maturation throughout remyelination.
... Many aspects of OL maturation are modulated by local extrinsic signals, including astrocytic and neuronal activity, as well as more global signals like hormones [13][14][15]. OPC and OL cells express a variety of neurotransmitter receptors and ion channels [16,17], and neural activity alters their maturation and consequently myelination of axons in both the developing and mature CNS. For example, some of the major neurotransmitters controlling arousal like Acetylcholine (ACh) and Norepinephrine (NE) regulate neurogenesis, but also OPC proliferation and survival [18][19][20][21]. ...
Oligodendrocytes (OL) are the only myelinating cells of the central nervous system thus interferences, either environmental or genetic, with their maturation or function have devastating consequences. Albeit so far neglected, one of the less appreciated, nevertheless possible, regulators of OL maturation and function is the circadian cycle. Yet, disruptions in these rhythms are unfortunately becoming a common "disorder" in the today's world. The temporal patterning of behaviour and physiology is controlled by a circadian timing system based in the anterior hypothalamus. At the molecular level, circadian rhythms are generated by a transcriptional/translational feedback system that regulates transcription and has a major impact on cellular function(s). Fundamental cellular properties/functions in most cell types vary with the daily circadian cycle: OL are unlikely an exception! To be clear, the presence of circadian oscillators or the cell-specific function(s) of the circadian clock in OL has yet to be defined. Furthermore, we wish to entertain the idea of links between the "thin" evidence on OL intrinsic circadian rhythms and their interjection(s) at different stages of lineage progression as well as in supporting/regulating OL crucial function: myelination. Individuals with intellectual and developmental syndromes as well as neurodegenerative diseases present with a disrupted sleep/wake cycle; hence, we raise the possibility that these disturbances in timing can contribute to the loss of white matter observed in these disorders. Preclinical and clinical work in this area is needed for a better understanding of how circadian rhythms influence OL maturation and function(s), to aid the development of new therapeutic strategies and standards of care for these patients.
After analyzing in Chap. 1 some of the most important features of the biological brain that underlie learning process and information storage, this Chapter provides a quick overview of optoelectronic and fully-optical neuromorphic techniques implemented to date. The operation and physical principles of each approach presented is described. These are grouped into three main categories called paradigms. Each neuromorphic paradigm is built on a main neural connotation. The first paradigm replicates neural excitability. The second paradigm focuses on the concept of connection between units. Finally, the third paradigm is an absolute novelty in the scientific panorama and encompasses only solitonic networks, which can replicate a tissue structure. The main difference from other neuromorphic hardware lies in their ability to self-organize according to the received stimuli, evolving accordingly over time.
Long-term potentiation is involved in physiological process like learning and memory, motor learning and sensory processing, and pathological conditions such as addiction. In contrast to the extensive studies on the mechanism of long-term potentiation on excitatory glutamatergic synapse onto excitatory neurons (LTPE→E), the mechanism of LTP on excitatory glutamatergic synapse onto inhibitory neurons (LTPE→I) remains largely unknown. In the central nervous system, astrocytes play an important role in regulating synaptic activity and participate in the process of LTPE→E, but their functions in LTPE→I remain incompletely defined. Using electrophysiological, pharmacological, confocal calcium imaging, chemogenetics and behavior tests, we studied the role of astrocytes in regulating LTPE→I in the hippocampal CA1 region and their impact on cognitive function. We show that LTPE→I in stratum oriens of hippocampal CA1 is astrocyte independent. However, in the stratum radiatum, synaptically released endocannabinoids increases astrocyte Ca2+ via type-1 cannabinoid receptors, stimulates D-serine release, and potentiate excitatory synaptic transmission on inhibitory neuron through the activation of (N-methyl-D-aspartate) NMDA receptors. We also revealed that chemogentic activation of astrocytes is sufficient to induce NMDA-dependent de novo LTPE→I in the stratum radiatum of hippocampus. Furthermore, we found that disrupt LTPE→I by knockdwon γCaMKII in interneurons of stratum radiatum resulted in dramatic memory impairment. Our findings suggest that astrocytes release D-serine, which activates NMDA receptors to regulate LTPE→I, and that cognitive function is intricately linked with the proper functioning of this LTPE→I pathway.
Neuroscientists have recognized the importance of astrocytes in regulating neurological function and their influence on the release of glial transmitters. Few studies, however, have focused on the effects of general anesthetic agents on neuroglia or astrocytes. Astrocytes can also be an important target of general anesthetic agents as they exert not only sedative, analgesic, and amnesic effects but also mediate general anesthetic-induced neurotoxicity and postoperative cognitive dysfunction. Here, we analyzed recent advances in understanding the mechanism of general anesthetic agents on astrocytes, and found that exposure to general anesthetic agents will destroy the morphology and proliferation of astrocytes, in addition to acting on the receptors on their surface, which not only affect Ca ²⁺ signaling, inhibit the release of brain-derived neurotrophic factor and lactate from astrocytes, but are even involved in the regulation of the pro- and anti-inflammatory processes of astrocytes. These would obviously affect the communication between astrocytes as well as between astrocytes and neighboring neurons, other neuroglia, and vascular cells. In this review, we summarize how general anesthetic agents act on neurons via astrocytes, and explore potential mechanisms of action of general anesthetic agents on the nervous system. We hope that this review will provide a new direction for mitigating the neurotoxicity of general anesthetic agents.
Long-term potentiation is involved in physiological process like learning and memory, motor learning and sensory processing, and pathological conditions such as addiction. In contrast to the extensive studies on the mechanism of long-term potentiation on excitatory glutamatergic synapse onto excitatory neurons (LTP E→E ), the mechanism of LTP on excitatory glutamatergic synapse onto inhibitory neurons (LTP E→I ) remains largely unknown. In the central nervous system, astrocytes play an important role in regulating synaptic activity and participate in the process of LTP E→E , but their functions in LTP E→I remain incompletely defined. Using electrophysiological, pharmacological, confocal calcium imaging, chemogenetics and behavior tests, we studied the role of astrocytes in regulating LTP E→I in the hippocampal CA1 region and their impact on cognitive function. We show that LTP E→I in stratum oriens of hippocampal CA1 is astrocyte independent. However, in the stratum radiatum, synaptically released endocannabinoids increases astrocyte Ca ²⁺ via type-1 cannabinoid receptors, stimulates D-serine release, and potentiate excitatory synaptic transmission on inhibitory neuron through the activation of (N-methyl-D-aspartate) NMDA receptors. We also revealed that chemogentic activation of astrocytes is sufficient to induce NMDA-dependent de novo LTP E→I in the stratum radiatum of hippocampus. Furthermore, we found that disrupt LTP E→I by knockdwon γCaMKII in interneurons of stratum radiatum resulted in dramatic memory impairment. Our findings suggest that astrocytes release D-serine, which activates NMDA receptors to regulate LTP E→I , and that cognitive function is intricately linked with the proper functioning of this LTP E→I pathway.
The intracellular sodium ion is one of the crucial elements for regulating physiological functions such as action potential and muscle contractions. However, detecting sodium ions in live cells is challenging because false signals may arise from the abundant sodium ions in the extracellular environment when introducing the detection agents. To minimize it, we report a DNAzyme-based detection of sodium ions in live cells via activation by endogenous mRNA. The substrate strand of DNAzyme first hybridizes to a blocking strand that prevents undesired cleavage of DNAzyme when delivered. Once entering cells, an endogenous mRNA biomarker binds to the toehold region of the blocking strand and displaces it, allowing the proper formation of the DNAzyme, which specifically catalyzes the cleavage of the substrate strand in the presence of intracellular Na⁺ and produces fluorescence signals. Using differentiating skeletal muscle cells as the model system, we demonstrated the successful delivery and phenotype-specific detection of intracellular sodium ions only in differentiated myotubes with highly-expressed myosin heavy chain mRNA. Moreover, using a drug cocktail to increase the permeability of the cell membrane, elevated levels of intracellular sodium ion was observed. This platform offers a broad and promising strategy for detecting intracellular metal ions, suggesting a great potential for understanding its role in cell/tissue physiology.
In an effort to develop the next generation of neural implants, current research has focused on solving the challenges associated with the foreign body reaction and promoting long-term device performance in vivo. Although the cutting edge of research in this field appears to be moving away from traditional metallic and semiconductor materials, the complex tissue dynamics which occur at the electrode–neural interface following device implantation are yet to be resolved. In particular, understanding the molecular processes of gliosis and the onset and persistence of scar formation is key in developing stable and specific neural recording/stimulation devices. Critically, it is recognized that neural device implantation leads to a significant disruption of tissue integrity at the peri-electrode site. Accordingly, an in-depth understanding of mechanotransduction in neuronal cell populations at the peri-implant region is required to better inform neural interface design. This perspective highlights the need for a comprehensive mechanobiological understanding of gliosis to enhance the development of neural implants with improved chronic functionality.
Excitation mechanisms in the nervous system and neuronal–glial interactions involved in this process are described on the basis of published data and original findings. Two processes, passive and active, form excitation in the nervous system. The active type of excitation requires energy support and is associated with the regulation of the membrane properties of neurons, leading to generation of variable spontaneous pulses. Spike activity generated by the passive process is highly stable and results from transmembrane movement of Na+ and K+ ions along their concentration gradients. The passive type of excitation is due to glutamatergic contacts; the active type of excitation is due to a diffuse release of acetylcholine from cholinergic nuclei of the brain and attenuation of K+ membrane permeability. Energy supply of the active excitation process involves glia. Glial cells directly interact with brain vessels, accumulate glucose in the form of glycogen, realise glycolysis as the first step of energy metabolism, and regulate local cerebral blood flow coupled with M-cholinergic excitation of neurons. A steady decrease in the rate of the M-cholinergic process (in terms of concentration, temperature, or energy) leads to a rapid outflow of K+ ions from neurons, and removing K+ from the intercellular environment is also a function of glia.
Nitric oxide (NO) has emerged as an ubiquitous signaling molecule in the central nervous system (CNS). NO is synthesised from molecular oxygen and the amino acid L-arginine (L- ARG) by the enzyme NO synthase (NOS), and the availability of L-ARG has been implicated as the limiting factor for NOS activity. Previous studies have indicated that L- ARG is localised in astrocytes in vitro and that the in vitro activation of non-N-methyl-D- aspartate (NMDA) receptors, as well as the presence of peroxynitrite (ONOO ), led to the release of L-ARG. Microdialysis was therefore used in this study to investigate whether this held true in vivo. The results indicated that, while L-ARG was localised in glia in vivo and the infusion of α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) caused the release of L-ARG, this increase in extracellular L-ARG levels did not drive the NOS reaction. The increased L-ARG levels were halved by the coinfusion of CNQX but not totally inhibited. NMDA receptor activation is recognised as a major pathway of NOS activation and hence NO production and, while the results of this study concur, the results herein also suggest no need for increased L-ARG in the extracellular space as a prerequisite for NOS activation. Interesting results were also achieved using the NOS inhibitors Nw- nitro-L-ARG methyl ester (L-NAME) and 7-nitroindazole (7-NI). Both inhibitors increased the basal activity of NOS and production of NO, while each drug had contrasting effects on the NMDA-stimulated response. L-NAME blocked the increased NOS activity with no effect on NO production while 7-NI had no effect on NOS but blocked the production of NO. In conclusion, the regulation of the supply of L-ARG for NOS in vivo is far more complicated than in vitro studies suggest.
Injury to the adult central nervous system cannot be effectively repaired, leading to chronic disability. In contrast, embryonic neurons possess extensive regenerative capabilities, permitting functional recovery after axonal damage. The embryonic chick can functionally recover from spinal injury at developmental stages prior to embryonic day 13, at which point the CNS undergoes transition from a permissive to restrictive environment for neuronal regeneration. The developmentally regulated molecular changes responsible for this transition remain to be fully characterised: the principle aim of this study was to advance understanding in this area. A number of markers have been investigated, using immunohistochemistry, RT-PCR, and Western Blotting to establish the contribution of neuronal and glial populations to the failure of regeneration in the embryonic chick and human. In addition, the relative contributions of primary and secondary tissue damage following spinal injury have been addressed by measuring the extent and duration to which apoptosis occurs during permissive or restrictive stages of development. The major findings of this research are that; 1.) Cell death following injury, particularly due to secondary injury mechanisms, plays a crucial, and perhaps principal role in establishing a non-permissive CNS environment during the restrictive period for regeneration: 2.) The myelin-associated inhibitor of neurite outgrowth, Nogo-A, and its receptor, do not contribute majorly to the transition from permissive to restrictive states during development, and seems to play an important, previously un-described, role during embryogenesis; 3.) Elevated levels of chondroitin sulphate proteoglycans through development, in addition to altered cellular localisation, are likely to contribute to the overall non-permissiveness of the mature spinal cord. To conclude, this study has clarified some of the issues concerning the generation of a non-permissive environment during development in the embryonic chick and human, and has laid foundation for further research concerning the novel role of Nogo proteins during embryogenesis.
Calcium (Ca2+) is an essential component in intracellular signaling of brain cells, and its control mechanisms are of great interest in biological systems. Ca2+ can signal differently in neurons and glial cells using the same intracellular pathways or cell membrane structural components. These types of machinery are responsible for entry, permanence, and removal of Ca2+ from the cellular environment and are of vital importance for brain homeostasis. This review highlights the importance of Ca2+ in neuronal and glial cell physiology as well as aspects of learning, memory, and Alzheimer's disease, focusing on the involvement of L-type voltage-gated Ca2+ channels.
We have analyzed the role of glutamate and its receptors (GluRs) in regulating the development of oligodendrocytes. Activation of AMPA- preferring GluRs with selective agonists inhibited proliferation of purified cortical oligodendrocyte progenitor (O-2A) cells cultured with different mitogens, as measured by [3H]thymidine incorporation or bromodeoxyuridine staining. In contrast, activation of GABA or muscarinic receptors did not affect O-2A proliferation. Cell viability and apoptosis assays demonstrated that the inhibition of O-2A proliferation was not attributable to a cytotoxic action of GluR agonists, and was reversible. Activation of GluRs prevented lineage progression from the O-2A (GD3+/nestin+) stage to the prooligodendroblast (O4+) stage, but did not affect O-2A migration. Additional experiments examined the membrane ionic channels mediating these GluR activation effects. We found that proliferating O-2A cells expressed functional delayed rectifier K+ channels, which were absent in pro-oligodendroblasts. GluR agonists and the K+ channel blocker tetraethylammonium (TEA) strongly inhibited delayed rectifier K+ currents in O-2A cells. TEA reproduced the effects of GluR activation on O-2A proliferation and lineage progression in the same concentration range that blocked delayed rectifier K+ currents. These results indicate that glutamate regulates oligodendrogenesis specifically at the O-2A stage by modulating K+ channel activity.
Glial–neuronal communication was studied by monitoring the effect of intercellular glial Ca ²⁺ waves on the electrical activity of neighboring neurons in the eyecup preparation of the rat. Calcium waves in astrocytes and Müller cells were initiated with a mechanical stimulus applied to the retinal surface. Changes in the light-evoked spike activity of neurons within the ganglion cell layer occurred when, and only when, these Ca ²⁺ waves reached the neurons. Inhibition of activity was observed in 25 of 53 neurons (mean decrease in spike frequency, 28 ± 2%). Excitation occurred in another five neurons (mean increase, 27 ± 5%). Larger amplitude Ca ²⁺ waves were associated with greater modulation of neuronal activity. Thapsigargin, which reduced the amplitude of the glial Ca ²⁺ increases, also reduced the magnitude of neuronal modulation. Bicuculline and strychnine, inhibitory neurotransmitter antagonists, as well as 6-Nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and d (−)-2-amino-7-phosphonoheptanoic acid (D-AP7), glutamate antagonists, reduced the inhibition of neuronal activity associated with glial Ca ²⁺ waves, suggesting that inhibition is mediated by inhibitory interneurons stimulated by glutamate release from glial cells. The results suggest that glial cells are capable of modulating the electrical activity of neurons within the retina and thus, may directly participate in information processing in the CNS.
Closely related K+ channels can coassemble to form heteromultimers in expression systems, as well as in vivo. Whether in vivo this coassembly is random and inevitable or whether highly homologous channels can be segregated and targeted independently within a given cell has not been determined. In this study, we address these questions by characterizing and localizing voltage-dependent K+ channels in Schwann cells. Transcripts for three closely related members of the Shaker-like family of K+ channels are found in adult rat sciatic nerve: Kv1.1, Kv1.2, and Kv1.5. We have examined two of these and observed that both Kv1.1 and Kv1.5 proteins are expressed in Schwann cells but differ in their distributions. Kv1.5 is localized on the Schwann cell membrane at the nodes of Ranvier and in bands that run along the outer surface of the myelin. It is also seen intracellularly in the vicinity of the nucleus. Schwann cell staining for Kv1.1, on the other hand, was seen only in perinuclear, intracellular compartments. These results provide evidence that closely related channels from the same family need not coassemble and can be localized differentially in the same cell. In addition, Kv1.1 was highly concentrated in the axonal membrane at juxtaparanodal regions. The distributions of these K+ channels in myelinated nerve highlight the elaborate molecular specializations of these membranes.
The effects of a variety of antiproliferative agents on voltage-dependent K ⁺ channel function in cortical oligodendrocyte progenitor (O-2A) cells were studied. Previously, we had shown that glutamate receptor activation reversibly inhibited O-2A cell proliferation stimulated by mitogenic factors and prevented lineage progression by attenuating outward K ⁺ currents in O-2A cells. We now show that the antiproliferative actions of glutamate receptor activation are Ca ²⁺ -independent and arise from an increase in intracellular Na ⁺ and subsequent block of outward K ⁺ currents. In support of this mechanism, agents that acted to depolarize O-2A cells or increase intracellular sodium similarly had an antiproliferative effect, attributable at least in part to a reduction in voltage-gated K ⁺ currents. Also, these effects were reversible and Ca ²⁺ -independent. Chronic treatment with glutamate agonists was without any long-term effect on K ⁺ current function. Cells cultured in elevated K ⁺ , however, demonstrated an upregulation of inward rectifier K ⁺ currents, concomitant with an hyperpolarization of the resting membrane potential. This culture condition therefore promoted a current phenotype typical of pro-oligodendroblasts. Finally, cells chronically treated with the mitotic inhibitor retinoic acid displayed a selective downregulation of outward K ⁺ currents. In conclusion, signals that affect O-2A cell proliferation do so by regulating K ⁺ channel function. These data indicate that the regulation of K ⁺ currents in cells of the oligodendrocyte lineage plays an important role in determining their proliferative potential and demonstrate that O-2A cell K ⁺ current phenotype can be modified by long-term depolarization of the cell membrane.
TWIK-1, TREK-1 and TASK K+ channels comprise a class of pore-forming subunits with four membrane-spanning segments and two P domains. Here we report the cloning of TRAAK, a 398 amino acid protein which is a new member of this mammalian class of K+ channels. Unlike TWIK-1, TREK-1 and TASK which are widely distributed in many different mouse tissues, TRAAK is present exclusively in brain, spinal cord and retina. Expression of TRAAK in Xenopus oocytes and COS cells induces instantaneous and non-inactivating currents that are not gated by voltage. These currents are only partially inhibited by Ba2+ at high concentrations and are insensitive to the other classical K+ channel blockers tetraethylammonium, 4-aminopyridine and Cs+. A particularly salient feature of TRAAK is that they can be stimulated by arachidonic acid (AA) and other unsaturated fatty acids but not by saturated fatty acids. These channels probably correspond to the functional class of fatty acid-stimulated K+ currents that recently were identified in native neuronal cells but have not yet been cloned. These TRAAK channels might be essential in normal physiological processes in which AA is known to play an important role, such as synaptic transmission, and also in pathophysiological processes such as brain ischemia. TRAAK channels are stimulated by the neuroprotective drug riluzole.
Four types of glial cells could be distinguished in the grey matter of rat spinal cord slices at postnatal days 1-19 (P1-P19), based on their pattern of membrane currents as revealed by the whole cell patch clamp technique, and by their morphological and immunocytochemical features. The recorded cells were labelled with Lucifer Yellow, which allowed the subsequent identification of cells using cell-type-specific markers. Astrocytes were identified by positive staining for glial fibrillary acidic protein (GFAP). These were morphologically characterized by multiple, very fine and short processes and electrophysiologically by symmetrical, non-decaying K+ selective currents. Oligodendrocytes were identified by a typical oligodendrocyte-like morphology, lack of GFAP staining and positive labelling with a combination of O1 and O4 antibodies (markers of the oligodendrocyte lineage), and their membrane was dominated by symmetrical, passive, decaying K+ currents. The third population of glial cells was also characterized by positive staining for O1/O4 or only for O4 antigens, lack of GFAP staining and, in some cells, oligodendrocyte-like morphology. However, these cells could be distinguished by the presence of inwardly rectifying (KDR), delayed outwardly rectifying (KDR) and A-type K+ currents (KA), representing the most likely glial precursor cells of the oligodendrocyte lineage. The fourth population of glial cells had small somata and a widespread network of long processes with no apparent orientation preference. In one case, processes were positively labelled with GFAP, while 30% were characterized by faint, diffuse staining. These cells expressed a complex pattern of voltage-gated channels, namely Na+, KDR, KA and KIR channels. In contrast to neurons, the amplitude of Na+ currents was at least one order of magnitude smaller than the K+ currents, and none of these cells showed the ability to generate action potentials in the current clamp mode. Since none of these cells could be labelled by oligodendrocyte markers we assume that they were either astrocytes or glial precursor cells of the astrocyte lineage. The four cell types were found in all regions of the grey matter. When randomly accessing the glial cells, the probability of recording from the oligodendrocyte precursor cells and the glial cells with Na+ currents decreased during development. At P1-P3, 50% of the cells revealed the Na+ current, while at P13-P15 only 18% did. Concomitantly, the number of glial cells with astrocyte- and oligodendrocyte-like membrane currents increased from 19 and 12% to 41 and 35.5% respectively. We conclude that the glial cells in the spinal cord slices possess distinct morphological, immunohistochemical and physiological properties, and that the glial populations undergo changes during postnatal development.
Anti-peptide antibodies that specifically recognize the alpha1 subunit of class A-D voltage-gated Ca2+ channels and a monoclonal antibody (MANC-1) to the alpha2 subunit of L-type Ca2+ channels were used to investigate the distribution of these Ca2+ channel subtypes in neurons and glia in models of brain injury, including kainic acid-induced epilepsy in the hippocampus, mechanical and thermal lesions in the forebrain, hypomyelination in white matter, and ischemia. Immunostaining of the alpha2 subunit of L-type Ca2+ channels by the MANC-1 antibody was increased in reactive astrocytes in each of these forms of brain injury. The alpha1C subunits of class C L-type Ca2+ channels were upregulated in reactive astrocytes located in the affected regions in each of these models of brain injury, although staining for the alpha1 subunits of class D L-type, class A P/Q-type, and class B N-type Ca2+ channels did not change from patterns normally observed in control animals. In all of these models of brain injury, there was no apparent redistribution or upregulation of the voltage-gated Ca2+ channels in neurons. The upregulation of L-type Ca2+ channels in reactive astrocytes may contribute to the maintenance of ionic homeostasis in injured brain regions, enhance the release of neurotrophic agents to promote neuronal survival and differentiation, and/or enhance signaling in astrocytic networks in response to injury.
Little is known of the molecular mechanisms that trigger oligodendrocyte death and demyelination in many acute central nervous system insults. Since oligodendrocytes express functional alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-type glutamate receptors, we examined the possibility that oligodendrocyte death can be mediated by glutamate receptor overactivation. Oligodendrocytes in primary cultures from mouse forebrain were selectively killed by low concentrations of AMPA, kainate or glutamate, or by deprivation of oxygen and glucose. This toxicity could be blocked by the AMPA/kainate receptor antagonist 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX). In vivo, differentiated oligodendrocytes in subcortical white matter expressed AMPA receptors and were selectively injured by microstereotaxic injection of AMPA but not NMDA. These data suggest that oligodendrocytes share with neurons a high vulnerability to AMPA/kainate receptor-mediated death, a mechanism that may contribute to white matter injury in CNS disease.
The actions of grayanotoxin I, veratrine, and tetrodotoxin on the membrane potential of the Schwann cell were studied in the giant nerve fiber of the squid Sepioteuthis sepioidea. Schwann cells of intact nerve fibers and Schwann cells attached to axons cut lengthwise over several millimeters were utilized. The axon membrane potential in the intact nerve fibers was also monitored. The effects of grayanotoxin I and veratrine on the membrane potential of the Schwann cell were found to be similar to those they produce on the resting membrane potential of the giant axon. Thus, grayanotoxin I (1-30 muM) and veratrine (5-50 mug-jl-1), externally applied to the intact nerve fiber or to axon-free nerve fiber sheaths, produce a Schwann cell depolarization which can be reversed by decreasing the external sodium concentration or by external application of tetrodotoxin. The magnitude of these membrane potential changes is related to the concentrations of the drugs in the external medium. These results indicate the existence of sodium pathways in the electrically unexcitable Schwann cell membrane of S. sepioidea, which can be opened up by grayanotoxin I and veratrine, and afterwards are blocked by tetrodotoxin. The sodium pathways of the Schwann cell membrane appear to be different from those of the axolemma which show a voltage-dependent conductance.
Some macroglial cells of the O-2A lineage express glutamate receptor channels of the quisqualate/kainate type and take up extracellular cobalt when activated by glutamate agonists. These cells can be identified both in vitro and in situ following precipitation and intensification of the intracellular cobalt. We have used this technique to characterize these cells in the developing and adult rat optic nerve. In purified cultures of optic nerve cells, O-2A progenitor cells and type 2 astrocytes took up cobalt in the presence of quisqualate, while oligodendrocytes, type 1 astrocytes, and microglial cells did not. When whole optic nerves of various postnatal ages were exposed to quisqualate and cobalt, a subpopulation of glial cells took up cobalt. Cobalt uptake in vitro and in situ was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. The number, morphology, and spatial distribution of cobalt-filled cells in situ varied with age. In perinatal nerves, 9% of glial cells took up cobalt. These cells had a simple unipolar or bipolar morphology and were two to three times more concentrated at the chiasm end than at the eye end of the nerve. During subsequent development, this gradient disappeared and the cobalt-filled cells became progressively more complex in morphology and increased in number and density, reaching a peak toward the end of the second postnatal week. The number subsequently declined to about 16,000 (7%) in the adult nerve. The processes of some cobalt-filled cells appeared to contact nodes of Ranvier. All cobalt-filled cells in 2 1/2-week-old optic nerves had a similar ultrastructural appearance and did not resemble either mature oligodendrocytes or astrocytes. Our results suggest that the cells stimulated by quisqualate to take up cobalt in the optic nerve are the in vivo counterpart of O-2A progenitor cells. We found no evidence that any of these cells are type 2 astrocytes.
Research over the past 25 years has identified specific ion transporters and channels that are activated by acute changes in cell volume and that serve to restore steady-state volume. The mechanism by which cells sense changes in cell volume and activate the appropriate transporters remains a mystery, but recent studies are providing important clues. A curious aspect of volume regulation in mammalian cells is that it is often absent or incomplete in anisosmotic media, whereas complete volume regulation is observed with isosmotic shrinkage and swelling. The basis for this may lie in an important role of intracellular Cl ⁻ in controlling volume-regulatory transporters. This is physiologically relevant, since the principal threat to cell volume in vivo is not changes in extracellular osmolarity but rather changes in the cellular content of osmotically active molecules. Volume-regulatory transporters are also closely linked to cell growth and metabolism, producing requisite changes in cell volume that may also signal subsequent growth and metabolic events. Thus, despite the relatively constant osmolarity in mammals, volume-regulatory transporters have important roles in mammalian physiology.
Since their discovery, glial cells have generally been considered to be passive elements with only a few of their properties being recognized as of great importance for the proper functioning of the nervous system, e.g., the formation of myelin by oligodendrocytes and Schwann cells (for reviews see Morell and Norton, 1980), the clearance of K + from the extracellular space by astrocytes (Orkand, 1977, 1980), the guidance of neurons during development (Rakic, 1981), and the uptake of neurotransmitters (Hertz, 1979). Neurons and glial cells are separated by the extracellular space and communication between these two cell populations requires that signals travel across the space. Release of K + into the extracellular space during neuronal activity and the response of glial cells, which take up the excess K + to regulate extracellular concentrations, is an example of such a signal between neurons and glial cells (e.g., Salem et al., 1975; Sykova and Orkand, 1980; Walz and Hertz, 1983b). The potassium uptake by glial cells is mediated by passive processes, namely spatial buffering and passive KC1 uptake, and/or by stimulation of the Na +/K + -ATPase (Kettenmann, 1987a). The efficiency of spatial buffering seems to be determined by the density and distribution of K + channels (Newman, 1985a,b, 1986; Orkand, 1977), that of KCl uptake by the relative density of K + and Cl− channels (Ballanyi et al., 1986; Kettenmann, 1987b). Thus, expression of Cl− channels in glia can play a functional role in K + homeostasis. Recent observations indicate that not only K + undergoes changes in the extracellular space during neuronal activity, but also Na +, Ca24, H +, and Cl−(Chesler, 1987; Dietzel et al., 1982; Nicholson, 1980a,b). Thus, glial cells may be involved in controlling the free concentration of other physiologically relevant ions including H + and Cl. This would not be surprising, since most ion transport systems across cell membranes function as co- or countertransporters. A well-known example is the countertransport of Na + and K + by the Na + /K + -ATPase, which is present in glial cells (Orkand, 1977). Other transport systems present in glial cells include Na +/H + and Cl−/HCO−3 exchangers, Na +/HCO−3 and K +/CI− cotransporters (Hoppe and Kettenmann, 1989a; Kettenmann and Schlue, 1988; Kimelberg et al.,1979). The combined activity of these carriers and the Na +, K +, Cl−, and HCO−3 channels which can be expressed by glial cells (Bevan et al., 1986; Gray et al., 1986; Kettenmann, 1987b; Astion et al., 1987; Tang et al., 1979) are likely to strongly influence nervous tissue extracellular ionic microenvironment.
Ions flow across cell membranes by two major classes of transport mechanisms. One class is represented by ion channels, which are best viewed as water-filled pathways through specific proteins embedded in the membrane lipid bilayer (Hille, 1984). Such channels show selectivity for different ions according to size and charge. The rate of movement of ions through these channels is determined by the electrochemical driving force on the ion and the individual conductances of the channels. Characteristically, channel-mediated fluxes are very large, in excess of 106 ions/sec per channel (Hille, 1984). The number of channels per unit area, the rate at which these channels open, and the duration of time they are open are important determinants of channel-mediated ion fluxes. Channel opening can be modified by transmembrane voltage, specific ligands, or when tension is applied to the membrane (Sachs, 1988). In the case of the various chloride channels, all these forces seem to operate as we shall learn later in this chapter as well as in other chapters in this volume.
The actions of grayanotoxin I, veratrine, and tetrodotoxin on the membrane potential of the Schwann cell were studied in the giant nerve fiber of the squid Sepioteuthis sepioidea. Schwann cells of intact nerve fibers and Schwann cells attached to axons cut lengthwise over several millimeters were utilized. The axon membrane potential in the intact nerve fibers was also monitored. The effects of grayanotoxin I and veratrine on the membrane potential of the Schwann cell were found to be similar to those they produce on the resting membrane potential of the giant axon. Thus, grayanotoxin I (1-30 muM) and veratrine (5-50 mug-jl-1), externally applied to the intact nerve fiber or to axon-free nerve fiber sheaths, produce a Schwann cell depolarization which can be reversed by decreasing the external sodium concentration or by external application of tetrodotoxin. The magnitude of these membrane potential changes is related to the concentrations of the drugs in the external medium. These results indicate the existence of sodium pathways in the electrically unexcitable Schwann cell membrane of S. sepioidea, which can be opened up by grayanotoxin I and veratrine, and afterwards are blocked by tetrodotoxin. The sodium pathways of the Schwann cell membrane appear to be different from those of the axolemma which show a voltage-dependent conductance.
: Transport and permeability properties of the blood-brain and blood-CSF barriers were determined by kinetic analysis of radioisotope uptake from the plasma into the CNS of the adult rat. Cerebral cortex and cerebellum uptake curves for 36Cl and 22Na were resolved into two components. The fast component (t½ 0.02–0.05 h, fractional volume 0.04–0.08) is comprised of the vascular compartment and a small perivascular space whereas the slow component (t½ 1.06–1.69 h, fractional volume 0.92–0.96) represents isotope movement across the blood-brain barrier into the brain extracellular and cellular compartments. Uptake curves of both 36Cl and 22Na into the CSF were also resolved into two components, a fast component (t½ 0.18 h, fractional volume 0.24) and a slow component (t½ 1.2 h, fractional volume 0.76). Evidence suggests that the fast component represents isotope movement across the blood-CSF barrier, i.e., the choroid plexuses, whereas the CSF slow component probably reflects isotope penetration primarily from the brain extracellular fluid into the CSF. The extracellular fluid volume of the cerebral cortex and cerebellum was estimated as ˜13% from the initial slope of the curve of brain space versus CSF space curve for both 36Cl and 22Na. Like the choroid plexuses, the glial cell compartment of the brain appears to accumulate Cl from 2 to 6 times that predicted for passive distribution. The relative permeability of the blood-CSF and blood-brain barriers to 36Cl, 22Na, and [3H]mannitol was determined by calculating permeability surface-area products (PA). Analysis of the PA values for all three isotopes indicates that the effective permeability of the choroidal epithelium (blood/CSF barrier) is significantly greater than that of the capillary endothelium in the cerebral cortex and cerebellum (blood-brain barrier).
The effect of glutamate on c-fos expression in oligodendrocyte progenitors was investigated by Northern blot analysis. Glutamate caused rapid and transient induction. Both 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX), two competitive non-NMDA ionotropic receptor antagonists, reduced glutamate-induced c-fos expression, whereas the NMDA antagonist MK-801 was ineffective. In addition, the glutamate receptor agonists (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) and kainate strongly induced c-fos. However, the metabotropic receptor agonist trans-(±)-1-amino-(1S,3R)-cyclopentanedicarboxylic acid (trans-(±)-ACPD) did not increase c-fos mRNA level and the antagonist L-(+)-2-amino-3-phosphonopropionic acid did not block glutamate-induced c-fos mRNA. These findings indicate that c-fos induction in oligodendrocyte progenitors is mediated through the AMPA/kainate receptors, while NMDA and metabotropic receptor subtypes are not involved. Chelation of extracellular calcium by EDTA prevented glutamate-induced c-fos expression. Similarly, the protein kinase C inhibitor 1-(5-isoquinoline-sulphonyl)-2-methylpiperazine dihydrochloride (H7) and down-regulation of protein kinase C by prolonged exposure to phorbol-12-myristate 13-acetate blocked c-fos induction. These results suggest that induction of c-fos through AMPA/kainate receptors is dependent on extracellular calcium influx and involves downstream activation of phorbol ester-sensitive protein kinase C. The effect of glutamate on oligodendrocyte progenitor proliferation was assessed by [3H]thymidine incorporation. Glutamate and the agonists kainate and AMPA, but not trans-(±)-ACPD, caused a dose-dependent decrease in [3H]thymidine incorporation. All these pharmacological agents were not toxic to oligodendrocyte progenitors. CNQX reversed the inhibitory effects produced by glutamate and the various agonists. These results suggest that glutamate may modulate the growth and differentiation of oligodendrocytes in the central nervous system.
Oligodendrocytes and their progenitors (O-2A) express functional kainate- and DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring glutamate receptors. The physiological consequences of activation of these receptors were studied in purified rat cortical O-2A progenitors and in the primary oligodendrocyte cell line CG-4. Changes in the mRNA levels of a set of immediate early genes were studied and were correlated to intracellular Ca2+ concentration, as measured by fura-2 Ca2+ imaging. Both in CG-4 and in cortical O-2A progenitors, basal mRNA levels of NGFI-A were much higher than c-fos, c-jun, or jun-b. Glutamate, kainate, and AMPA greatly increased NGFI-A mRNA and protein by activation of membrane receptors in a Ca2+-dependent fashion. Agonists at non-N-methyl-D-aspartate receptors promoted transmembrane Ca2+ influx through voltage-dependent channels as well as kainate and/or AMPA channels. The influx of Ca2+ ions occurring through glutamate-gated channels was sufficient by itself to increase the expression of NGFI-A mRNA. AMPA receptors were found to be directly involved in intracellular Ca2+ and NGFI-A mRNA regulation, because the effects of kainate were greatly enhanced by cyclothiazide, an allosteric modulator that selectively suppresses desensitization of AMPA but not kainate receptors. Our results indicate that glutamate acting at AMPA receptors regulates immediate early gene expression in cells of the oligodendrocyte lineage by increasing intracellular calcium. Consequently, modulation of these receptor channels may have immediate effects at the genomic level and regulate oligodendrocyte development at critical stages.
The cell-attached and excised patch configurations of the patch clamp technique were used to characterize Ca(2+)-activated maxi-K+ channels in freshly-isolated Müller glial cells. The cells were dissociated from postmortem adult human and porcine retinas. The maxi-K+ channels in Müller cells of both species display a single channel conductance of 175 pS in cell-attached and inside-out patches (125/110 mM K+). The channels are activated by membrane depolarization and by elevation of intracellular Ca2+. In the presence of 10(-5), 10(-4), and 10(-3) M intracellular free Ca2+, the half-activation voltages are +7.2, -26.6, and -47.5 mV, respectively. The half-activation-[Ca2+] at +10 mV is 8.1 microM, and the Hill coefficient of Ca2+ binding is 1.7, Ba2+ exerts a voltage-dependent block of the open-state probability. The maxi-K+ channels of Müller cells are activated by raising of the intracellular pH as well as by Mg2- ions at the cytosolic face of the channels. Phosphorylation of the channel after cytosolic addition of the catalytic subunit of a cAMP dependent protein kinase in the presence of Mg-ATP caused a shift of the activation curve to negative membrane potentials. Between -40 and -80 mV membrane potentials, the open-state probability rose to 190.3% of the control value (100%) after phosphorylation of the channel. Therefore, phosphorylation enhances sensitivity of the channels to Ca2+ and voltage. The maxi-K+ channels may provide a link between second messenger systems and membrane conductance of retinal Müller cells and may have an important function in repolarization of the Müller cell membrane and, therefore, in the maintainance of the retinal spatial K+ buffering mechanisms.
The past three years have seen remarkable progress in research on the molecular basis of inward rectification, with significant implications for basic understanding and pharmacological manipulation of cellular excitability. Expression cloning of the first inward rectifier K channel (Kir) genes provided the necessary break-through that has led to isolation of a family of related clones encoding channels with the essential functional properties of classical inward rectifiers, ATP-sensitive K channels, and muscarinic receptor-activated K channels. High-level expression of cloned channels led to the discovery that classical inward so-called anomalous rectification is caused by voltage-dependent block of the channel by polyamines and Mg2+ ions, and it is now clear that a similar mechanism results in inward rectification of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-kainate receptor channels. Knowledge of the primary structures of Kir channels and the ability to mutate them also has led to the determination of many of the structural requirements of inward rectification.
To understand the physiology of Schwann cells and myelinated nerve, we have been engaged in identifying K+ channels in sciatic nerve and determining their subcellular localization. In the present study, we examined the slo family of Ca2+-activated K+ channels, a class of channel that had not previously been identified in myelinated nerve. We have determined that these channels are indeed expressed in peripheral nerve, and have cloned rat homologues of slo that are more than 95% identical to the murine slo. We found that sciatic nerve RNA contained numerous alternatively spliced variants of the slo homologue, as has been seen in other tissues. We raised a polyclonal antibody against a peptide from the carboxyl terminal of the channels. Immunocytochemistry revealed that the channel proteins are in Schwann cells and are associated with canaliculi that run along the outer surface of the cells. They are also relatively concentrated near the node of Ranvier in the Schwann cell outer membrane. This staining pattern is quite similar to what we previously reported for the voltage-dependent K+ channel Kv1.5. We did not observe staining of axons or connective tissue in the nerve and so it seems likely that most or all of the splicing variants are located in the Schwann cells. The localization of these channels also suggests that they may participate in maintaining the resting potential of the Schwann cells during K+ buffering. GLIA 26:166–175, 1999. © 1999 Wiley-Liss, Inc.
Expression cloning revealed a chloride channel (ICln) that we found to be fundamental for the regulatory volume decrease in a variety of cells. The chromosomal localization of the human ICln-gene showed two loci, one at chromosome 11 in position q13.5–q14.1, termed CLNS1A, and a second one at chromosome 6 in position p12.1–q13, termed CLNS1B. In this study, we offer a detailed characterization of the CLNS1A gene and provide the exact position (6p12) and sequence data of CLNS1B, an intronless gene 91.3% homologous to the coding region of CLNS1A.
Glioma cells in acute slices and in primary culture, and glioma-derived human cell lines were screened for the presence of functional GABAA receptors. Currents were measured in whole-cell voltage clamp in response to γ-aminobutyric acid (GABA). While cells from the most malignant glioma, the glioblastoma multiforme, did not respond to GABA, an inward current (under our experimental conditions with high Cl− concentration in the pipette) was induced in gliomas of lower grades, namely in 71% of oligodendroglioma cells and in 62% of the astrocytoma cells. Glioma cell lines did not express functional GABAA receptors, irrespective of the malignancy of the tumour they originate from. The currents elicited by application of GABA were due to activation of GABAA receptors; the specific agonist muscimol mimicked the response, the antagonists bicuculline and picrotoxin blocked the GABA-activated current and the benzodiazepine receptor agonist flunitrazepam augmented the GABA-induced current and the benzodiazepine inverse agonist DMCM decreased the GABA current. Cells were heterogeneous with respect to the direction of the current flow as tested in gramicidin perforated patches: in some cells GABA hyperpolarized the membrane, while in the majority it triggered a depolarization. Moreover, GABA triggered an increase in [Ca2+]i in the majority of the tumour cells due to the activation of Ca2+ channels. Our results suggest a link between the expression of GABA receptors and the growth of glioma cells as the disappearance of functional GABAA receptors parallels unlimited growth typical for malignant tumours and immortal cell lines.
Glutamate is an excitatory receptor agonist in both neurones and glial cells, and, in addition, glutamate is also a substrate for glutamate transporter in glial cells. We have measured intracellular and extracellular pH changes induced by bath application of glutamate, its receptor agonist kainate, and its transporter agonist aspartate, in the giant neuropile glial cell in the central nervous system of the leech Hirudo medicinalis, using double-barrelled pH-sensitive microelectrodes. The giant glial cells responded to glutamate and aspartate (100–500 μM), and kainate (5–20 μM) with a membrane depolarization or an inward current, and with a distinct intracellular acidification. Glutamate and aspartate (both 500 μM) evoked a decrease in intracellular pH (pHi) by 0.187 ± 0.081 (n = 88) and 0.198 ± 0.067 (n = 86) pH units, respectively. With a resting pHi of 7.1 or 80 nM H+, these acidifications correspond to a mean increase of the intracellular H+ activity by 42 nM and 45 nM. Kainate caused a decrease of pHi by 0.1 − 0.35 pH units (n = 15). The glutamate/aspartate-induced decrease in pHi was not significantly affected by the glutamate receptor blockers kynurenic acid (1 mM) and 6-cyano-7-dinitroquinoxaline-2,3-dione (CNQX, 50–100 μM), which greatly reduced the kainate-induced change in pHi. Extracellular alkalinizations produced by glutamate and aspartate were not affected by CNQX. Reduction of the external Na+ concentration gradually decreased the intracellular pH change induced by glutamate/aspartate, indicating half maximal activation of the acidifying process at 5–10 mM external Na+ concentration. When all external Na+ was replaced by NMDG+, the pHiresponses were completely suppressed (glutamate) or reduced to 10% (aspartate). When Na+ was replaced by Li+, the glutamate- and aspartate-evoked pHi responses were reduced to 18% and 14%, respectively. Removal of external Ca2+ reduced the glutamate- and aspartate-induced pHi responses to 93 and 72%, respectively. The glutamate/aspartate-induced intracellular acidifications were not affected by the putative glutamate uptake inhibitor amino-adipidic acid (1 mM). DL-aspartate-β-hydroxamate (1 mM), and dihydrokainate (2 mM), which caused some pHi decrease on its own, reduced the glutamate/aspartate-induced pHi responses by 40 and 69%, respectively. The putative uptake inhibitor DL-threo-β-hydroxyaspartate (THA, 1 mM) induced a prominent intracellular acidification (0.36 ± 0.05 pH units, n = 9), and the pHi change evoked by glutamate or aspartate in the presence of THA was reduced to less than 10%. The results indicate that glutamate, aspartate, and kainate produce substantial intracellular acidifications, which are mediated by at least two independent mechanisms: 1) via activation of non-NMDA glutamate receptors and 2) via uptake of the excitatory amino acids into the leech glial cell. © 1997 Wiley-Liss Inc.
The voltage-gated currents of the astrocytes associated with the retinal capillaries of the rabbit retina were studied using whole-cell patch clamp recording. The resting potential of these cells was −70 ± 4.8 mV (mean ± SEM; n= 54), and the input resistance and cell capacitance were 558 ± 3.6 MΩ and 19.5 ± 1.8 pF respectively. Depolarization to potentials positive to −50 mV evoked rapidly activating inward and outward currents. The inward current was transient, eliminated by substitution of choline for Na+ in the bathing solution, and reduced by 50% in the presence of 1 μM tetrodotoxin. The time-to-peak of the Na+ current was more than twice that for the Na+ current found in retinal neurons. The glial Na+ current was half-inactivated at −55 mV. A transient component of the outward K+ current was blocked by external 4-aminopyridine while a more sustained component was blocked by external tetraethylammonium. At potentials between −150 and −50 mV the membrane behaved Ohmically. Voltage-gated currents in retinal astrocytes recorded in situ appear qualitatively similar to those described for some glial cells in vitro.
We previously demonstrated that the inhibitory neurotransmitter glycine induced membrane currents in glial cells from rat spinal cord. In the present study, the patch-clamp technique was combined with the reverse transcription-mediated PCR to analyze the glycine receptor-subunit expression in individual glial cells of rats age 3–18 days. Using the patch-clamp technique in the whole-cell configuration, glial cells were identified by their membrane current pattern and tested for responsiveness to glycine. Subsequently, the cytoplasm was harvested followed by reverse transcription of total cytoplasmic RNA. Subunit-specific cDNA fragments were amplified and analyzed by agarose gel electrophoresis, Southern blotting, and sequencing. In all glial cell types investigated, transcripts of the α1 subunit, but not of α2 or α3 subunits, were detected. In addition, about one-half the glial cells analyzed contained β-subunit mRNA. These results illustrate that glial cells of rat spinal cord express functional glycine receptors in contrast to cultured glial cells. Glial cells are in intimate contact with synaptic regions making it likely that these nonneuronal receptors may be activated during glycinergic transmission and may trigger yet unknown responses in the glial cells.
Recently, we could demonstrate that ‘complex’ glial cells in mouse hippocampal slices express glutamate receptor channels of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate subtypes. In the present study, we further characterized this glial receptor. Since voltage-clamp control is imperfect and diffusion barriers hinder the quantitative analysis of the receptor currents in situ, the patch-clamp technique was applied to glial cells acutely isolated from the mouse hippocampal CA1 stratum radiatum subregion. A concentration-clamp technique was used which enabled very fast exchange of the extracellular solutions. Thus, it was possible to characterize the transient receptor currents with high time resolution. Application of L-glutamate, AMPA and L-homocysteate induced rapidly activating and fast desensitizing receptor currents in the suspended glial cells. In contrast, kainate induced non-desensitizing currents. The corresponding dose-response curve revealed a half-maximum of current activation at 350 μM. The current/voltage relationship of the kainate-evoked response was linear, with a reversal potential at ∼9 mV. Analysis of the reversal potential in solutions containing high concentrations of CaCl2 confirmed earlier in situ data by demonstrating significant Ca2+ permeability of the glial glutamate receptor channels in the hippocampus. The kainate-induced receptor currents were markedly increased by cyclothiazide, a substance which selectively potentiates glutamate receptors of the AMPA subtype. We conclude that glial cells of the juvenile hippocampus mainly express heteromeric high-affinity AMPA receptors. Most probably, the receptor channels are assembled from the low Ca2+-permeable glutamate receptor-2 subunit together with Ca2+-permeable AMPA-preferring subunits.
We have used postnatal rat cerebellar astrocyte-enriched cultures to study the excitatory amino acid receptors present on these cells. In the cultures used, type-2 astrocytes (recognized by the monoclonal antibodies A2B5 and LB1) selectively took up γ-[3H]aminobutyric acid ([3H]GABA) and released it when incubated in the presence of micromolar concentrations of kainic and quisqualic acids. The releasing effect of kainic acid was concentration dependent in the range of 5–100 μM. Quisqualate was more effective than kainate in the lower concentration range but less effective at concentrations at which its releasing activity was maximal (∼50 μM). N-Methyl-d-aspartic acid and dihydrokainate (100 μM) did not stimulate [3H]GABA release from cultured astrocytes. l-Glutamic acid (20–100 μM) stimulated [3H]GABA release as effectively as kainate. The stimulatory effects of kainate and quisqualate on [3H]GABA release were completely Na+ dependent; that of kainate was also partially Ca2+ dependent. Kynurenic acid (50–200 μM) selectively antagonized the releasing effects of kainic acid and also that of l-glutamate; quisqualate was unaffected. Quisqualic acid inhibited the releasing effects of kainic acid when both agonists were used at equimolar concentrations (50 μM). d-[3H]aspartate was taken up by both type-1 and type-2 astrocytes, but only type-2 astrocytes released it in the presence of kainic acid. Excitatory amino acid receptors with a pharmacology similar to that of the receptors present in type-2 astrocytes were also expressed by the immature, bipotential progenitors of type-2 astrocytes and oligodendrocytes.
This lecture is dedicated to Max Delbrück and Seymour Benzer. Max Delbrück was our graduate advisor. He introduced us to a variety of biophysical problems, and taught us ways of thinking about these problems by example. Potassium channels was one of the topics included in his journal club in the early seventies; Max also carefully considered the feasibility of purifying potassium channels then. It was in Seymour Benzer's laboratory that we began to look for Drosophila mutants that aÍfect synaptic transmission at the larval neuromuscular junction. Shaker was the first behavioural mutant we tested that gave a robust phenotype, a phenotype that could be mimicked by treating wild‐type preparations with a potassium channel blocker. This mutant fly has led us to our subsequent molecular studies of potassium channels.
Since we settled in the University of California, San Francisco, and began to study neural development as well as potassium channels, we have settled into the pattern of each attending meetings and presenting our studies on one of these two areas so as to avoid both being away from home and our children at the same time. In following this pattern, I will be presenting the studies of potassium channels as part of our long‐term collaboration. In this talk I will first briefly take you through the path that led us to the molecular studies of potassium channels and then discuss the diversity and modulation of these potassium channels at the molecular and physiological level.
1. Whole-cell recordings were obtained from Bergmann glial cells in rat cerebellar slices. 2. The cells had low input resistances (70 +/- 38 M omega; n = 13) and a mean resting potential of -82 +/- 6 mV (n = 12) with a potassium-based internal solution. Electrical and dye coupling between Bergmann glia were confirmed. 3. Stimulation of parallel fibres induced a complex, mostly inward current which could be decomposed pharmacologically. 4. The ionotropic glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM), but not DL-2-amino-5-phosphonopentanoic acid (DL-APV; 100 microM) consistently blocked an early inward current component that may reflect synaptic activation of AMPA/kainate receptors in Bergmann glia. 5. Addition of cadmium ions (100 microM) to inhibit transmitter release blocked most of the CNQX-APV-insensitive current. This component probably reflects electrogenic uptake of the synaptically released glutamate. 6. Tetrodotoxin (TTX; 1 microM) blocked the remaining inward current: a slow component, possibly produced by the potassium ion efflux during action potential propagation in parallel fibres. An initial triphasic component of the response was also TTX sensitive and reflected passage of the parallel fibre action potential volley. 7. The putative glutamate uptake current was further characterized; it was blocked by the competitive uptake blockers D-aspartate (0.5 mM) and L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC; 0.5 mM), and by replacement of sodium with lithium. Monitoring the triphasic TTX-sensitive component showed that this inhibition did not result from changes of action potential excitation and propagation. 8. Intracellular nitrate ions increased the putative uptake current, consistent with the effect of this anion on glutamate transporters. 9. The putative uptake current was reduced by depolarization, consistent with the voltage dependence of glutamate uptake. 10. It is concluded that a large fraction of the current induced by parallel fibre stimulation reflects the uptake of synaptically released glutamate. The uptake current activated rapidly, with a 20-80% rise time of 2.3 +/- 0.7 ms (n = 10), and decayed with a principal time constant of 25 +/- 6 ms (n = 10).
The distribution of AMPA-selective subunits, GluR1–4, was determined in the human hippocampus and cerebellum by in situ hybridization and immunocytochemistry. In the hippocampus, in situ hybridization revealed that GluRl and GluR2 mRNAs were similarly distributed and highly expressed in the dentate gyrus, with lower levels in the CA regions. GluR3 and GluR4 mRNAs were expressed at very low levels. Immunocytochemical studies showed that GluRl- and GluR2/3-immunoreactivity were highest in the dentate molecular and granular layers. In the CA regions, GluRl and GluR2/3 staining was observed in pyramidal cell bodies and surrounding neuropil and was more intense in CA4/3/2 compared with CA1. GIuR4-immunoreactivity was low throughout the hippocampus. In the cerebellum, GluRl and G1uR4 transcripts were expressed in the granular and Purkinje cell/Bergmann glia layers. GluR2 mRNA was highly expressed in the granular layer and individual Purkinje cells, while GluR3 mRNA was not detectable in the cerebellum. GluR1- and GluR4-immunoreactivity were localized to Purkinje cells and putative Bergmann glia, as well as their processes extending into the molecular layer. GluR2/3 staining was intense in Purkinje cells, with moderate staining in the granular layer. Thus, GluR1–4 subunits are differentially distributed in the hippocampus and cerebellum. In addition, the distribution of subunit mRNA and protein correlate well with each other and with the glutamatergic neuroanatomy of the hippocampus and cerebellum.
For many years acetylcholine was considered merely as a neurotransmitter passing ‘information’ from one neurone to the next. Then came the finding that the Schwann cell also reacted to the passage of impulses along the axon which it was engulfing, in that the Schwann cell became hyperpolarized. The involvement of acetylcholine in a chemical coupling between the axon and its Schwann cell appears to be involved in the mechanism behind this hyperpolarization. However, as is explained in this article, this is probably not the whole story.
Mitogenesis in a variety of tissues is known to be inhibited by K+ channel blockers such as tetraethylammonium (TEA) and 4-aminopyridine (4-AP). Using radiolabeled thymidine as a proliferation index we have examined what role, if any, specific K+ channels have in cultured Schwann cells that have been induced to proliferate by pre-exposure to mitogens. TEA and 4-AP are "broad-spectrum" in that they block a variety of different types of K+ channel. In contrast, we found that alpha-dendrotoxin (alpha-DTX), a specific blocker of the type 1 fast delayed rectifier current (the largest component of Schwann cell K+ current) does not affect proliferation, suggesting that type 1 current may not be involved in mitogenesis. This suggestion is supported by our finding that the values of the KD for the mitogenic effect (722 nM, 4-AP; 13 mM, TEA) are much larger than the corresponding electrophysiological values for type 1 channels (0.1 mM, 4-AP; 0.2 mM, TEA). Charybdotoxin (200 nM) and iberiotoxin (100 nM), inhibitors of Ca2+-activated K+ channels, cesium (5 mM), an inhibitor of inward rectifier channels, and furosemide (100 pM), which blocks Na+/K+/Cl- cotransport, all had no effect on proliferation. Interestingly, 4,4'-diisothiocyanatostilbene 2,2'-disulphonate (DIDS), which blocks voltage-gated Cl- channels, reduced proliferation. In summary, broad-spectrum K+ channel blockers inhibit Schwann cell proliferation, but inhibitors specific for type 1, Ca2+-activated, and inward rectifier K+ channels do not. Whether the inhibition is mediated by type 2 K- channels, by an as yet unidentified Schwann cell K+ channel, or by another mechanism remains unclear.
Two distinct morphological subtypes of astrocytes have been shown to express Na+ currents that differ biophysically and pharmacologically. Using an in vitro model for reactive gliosis, we recently reported marked changes in Na+ and K+ channel expression by astrocytes induced to proliferate. Using this in vitro assay in which a confluent monolayer of astrocytes is mechanically scarred to induce gliosis, we now demonstrate that sodium currents of scar-associated cells, in addition to doubling in current density, also switch from being tetrodotoxin-sensitive(TTX-S, IC50 8 nM) to being approximately 40-fold more TTX-resistant (TTX-R,IC50 314 nM). These changes occurred within 6 h after injury and were not associated with any notable changes in cell morphology. Changes in biophysical properties were analyzed for the two current types. The activation curve for TTX-R currents demonstrated a significant depolarized shift versus that of TTX-S currents (P </= 0. 003), and TTX-R currents have more depolarized V1/2 of activation (-33 vs. -23 mV). The V1/2 of inactivation was slightly, but not significantly, more depolarized for TTX-R currents as compared to TTX-S (-63 vs. -68 mV). Most notably, TTX-R currents showed significantly slower inactivation kinetics at depolarized voltage potentials than TTX-S sodium currents (0.76 vs. 1.128 ms, at -10 mV; P < 0.0004).
Using whole-cell patch-clamp recordings, we identified a novel voltage-activated chloride current that was selectively expressed in glioma cells from 23 patient biopsies. Chloride currents were identified in 64% of glioma cells studied in acute slices of nine patient biopsies. These derived from gliomas of various pathological grades. In addition, 98% of cells acutely isolated or in short-term culture from 23 patients diagnosed with gliomas showed chloride current expression. These currents, which we termed glioma chloride currents activated at potentials >45 mV, showed pronounced outward rectification, and were sensitive to bath application of the presumed Cl- channel specific peptide chlorotoxin (approximately 600 nM) derived from Leiurus scorpion venom. Interestingly, low grade tumours (e.g., pilocytic astrocytomas), containing more differentiated, astrocyte-like cells showed expression of glioma chloride currents in concert with voltage-activated sodium and potassium currents also seen in normal astrocytes. By contrast, high grade tumours (e.g., glioblastoma multiforme) expressed almost exclusively chloride currents, suggesting a gradual loss of Na+ currents and gain of Cl- currents with increasing pathological tumour grade. To expand on the observation that these chloride currents are glioma-specific, we introduced experimental tumours in scid mice by intracranial injection of D54MG glioma cells and subsequently recorded from tumour cells and adjacent normal glial cells in acute slices. We consistently observed expression of chlorotoxin-sensitive chloride channels in implanted glioma cells, but without evidence for expression of chloride channels in surrounding "normal" host glial cells, suggesting that these chloride channels are probably a glioma-specific feature. Finding of this novel glioma specific Cl- channel in gliomas in situ and it's selective binding of chlorotoxin may provide a way to identify or target glioma cells in the future.
Recording of glutamate-activated currents in membrane patches was combined with RT-PCR-mediated AMPA receptor (AMPAR) subunit mRNA analysis in single identified cells of rat brain slices. Analysis of AMPARs in principal neurons and interneurons of hippocampus and neocortex and in auditory relay neurons and Bergmann glial cells indicates that the GluR-B subunit in its flip version determines formation of receptors with relatively slow gating, whereas the GluR-D subunit promotes assembly of more rapidly gated receptors. The relation between Ca2+ permeability of AMPAR channels and the relative GluR-B mRNA abundance is consistent with the dominance of this subunit in determining the Ca2+ permeability of native receptors. The results suggest that differential expression of GluR-B and GluR-D subunit genes, as well as splicing and editing of their mRNAs, account for the differences in gating and Ca2+ permeability of native AMPAR channels.
Functionally diverse GIuR channels of the AMPA subtype are generated by the assembly of GIuR-A, -B, -C, and -D subunits into homo- and heteromeric channels. The GIuR-B subunit is dominant in determining functional properties of heteromeric AMPA receptors. This subunit exists in developmentally distinct edited and unedited forms, GIuR-B(R) and GIuR-B(Q), which differ in a single amino acid in transmembrane segment TM2 (Q/R site). Homomeric GIuR-B(R) channels expressed in 293 cells display a low divalent permeability, whereas homomeric GluR-B(Q) and GIuR-D channels exhibit a high divalent permeability. Mutational analysis revealed that both the positive charge and the size of the amino acid side chain located at the Q/R site control the divalent permeability of homomeric channels. Coexpression of Q/R site arginine- and glutamine-containing subunits generates cells with varying divalent permeabilities depending on the amounts of expression vectors used for cell transfection. Intermediate divalent permeabilities were traced to the presence of both divalent permeant homomeric and impermeant heteromeric channels. It is suggested that the positive charge contributed by the arginine of the edited GIuR-B(R) subunit determines low divalent permeability in heteromeric GIuR channels and that changes in GIuR-B(R) expression regulate the AMPA receptor-dependent divalent permeability of a cell.
gamma-Aminobutyric acid (GABA)-activated Cl- currents in neonatal rat cortical neurons and in cultured cells engineered for the expression of specific molecular forms of the GABAA receptor alpha, beta, and gamma subunits, were recorded with the patch-clamp technique in the whole- cell configuration. The effects of various allosteric modulators of GABAA receptors were determined. Diazepam and clonazepam showed greater efficacy as positive modulators of GABA-elicited currents in alpha 2 beta 1 gamma 2 or alpha 3 beta 1 gamma 2 receptors than in alpha 1 beta 1 gamma 2 or alpha 5 beta 1 gamma 2 receptors or in cortical neurons. Alpidem was more efficacious at alpha 1 beta 1 gamma 2 or alpha 2 beta 1 gamma 2 receptors than at alpha 1 beta 1 gamma 2 or alpha 5 beta 1 gamma 2 receptors or in cortical neurons. Conversely, zolpidem was equally efficacious for all these receptors except for alpha 5 beta 1 gamma 2. Both imidazopyridines (alpidem and zolpidem) were virtually ineffective at modulating the GABA response of alpha 5 beta 1 gamma 2 receptors and in almost all the receptors assembled from alpha 1, alpha 2, alpha 3 or alpha 5 subunits together with beta 1 and gamma 1 subunits. The beta-carboline derivatives methyl-6,7-dimethoxy-4-ethyl- beta-carboline-3-carboxylate (DMCM) and methyl-beta-carboline-3- carboxylate (beta-CCM) elicited a positive allosteric modulation of alpha 1 beta 1 gamma 1 or alpha 2 beta 1 gamma 1 receptors, whereas they acted as negative allosteric modulators at nearly all other receptors tested, as they do in cortical neurons. Although the positive allosteric modulation by beta-carbolines never exceeded a doubling of the GABA response, DMCM was more efficacious at alpha 1 beta 1 gamma 1 receptors and beta-CCM was more efficacious at alpha 2 beta 1 gamma 1 receptors. DMCM was inactive at alpha 3 beta 1 gamma 1 receptors, whereas beta-CCM was virtually inactive at alpha 5 beta 1 gamma 1 receptors. The benzodiazepine 4-chlorodiazepam, which is a negative modulator resistent to flumazenil inhibition, acted at all the various GABAA receptors that contained a gamma subunit.
1. Glutamate receptor (GluR) channels were studied in basket cells in the dentate gyrus of rat hippocampal slices. Basket cells were identified by their location, dendritic morphology and high frequency of action potentials generated during sustained current injection. 2. Dual-component currents were activated by fast application of glutamate to outside-out membrane patches isolated from basket cell somata (10 microM glycine, no external Mg2+). The fast component was selectively blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the slow component by D-2-amino-5-phosphonopentanoic acid (D-AP5). This suggests that the two components were mediated by alpha-amino-3- hydroxy-5- methyl-4-isoxazolepropionate receptor (AMPAR)/kainate receptor and N- methyl-D-aspartate receptor (NMDAR) channels, respectively. The mean ratio of the peak current of the NMDAR component to that of the AMPAR/kainate receptor component was 0.22 (1 ms pulses of 10 mM glutamate). 3. The AMPAR/kainate receptor component, which was studied in isolation in the presence of D-AP5, was identified as AMPAR mediated on the basis of the preferential activation by AMPA as compared with kainate, the weak desensitization of kainate-activated currents, the cross-desensitization between AMPA and kainate, and the reduction of desensitization by cyclothiazide. 4. Deactivation of basket cell AMPARs following 1 ms pulses of glutamate occurred with a time constant (tau) of 1.2 +/- 0.1 ms (mean +/- S.E.M.). During 100 ms glutamate pulses AMPARs desensitized with a tau of 3.7 +/- 0.2ms. 5. The peak current- voltage (I-V) relation of AMPAR-mediated currents in Na(+)-rich extracellular solution showed a reversal potential of -4.0 +/- 2.6 mV and was characterized by a a doubly rectifying shape. The conductance of single AMPAR channels was estimated as 22.6 +/- 1.6 pS using non- stationary fluctuation analysis. AMPARs expressed in hippocampal basket cells were highly Ca2+ permeable (PCa/PK = 1.79). 6. NMDARs in hippocampal basket cells were studied in isolation in the presence of CNQX. Deactivation of NMDARs activated by glutamate pulses occurred bi- exponentially with mean tau values of 266 +/- 23 ms (76%) and 2620 +/- 383 ms (24%). 7. The peak I-V relation of the NMDAR-mediated component in Na(+)-rich extracellular solution showed a reversal potential of 1.5 +/- 0.6 mV and a region of negative slope at negative membrane potentials in the presence of external Mg2+, due to voltage-dependent block by these ions. The conductance of single NMDAR channels in the main open state was 50.2 +/- 1.8 pS.
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1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.
Many physiologically important activities of oligodendrocyte progenitor cells (O‐2A cells), including proliferation, migration and differentiation, are regulated by cytosolic Ca ²⁺ signals. However, little is known concerning the mechanisms of Ca ²⁺ signalling in this cell type. We have studied the interactions between Ca ²⁺ entry, Ca ²⁺ release from endoplasmic reticulum and Ca ²⁺ regulation by mitochondria in influencing cytosolic Ca ²⁺ responses in O‐2A cells.
Methacholine (MCh; 100 μM) activated Ca ²⁺ waves that propagated from several initiation sites along O‐2A processes.
During a Ca ²⁺ wave evoked by MCh, mitochondrial membrane potential was often either depolarized (21 % of mitochondria) or hyperpolarized (20 % of mitochondria), as measured by changes in the fluorescence of 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazole carbocyanine iodide (JC‐1).
Stimulation with kainate (100 μM) evoked a slowly rising, sustained cytosolic Ca ²⁺ elevation in O‐2A cells. This also, in some cases, resulted in either a depolarization (15 % of mitochondria) or hyperpolarization (12 % of mitochondria) of mitochondrial membrane potential.
Simultaneous measurement of cytosolic (fluo‐3 AM) and mitochondrial (rhod‐2 AM) Ca ²⁺ responses revealed that Ca ²⁺ elevations in the cytosol evoked by either MCh or kainate were translated into long‐lasting Ca ²⁺ elevations in subpopulations of mitochondria. In some mitochondria, Ca ²⁺ signals appeared to activate Ca ²⁺ release into the cytosol.
Inhibition of the mitochondrial Na ⁺ ‐Ca ²⁺ exchanger by CGP‐37157 (25 μM) decreased kainate Ca ²⁺ response amplitude and increased the rate of return of the response to basal Ca ²⁺ levels.
Thus, both ionotropic and metabotropic stimulation evoke changes in mitochondrial membrane potential and Ca ²⁺ levels in O‐2A cells. Ca ²⁺ uptake into some mitochondria is activated by Ca ²⁺ entry into cells or release from stores. Mitochondrial Ca ²⁺ release appears to play a key role in shaping kainate‐evoked Ca ²⁺ responses.
Glutamate-operated ion channels (GluR channels) of the L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-kainate subtype are found in both neurons and glial cells of the central nervous system. These channels are assembled from the GluR-A, -B, -C, and -D subunits; channels containing a GluR-B subunit show an outwardly rectifying current-voltage relation and low calcium permeability, whereas channels lacking the GluR-B subunit are characterized by a doubly rectifying current-voltage relation and high calcium permeability. Most cell types in the central nervous system coexpress several subunits, including GluR-B. However, Bergmann glia in rat cerebellum do not express GluR-B subunit genes. In a subset of cultured cerebellar glial cells, likely derived from Bergmann glial cells. GluR channels exhibit doubly rectifying current-voltage relations and high calcium permeability, whereas GluR channels of cerebellar neurons have low calcium permeability. Thus, differential expression of the GluR-B subunit gene in neurons and glia is one mechanism by which functional properties of native GluR channels are regulated.
Glutamate receptors, the most abundant excitatory transmitter receptors in the brain, are not restricted to neurons; they
have also been detected on glial cells. Bergmann glial cells in mouse cerebellar slices revealed a kainate-type glutamate
receptor with a sigmoid current-to-voltage relation, as demonstrated with the patch-clamp technique. Calcium was imaged with
fura-2, and a kainate-induced increase in intracellular calcium concentration was observed, which was blocked by the non-N-methyl-D-aspartate
(NMDA) glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and by low concentrations of external calcium,
indicating that there was an influx of calcium through the kainate receptor itself. The entry of calcium led to a marked reduction
in the resting (passive) potassium conductance of the cell. Purkinje cells, which have glutamatergic synapses, are closely
associated with Bergmann glial cells and therefore may provide a functionally important stimulus.
Immunocytochemical and electrophysiological methods were used to examine the effect of retinal ablation on the expression of sodium channels within optic nerve astrocytes in situ and in vitro. Enucleation was performed at postnatal day 3 (P3), and electron microscopy of the enucleated optic nerves at P28-P40 revealed complete degeneration of retinal ganglion axons, resulting in optic nerves composed predominantly of astrocytes. In contrast to control (non-enucleated) optic nerve astrocytes, which exhibited distinct sodium channel immunoreactivity following immunostaining with antibody 7493, the astrocytes in enucleated optic nerves did not display sodium channel immunoreactivity in situ. Cultures obtained from enucleated optic nerves consisted principally (greater than 90%) of glial fibrillary acidic protein (GFAP)+/A2B5- ("type-1") astrocytes, as determined by indirect immunofluorescence; GFAP+/A2B5+ ("type-2") astrocytes were not present, nor were GFAP-/A2B5+ (O-2A) progenitor cells. Sodium channel immunoreactivity was not present in GFAP+/A2B5- astrocytes obtained from enucleated optic nerves; in contrast, GFAP+/A2B5- astrocytes from control optic nerves exhibited 7493 immunostaining for the first 4-6 days in culture. Sodium current expression, studied using whole-cell patch-clamp recording, was attenuated in cultured astrocytes derived from enucleated optic nerves. Whereas 39 of 50 type-1 astrocytes cultured from intact optic nerves showed measurable sodium currents at 1-7 days in vitro, sodium currents were present in only 6 of 38 astrocytes cultured from enucleated optic nerves. Mean sodium current densities in astrocytes from the enucleated optic nerves (0.66 +/- 0.3 pA/pF) were significantly smaller than in astrocytes from control optic nerves (7.15 +/- 1.1 pA/pF). The h infinity-curves of sodium currents were similar in A2B5- astrocytes from enucleated and control rat optic nerves. These results suggest that there is neuronal modulation of sodium channel expression in type-1 optic nerve astrocytes, and that, following chronic loss of axonal association in vivo, sodium channel expression is down-regulated in this population of optic nerve astrocytes.
1. Na+ and K+ channel expression was studied in cultured astrocytes derived from P--0 rat spinal cord using whole cell patch-clamp recording techniques. Two subtypes of astrocytes, pancake and stellate, were differentiated morphologically. Both astrocyte types showed Na+ channels and up to three forms of K+ channels at certain stages of in vitro development. 2. Both astrocyte types showed pronounced K+ currents immediately after plating. Stellate but not pancake astrocytes additionally showed tetrodotoxin (TTX)-sensitive inward Na+ currents, which displayed properties similar to neuronal Na+ currents. 3. Within 4-5 days in vitro (DIV), pancake astrocytes lost K(+)-current expression almost completely, but acquired Na+ currents in high densities (estimated channel density approximately 2-8 channels/microns2). Na+ channel expression in these astrocytes is approximately 10- to 100-fold higher than previously reported for glial cells. Concomitant with the loss of K+ channels, pancake astrocytes showed significantly depolarized membrane potentials (-28.1 +/- 15.4 mV, mean +/- SD), compared with stellate astrocytes (-62.5 +/- 11.9 mV, mean +/- SD). 4. Pancake astrocytes were capable of generating action-potential (AP)-like responses under current clamp, when clamp potential was more negative than resting potential. Both depolarizing and hyperpolarizing current injections elicited overshooting responses, provided that cells were current clamped to membrane potentials more negative than -70 mV. Anode-break spikes were evoked by large hyperpolarizations (less than -150 mV). AP-like responses in these hyperpolarized astrocytes showed a time course similar to neuronal APs under conditions of low K+ conductance. 5. In stellate astrocytes, AP-like responses were not observed, because the K+ conductance always exceeded Na+ conductance by at least a factor of 3. Thus stellate spinal cord astrocyte membranes are stabilized close to EK as previously reported for hippocampal astrocytes. 6. It is concluded that spinal cord pancake astrocytes are capable of synthesizing Na+ channels at densities that can, under some conditions, support electrogenesis. In vivo, however, AP-like responses are unlikely to occur because the cells' resting potential is too depolarized to allow current activation. Thus the absence of electrogenesis in astrocytes may be explained by two mechanisms: 1) a low Na-to-K conductance ratio, as in stellate spinal cord astrocytes and in other previously studied astrocyte preparations; or, 2) as described in detail in the companion paper, a mismatch between the h infinity curve and resting potential, which results in Na+ current inactivation in spinal cord pancake astrocytes.
1. Na+ currents expressed in astrocytes cultured from spinal cord were studied by whole cell patch-clamp recording. Two subtypes of astrocytes, pancake and stellate cells, were morphologically differentiated and showed expression of Na+ channels at densities that are unusually high for glial cells (2-8 channels/microns2) and comparable to cultured neurons. 2. Na+ currents in stellate and pancake astrocytes were comparable to neuronal Na+ currents with regard to Na(+)-current activation (tau m) and inactivation (tau h) time constants, which were equally fast in both astrocyte types. However, they differed with respect to voltage dependence of activation, and current-voltage (I-V) curves were approximately 10 mV more positive in stellate cells (-11.1 +/- 5.6 mV, mean +/- SD) than in pancake cells (19.7 +/- 4.5 mV). Steady-state activation (m infinity curves) was 16 mV more negative in pancake (mean V1/2 = -48.8 mV) than in stellate cells (mean V1/2 = -32.7 mV). 3. Steady-state inactivation (h infinity curves) of Na+ currents was distinctly different in the two astrocyte types. In stellate astrocytes h infinity curves had midpoints close to -65 mV (-64.6 +/- 6.5 mV), similar to most cultured neurons. In pancake astrocytes h infinity-curves were approximately 25 mV more negative, with midpoints close to -85 mV (84.5 +/- 9.5 mV). 4. The two forms of Na+ currents were additionally distinguishable by their sensitivity to tetrodotoxin (TTX). Na+ currents in stellate astrocytes were highly TTX sensitive [half-maximal inhibition (Kd) = 5.7 nM] whereas Na+ currents in pancake astrocytes were relatively TTX resistant, requiring 100- to 1,000-fold higher concentrations for blockage (Kd = 1,007 nM). 5. Na+ currents were fit by the Hodgkin-Huxley (HH) model. In pancake astrocytes, as in squid gigant axons, Na(+)-current kinetics could be well described with an m3h model, whereas in stellate astrocytes Na+ currents were better described with higher-order power terms for activation (m). On average, best fits were obtained using an m4h model. 6. Pancake astrocytes were capable of generating action-potential (AP)-like responses under current clamp whereas stellate astrocytes were not. The h infinity curve for APs shows that membrane potentials more negative than -70 mV are required to allow these responses to occur.(ABSTRACT TRUNCATED AT 400 WORDS)