No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Potassium buffering in the central nervous system
Abstract
Rapid changes in extracellular K+ concentration ([K+](o)) in the mammalian CNS are counteracted by simple passive diffusion as well as by cellular mechanisms of K+ clearance. Buffering of [K+](o) can occur via glial or neuronal uptake of K+ ions through transporters or K+-selective channels. The best studied mechanism for [K+](o) buffering in the brain is called K+ spatial buffering, wherein the glial syncytium disperses local extracellular K+ increases by transferring K+ ions from sites of elevated [K+](o) to those with lower [K+](o). In recent years, K+ spatial buffering has been implicated or directly demonstrated by a variety of experimental approaches including electrophysiological and optical methods. A specialized form of spatial buffering named K+ siphoning takes place in the vertebrate retina, where glial Muller cells express inwardly rectifying K+ channels (Kir channels) positioned in the membrane domains near to the vitreous humor and blood vessels. This highly compartmentalized distribution of Kir channels in retinal glia directs K+ ions from the synaptic layers to the vitreous humor and blood vessels. Here, we review the principal mechanisms of [K+](o) buffering in the CNS and recent molecular studies on the structure and functions of glial Kir channels. We also discuss intriguing new data that suggest a close physical and functional relationship between Kir and water channels in glial cells.
... Potassium-The Na + /K + -ATPase pumps Na + ions out of neurons in exchange for K + ions thereby progressively reversing the changes in ionic balance induced by neuronal activity; however, this process in neurons alone is not adequate for timely clearance of extracellular K + , particularly during seizures (Heinemann et al., 1977;Hodgkin and Huxley, 1952). Astrocytes regulate extracellular K + levels by (1) direct K + uptake from the extracellular space and (2) K + spatial buffering within astrocytes linked by gap junctions (Amédée et al., 1998;Kofuji and Newman, 2004). Astrocytes, like neurons, also express Na + /K + -ATPases; however, due to a difference in subunit composition (Cameron et al., 1994;McGrail et al., 1991), the function of Na + /K + -ATPases in astrocytes is enhanced by increased extracellular K + concentrations (Henn et al., 1972;Hertz and Chen, 2016). ...
... Additionally, Na + /K + /Cl − cotransporters pump Na + /K + /Cl − into the intracellular space of cells, including astrocytes (MacVicar et al., 2002;Walz, 1992). Lastly, inwardly rectifying K + channels, specifically Kir4.1, allow the inward and outward passage of K + ions with a preference towards inward flow (Doupnik et al., 1995;Kofuji and Newman, 2004). K + ions taken up from the extracellular space can be transferred between astrocytes connected by gap junctions in a process called spatial buffering (Holthoff and Witte, 2000;Kofuji and Newman, 2004). ...
... Lastly, inwardly rectifying K + channels, specifically Kir4.1, allow the inward and outward passage of K + ions with a preference towards inward flow (Doupnik et al., 1995;Kofuji and Newman, 2004). K + ions taken up from the extracellular space can be transferred between astrocytes connected by gap junctions in a process called spatial buffering (Holthoff and Witte, 2000;Kofuji and Newman, 2004). K + that was taken up by astrocytes is slowly released back into the extracellular space through Kir4.1 channels (Bay and Butt, 2012). ...
The epilepsies are a diverse spectrum of disease states characterized by spontaneous seizures and associated comorbidities. Neuron-focused perspectives have yielded an array of widely used anti-seizure medications and are able to explain some, but not all, of the imbalance of excitation and inhibition which manifests itself as spontaneous seizures. Furthermore, the rate of pharmacoresistant epilepsy remain high despite the regular approval of novel anti-seizure medications. Gaining a more complete understanding of the processes that turn a healthy brain into an epileptic brain (epileptogenesis) as well as the processes which generate individual seizures (ictogenesis) may necessitate broadening our focus to other cell types. As will be detailed in this review, astrocytes augment neuronal activity at the level of individual neurons in the form of gliotransmission and the tripartite synapse. Under normal conditions, astrocytes are essential to the maintenance of blood-brain barrier integrity and remediation of inflammation and oxidative stress, but in epilepsy these functions are impaired. Epilepsy results in disruptions in the way astrocytes relate to each other by gap junctions which has important implications for ion and water homeostasis. In their activated state, astrocytes contribute to imbalances in neuronal excitability due their decreased capacity to take up and metabolize glutamate and an increased capacity to metabolize adenosine. Furthermore, due to their increased adenosine metabolism, activated astrocytes may contribute to DNA hypermethylation and other epigenetic changes that underly epileptogenesis. Lastly, we will explore the potential explanatory power of these changes in astrocyte function in detail in the specific context of the comorbid occurrence of epilepsy and Alzheimer's disease and the disruption in sleep-wake regulation associated with both conditions.
... Reduced synaptically-evoked K + uptake through Kir4.1 channels in FMRP-deficient astrocytes Synaptic activity is the main source of neuronal K + release, whereas astrocytes are the major players in the clearance of excess K + from the extracellular environment 29,34 . This astroglial function is mediated by the inwardly rectifying potassium channel Kir4.1, which is highly expressed in perisynaptic astroglial processes [35][36][37][38] . ...
... Astrocytes are essential elements of the tripartite synapse that dynamically regulate the extracellular environment. Specifically, they control elevated [K + ] o following neuronal activity through K + clearance 29,34 . Here we demonstrate that astrocyte K + uptake through Kir4.1 channels during synaptic activity is markedly reduced in Fmr1 KO male mice. ...
Fragile X syndrome (FXS) is an inherited form of intellectual disability caused by the loss of the mRNA-binding fragile X mental retardation protein (FMRP). FXS is characterized by neuronal hyperexcitability and behavioral defects, however the mechanisms underlying these critical dysfunctions remain unclear. Here, using male Fmr1 knockout mouse model of FXS, we identify abnormal extracellular potassium homeostasis, along with impaired potassium channel Kir4.1 expression and function in astrocytes. Further, we reveal that Kir4.1 mRNA is a binding target of FMRP. Finally, we show that the deficit in astroglial Kir4.1 underlies neuronal hyperexcitability and several behavioral defects in Fmr1 knockout mice. Viral delivery of Kir4.1 channels specifically to hippocampal astrocytes from Fmr1 knockout mice indeed rescues normal astrocyte potassium uptake, neuronal excitability, and cognitive and social performance. Our findings uncover an important role for astrocyte dysfunction in the pathophysiology of FXS, and identify Kir4.1 channel as a potential therapeutic target for FXS.
... However, accumulating evidence indicates that glial cells are prominent contributors to many neurodegenerative diseases, such as Alzheimer's disease (Phillips et al., 2014;Fakhoury, 2018;Nanclares et al., 2021), Huntington's disease (Shin et al., 2005;Jiang et al., 2016;Meunier et al., 2016), Parkinson's disease (Halliday and Stevens, 2011;Díaz et al., 2019), amyotrophic lateral sclerosis (Yamanaka et al., 2008), and some forms of ataxia (Kretzschmar et al., 2005;Custer et al., 2006;Furrer et al., 2011), including SCA1 . Astroglial cells play key roles in numerous functions within the brain, such as regulation of regional blood flow, ionic balance of the extracellular space, and modulation of synaptic transmission (Kofuji and Newman, 2004;Perea et al., 2014;Sloan and Barres, 2014). The cerebellar cortex contains three main types of astroglial cells: Bergmann glia (BG), a specialized type of cerebellar astrocytes, protoplasmic velate astrocytes, and white matter fibrous astrocytes (Araujo et al., 2019). ...
... Bergmann glia are responsible for glutamate uptake and extracellular K + homeostatic clearance (Bellamy, 2006). Astrocytic Glutamate transporter-1 (GLT-1) and inwardly rectifying potassium channels (Kir) play a major role in glutamate removal from the synapses and spatial buffering of K + released by neurons during action potential propagation (Kofuji and Newman, 2004;Orkand et al., 1966;Newman et al., 1984;Newman, 1986;Newman, 1993;Wang et al., 2012b). In spite of a plethora of subtypes of Kir in glial cells, the evidence suggests that Kir4.1 is the principal pore forming subunit in glial cells (Neusch et al., 2001;Neusch et al., 2006;Olsen et al., 2006;Djukic et al., 2007). ...
Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by an abnormal expansion of glutamine (Q) encoding CAG repeats in the ATAXIN1 (ATXN1) gene and characterized by progressive cerebellar ataxia, dysarthria, and eventual deterioration of bulbar functions. SCA1 shows severe degeneration of cerebellar Purkinje cells (PCs) and activation of Bergmann glia (BG), a type of cerebellar astroglia closely associated with PCs. Combining electrophysiological recordings, calcium imaging techniques, and chemogenetic approaches, we have investigated the electrical intrinsic and synaptic properties of PCs and the physiological properties of BG in SCA1 mouse model expressing mutant ATXN1 only in PCs. PCs of SCA1 mice displayed lower spontaneous firing rate and larger slow afterhyperpolarization currents (sIAHP) than wildtype mice, whereas the properties of the synaptic inputs were unaffected. BG of SCA1 mice showed higher calcium hyperactivity and gliotransmission, manifested by higher frequency of NMDAR-mediated slow inward currents (SICs) in PC. Preventing the BG calcium hyperexcitability of SCA1 mice by loading BG with the calcium chelator BAPTA restored sIAHP and spontaneous firing rate of PCs to similar levels of wildtype mice. Moreover, mimicking the BG hyperactivity by activating BG expressing Gq-DREADDs in wildtype mice reproduced the SCA1 pathological phenotype of PCs, i.e., enhancement of sIAHP and decrease of spontaneous firing rate. These results indicate that the intrinsic electrical properties of PCs, but not their synaptic properties, were altered in SCA1 mice and that these alterations were associated with the hyperexcitability of BG. Moreover, preventing BG hyperexcitability in SCA1 mice and promoting BG hyperexcitability in wildtype mice prevented and mimicked, respectively, the pathological electrophysiological phenotype of PCs. Therefore, BG plays a relevant role in the dysfunction of the electrical intrinsic properties of PCs in SCA1 mice, suggesting that they may serve as potential targets for therapeutic approaches to treat the spinocerebellar ataxia type 1.
... However, accumulating evidence indicate that glial cells are prominent contributors to many neurodegenerative diseases, such as Alzheimer´s disease (Phillips et al., 2014;Fakhoury, 2018;Nanclares et al., 2021), Huntington´s disease (Shin et al., 2005;Jiang et al., 2016;Meunier et al., 2016), Parkinson´s disease (Halliday and Stevens, 2011;Díaz et al., 2019), amyotrophic lateral sclerosis (Yamanaka et al., 2008), and some forms of ataxia (Kretzschmar et al., 2005;Custer et al., 2006;Furrer et al., 2011), including SCA1 . Astroglial cells play key roles in numerous functions within the brain, such as regulation of regional blood flow, ionic balance of the extracellular space, and modulation of synaptic transmission (Kofuji and Newman, 2004;Perea et al., 2014;Sloan and Barres, 2014). The cerebellar cortex contains three main types of astroglial cells: Bergmann glia (BG), a specialized type of cerebellar astrocytes, protoplasmic velate astrocytes, and white matter fibrous astrocytes (Araujo et al., 2019). ...
... Bergmann glia is responsible for glutamate uptake and extracellular K + homeostatic clearance (Bellamy, 2006). Astrocytic Glutamate transporter-1 (GLT-1) and inwardly rectifying potassium channels (Kir) play a major role in glutamate removal from the synapses and spatial buffering of K + released by neurons during action potential propagation (Orkand et al., 1966;Newman et al., 1984;Newman, 1986Newman, , 1993Kofuji and Newman, 2004;Wang et al., 2012). In spite of a plethora subtypes of Kir in glial cells, the evidence suggests that Kir4.1 is the principal pore forming subunit in glial cells (Neusch et al., 2001(Neusch et al., , 2006Olsen et al., 2006;Djukic et al., 2007). ...
Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by an abnormal expansion of glutamine (Q) encoding CAG repeats in the ATAXIN1 (ATXN1) gene and characterized by progressive cerebellar ataxia, dysarthria, and eventual deterioration of bulbar functions. SCA1 shows severe degeneration of cerebellar Purkinje cells (PCs) and activation of Bergmann glia (BG), a type of cerebellar astroglia closely associated with PCs. Using electrophysiological recordings, confocal imaging and chemogenetic approaches, we have investigated the electrical intrinsic and synaptic properties of PCs and the physiological properties of BG in SCA1 mouse model expressing mutant ATXN1 only in PCs. PCs of SCA1 mice displayed lower spontaneous firing rate and larger medium and slow afterhyperpolarization currents (mIAHP and sIAHP) than wildtype mice, whereas the properties of the synaptic inputs were unaffected. BG of SCA1 mice showed higher calcium hyperactivity and gliotransmission, manifested higher frequency of NMDAR-mediated slow inward currents (SICs) in PC. Preventing the BG calcium hyperexcitability of SCA1 mice by loading BG with the calcium chelator BAPTA restored mIAHP and sIAHP and spontaneous firing rate of PCs to similar levels of wildtype mice. Moreover, mimicking the BG hyperactivity by activating BG expressing Gq-DREADDs in wildtype mice reproduced the SCA1 pathological phenotype of PCs, i.e., enhancement of mIAHP and sIAHP and decrease of spontaneous firing rate. These results indicate that the intrinsic electrical properties of PCs, but not their synaptic properties, were altered in SCA1 mice, and that these alterations were associated with the hyperexcitability of BG. Moreover, preventing BG hyperexcitability in SCA1 mice and promoting BG hyperexcitability in wildtype mice prevented and mimicked, respectively, the pathological electrophysiological phenotype of PCs. Therefore, BG plays a relevant role in the dysfunction of the electrical intrinsic properties of PCs in SCA1 mice, suggesting that they may serve as potential targets for therapeutic approaches to treat the spinocerebellar ataxia type 1.
... Astrocytes sustain numerous functions in the central nervous system, from controlling the water homeostasis and extracellular ion concentrations [1,2], regulating the blood flow [3], secreting humoral and trophic factors [4] providing metabolic support for neurones [5] and maintaining glutamate, GABA and adenosine homeostasis [6,7]. Furthermore, astroglia contributes to synaptogenesis, synaptic development, regulation and remodelling [8,9]. ...
... Indeed, S100β-positive astrocytes occupy the entire extent of this cortical region. S100β is also expressed in some rat and mouse neurons and oligodendrocytes, but its expression is very low and concerns only some brain areas, thus the non-astrocytic cells S100β-positive can be considered negligible in Table 2 S100β morphometric values in the 3xTg-AD mice at 1,9,12 between of s100β expressing astrocytes located around the senile plaques and those placed distant to the plaques in the EC of the 3xTg-AD, compared to s100β expressing astrocytes in the EC of the non-Tg animals. Morphometric analysis was performed on the entire astrocyte, but also cells' counterparts, the processes and the somata. ...
Astrocytes contribute to the progression of neurodegenerative diseases, including Alzheimer's disease (AD). Here, we report the neuroanatomical and morphometric analysis of astrocytes in the entorhinal cortex (EC) of the aged wild type (WT) and triple transgenic (3xTg-AD) mouse model of AD. Using 3D confocal microscopy, we determined the surface area and volume of positive astrocytic profiles in male mice (WT and 3xTg-AD) from 1 to 18 months of age. We showed that S100β-positive astrocytes were equally distributed throughout the entire EC in both animal types and showed no changes in Nv (number of cells/mm3) nor in their distribution at the different ages studied. These positive astrocytes, demonstrated an age-dependent gradual increase in their surface area and in their volume starting at 3 months of age, in both WT and 3xTg-AD mice. This last group demonstrated a large increase in both surface area and volume at 18 months of age when the burden of pathological hallmarks of AD is present (69.74% to 76.73% in the surface area and the volume, for WT and 3xTg-AD mice respectively). We observed that these changes were due to the enlargement of the cell processes and to less extend the somata. In fact, the volume of the cell body was increased by 35.82% in 18-month-old 3xTg-AD compared to WT. On the other hand, the increase on the astrocytic processes were detected as soon as 9 months of age where we found an increase of surface area and volume (36.56% and 43.73%, respectively) sustained till 18 month of age (93.6% and 113.78%, respectively) when compared age-matched non-Tg mice. Moreover, we demonstrated that these hypertrophic S100β-positive astrocytes were mainly associated with Aβ plaques. Our results show a severe atrophy in GFAP cytoskeleton in all cognitive areas; whilst within the EC astrocytes independent to this atrophy show no changes in GS and S100β; which can play a key role in the memory impairment.
... Indeed, SD induces significant K + uptake into astrocytes, leading to astrocyte swelling [12], whereas pronounced Ca 2+ waves are propagated through astrocyte networks with SDs [13][14][15]. Intercellular communication mediated by connexin-based channels is critical not only for the propagation of Ca 2+ waves among astrocytes but also for the spatial buffering of K + in the brain [9,[16][17][18]. Connexins are transmembrane proteins that create two pathways for intercellular communication: (1) gap junctional channels (GJCs), which are formed by the docking of two connexons or hemichannels positioned at opposite membranes between adjacent cells, and (2) hemichannels, which are situated at unopposed regions of cell surfaces [19]. ...
Background
Spreading depression (SD) is an intriguing phenomenon characterized by massive slow brain depolarizations that affect neurons and glial cells. This phenomenon is repetitive and produces a metabolic overload that increases secondary damage. However, the mechanisms associated with the initiation and propagation of SD are unknown. Multiple lines of evidence indicate that persistent and uncontrolled opening of hemichannels could participate in the pathogenesis and progression of several neurological disorders including acute brain injuries. Here, we explored the contribution of astroglial hemichannels composed of connexin-43 (Cx43) or pannexin-1 (Panx1) to SD evoked by high-K ⁺ stimulation in brain slices.
Results
Focal high-K ⁺ stimulation rapidly evoked a wave of SD linked to increased activity of the Cx43 and Panx1 hemichannels in the brain cortex, as measured by light transmittance and dye uptake analysis, respectively. The activation of these channels occurs mainly in astrocytes but also in neurons. More importantly, the inhibition of both the Cx43 and Panx1 hemichannels completely prevented high K ⁺ -induced SD in the brain cortex. Electrophysiological recordings also revealed that Cx43 and Panx1 hemichannels critically contribute to the SD-induced decrease in synaptic transmission in the brain cortex and hippocampus.
Conclusions
Targeting Cx43 and Panx1 hemichannels could serve as a new therapeutic strategy to prevent the initiation and propagation of SD in several acute brain injuries.
... These factors can interact and create a cascade of cellular dysfunctions, increasing the vulnerability of dopaminergic (DA) neurons to neurodegeneration. DA neurons are known for their intrinsic pacemaker activity, a self-generated electrical rhythm crucial for their function, dependent on the coordinated activities of various ion channels including Na + , Ca 2+ , and K + channels (Cantrell and Catterall 2001, Pietrobon 2002, Kofuji and Newman 2004, Vaidya et al. 2024. ...
Parkinson's disease (PD) is a complex progressive neurodegenerative disorder involving multiple pathogenetic factors, including oxidative stress, mitochondria dysfunction, neuroinflammation, and ion imbalance. Emerging evidence underscores the significant role of potassium channels in multiple aspects of PD etiology. We recently identified a PD-linked genetic mutation in the KCNJ15 gene (KCNJ15p.R28C), encoding the inwardly rectifying potassium channel Kir4.2, within a four-generation family with familial PD. The role of the Kir4.2 channel, especially in neurodegenerative diseases, remains largely unexplored. This study aimed to elucidate the impact of the KCNJ15p.R28C (Kir4.2R28C) mutation on the biophysical and biochemical properties of Kir4.2. Employing Kir4.2-overexpressing HEK293T cells as our model, we investigated how the mutation affects the channel's biophysical properties, total protein expression, endoplasmic reticulum and lysosome processing, and plasma membrane trafficking. Patch clamp studies revealed that the Kir4.2R28C mutation results in loss of channel function, exhibiting a strong dominant-negative effect. This can be partially attributed to the significantly diminished overall expression of the mutant channel protein compared to the wild-type (Kir4.2WT). We observed that both Kir4.2WT and Kir4.2R28C proteins undergo glycosylation during the post-translational modification process, albeit with differing protein turnover efficiencies. Furthermore, the KCNJ15p.R28C mutation exhibits reduced stability compared to Kir4.2WT and is more susceptible to protein recycling through the lysosomal degradation pathway. Additionally, Kir4.2R28C displayed reduced plasma membrane trafficking capacity compared to Kir4.2WT. These findings suggest that the Kir4.2R28C mutant possesses unique biomolecular and biophysical characteristics distinct from the Kir4.2WT channel, which potentially elucidates its role in the pathogenesis of PD.
... through the synapses. During repolarization, neurons release K+ in the synaptic cleft, which are then taken in by the astrocytes from their increased K+ channels [1][2]. The K+ ions are then released into other parts of the body [3]. ...
Astrocytes are cells in the central nervous system (CNS) that are responsible for many things, such as maintaining blood brain barrier (BBB), regulating synapses in the spinal cord, and responding to spinal cord injury (SCI). Astrogliosis, the astrocytic response to spinal cord injuries (SCIs), helps repair CNS damages by regulating different protein filaments, thus limiting axonal growth. Former studies that were demonstrated through laser capture microdissection and immunohistochemistry (IHC) helped to identify important genes involved in experimental therapies for SCIs. Additionally, there are potential clinical treatments options for SCIs such as hydrogels, mesenchymal stem cells and steroids. Increased imaging modalities indicate that excessive astrogliosis can have adverse effects. These imaging techniques include positron emission tomography (PET), magnetic resonance imaging (MRI), and two-photon laser-scanning microscopy (TPLSM). These techniques illuminate greater details of the astrocytic response to SCIs. Despite these findings, astrogliosis is not well understood by the research community. Many of the studies presented in this literature review are experimental attempts to understand the mechanisms of astrogliosis in SCIs. This literature review aims to summarize the methods of each study in visualizing the mechanisms of astrogliosis and how they play a role in SCIs. Furthermore, this paper is aimed to comprehensively bridge the developments in the treatment for SCI patients based on innovative imaging modalities. Compared to prior studies, this review utilizes more recent understandings of the astrogliosis mechanisms to highlight insights into targeted developments, both clinically and preclinically. Some limitations of this literature review include the limited studies on astrogliosis and its impact on SCIs. Nonetheless, there is ongoing potential in the search for treatments for SCIs.
... Transporters in the Müller glia cell control extracellular levels of neurotransmitters such as the excitatory amino acid glutamate and GABA (Bringmann et al., 2013). Neuronal waste products ions such as ammonia, and potassium, are removed from the interstitial space by Müller glia cells (Kofuji and Newman, 2004;Kuo et al., 2020), while pyruvate is supplied to retinal neurons. While Müller cells express many voltage-gated channels, large inward rectifier conductances to potassium ions and leak channels are often found on their end feet processes (e.g., Solessio et al., 2000;Newman, 2005;Hughes et al., 2017). ...
Introduction
We examined how pulse train electrical stimulation of the inner surface of the rabbit retina effected the resident glial cells. We used a rabbit retinal eyecup preparation model, transparent stimulus electrodes, and optical coherence tomography (OCT). The endfeet of Müller glia processes line the inner limiting membrane (ILM).
Methods
To examine how epiretinal electrode stimulation affected the Müller glia, we labeled them post stimulation using antibodies against soluble glutamine synthetase (GS). After 5 min 50 Hz pulse train stimulation 30 μm from the surface, the retina was fixed, immunostained for Müller glia, and examined using confocal microscopic reconstruction. Stimulus pulse charge densities between 133–749 μC/cm2/ph were examined.
Results
High charge density stimulation (442–749 μC/cm2/ph) caused significant losses in the GS immunofluorescence of the Müller glia endfeet under the electrode. This loss of immunofluorescence was correlated with stimuli causing ILM detachment when measured using OCT. Müller cells show potassium conductances at rest that are blocked by barium ions. Using 30 msec 20 μA stimulus current pulses across the eyecup, the change in transretinal resistance was examined by adding barium to the Ringer. Barium caused little change in the transretinal resistance, suggesting under low charge density stimulus pulse conditions, the Müller cell radial conductance pathway for these stimulus currents was small. To examine how epiretinal electrode stimulation affected the microglia, we used lectin staining 0–4 h post stimulation. After stimulation at high charge densities 749 μC/cm2/ph, the microglia under the electrode appeared rounded, while the local microglia outside the electrode responded to the stimulated retina by process orientation inwards in a ring by 30 min post stimulation.
Discussion
Our study of glial cells in a rabbit eyecup model using transparent electrode imaging suggests that epiretinal electrical stimulation at high pulse charge densities, can injure the Müller and microglia cells lining the inner retinal surface in addition to ganglion cells.
... This model requires the coordinated activity of strongly rectifying Kir2.1 channels, which mediate K + influx from neuronal "sources" into glial cells, and weakly rectifying Kir4.1 channels, which allow K + to leave the glial cells and enter extracellular "sinks." [65][66][67][68] In the CNS, the repolarization of neurons after sustained action potential firing tends to raise the K + concentration in the F I G U R E 8 Effect of short and long-term exposure to pro-oxidants on Kir2.1 activity. Current density-to-voltage relationship of (A) U-87 MG cells endogenously expressing Kir2.1 and (B) Xenopus oocytes ectopically expressing Kir2.1 before and after perfusion with a bath solution containing 1 mM tert-butyl hydroperoxide (TBHP) for 5 min, (C) HEK293T cells transfected with Kir2.1 before and after perfusion with a bath solution containing 500 μM chloramine T (Cl-T) for 5 min, and (D) U-87 MG cells endogenously expressing Kir2.1 incubated with 1 mM TBHP in the cell culture medium for 2 h. ...
Melatonin is a pleiotropic biofactor and an effective antioxidant and free radical scavenger and, as such, can be protective in oxidative stress‐related brain conditions including epilepsy and aging. To test the potential protective effect of melatonin on brain homeostasis and identify the corresponding molecular targets, we established a new model of oxidative stress‐related aging neuroglia represented by U‐87 MG cells exposed to D‐galactose (D‐Gal). This model was characterized by a substantial elevation of markers of oxidative stress, lipid peroxidation, and protein oxidation. The function of the inward rectifying K ⁺ channel Kir2.1, which was identified as the main Kir channel endogenously expressed in these cells, was dramatically impaired. Kir2.1 was unlikely a direct target of oxidative stress, but the loss of function resulted from a reduction of protein abundance, with no alterations in transcript levels and trafficking to the cell surface. Importantly, melatonin reverted these changes. All findings, including the melatonin antioxidant effect, were reproduced in heterologous expression systems. We conclude that the glial Kir2.1 can be a target of oxidative stress and further suggest that inhibition of its function might alter the extracellular K ⁺ buffering in the brain, therefore contributing to neuronal hyperexcitability and epileptogenesis during aging. Melatonin can play a protective role in this context.
... Вначале в эпилептогенных регионах накапливается избыток калия, который затем транспортируется в связанные с ними здоровые регионы. Наиболее хорошо изученным механизмом распределения калия по областям головного мозга является механизм его пространственной буферизации [42], заключающийся в следующем. Из кровеносных сосудов и спинномозговой жидкости калий закачивается в глию с помощью белковтранспортеров и больших пор в мембранах астроцитов. ...
The paper presents novel developed modular mathematical model of epileptic seizures, obtained by combining and modifying existing models of epilepsy, which for the first time makes it possible to simulate the dynamics of seizure onset, propagation and termination simultaneously at the cellular and regional levels of brain organization. At the level of individual cells, the dynamics of AMPA receptor trafficking, changes in the concentrations of intra- and extracellular ions, membrane depolarization, and other biophysical processes responsible for the development of ictal activity were calculated. Local field potentials of brain regions were modeled at the regional level taking into account cellular processes and the large-scale structure of the brain network. It is shown that the dynamics of submodels used in the structure of the multilevel model corresponds to the dynamics of the original models, the authors of which validated them with experimental data. The theoretical justification of the connections between submodels was given.
... Astroglial K + buffering is a complex process which comprises removal of the excess of [K + ] o with subsequent return of K + back to the extracellular space for restoration of neuronal ionic gradients. For a long time, passive influx of K + into astrocytes through inward rectifying K ir 4.1 channels was believed to be responsible for K + removal from the extracellular space [49][50][51] (Fig. 3). Recently however, the primary role of an active K + removal through NKA, first suggested by Leif Hertz in 1965 [52], became universally accepted (Fig. 3). ...
... 3 Narrow space, high tortuosity, local proteome complexity, 4 and low diffusion coefficient restrict the capacity of the perisynaptic extracellular space to buffer [K + ] o fluctuations during neuronal activity, 5,6 causing increases in [K + ] o , temporarily way beyond physiological levels. [7][8][9] Outward co-transport of K + and Cl À by neuronal KCC2 may contribute to [K + ] o when intracellular Cl À [Cl À ] i is elevated. So far, research on KCC2 has been mainly focused on this ''canonical'' outward transport that is linked to synaptic inhibition, keeping [Cl À ] i at low levels and therewith GABA A receptor activity hyperpolarizing. ...
Extracellular potassium [K+]o elevation during synaptic activity retrogradely modifies presynaptic release and astrocytic uptake of glutamate. Hence, local K+ clearance and replenishment mechanisms are crucial regulators of glutamatergic transmission and plasticity. Based on recordings of astrocytic inward rectifier potassium current IKir and K+-sensitive electrodes as sensors of [K+]o as well as on in silico modeling, we demonstrate that the neuronal K+-Cl- co-transporter KCC2 clears local perisynaptic [K+]o during synaptic excitation by operating in an activity-dependent reversed mode. In reverse mode, KCC2 replenishes K+ in dendritic spines and complements clearance of [K+]o, therewith attenuating presynaptic glutamate release and shortening LTP. We thus demonstrate a physiological role of KCC2 in neuron-glial interactions and regulation of synaptic signaling and plasticity through the uptake of postsynaptically released K+.
... While audiogenic seizures are reportedly associated with increased glutamate in the inferior colliculus and midbrain nucleus of the auditory pathway in other rodent models [27,28], we previously demonstrated that the hippocampus was one of the audiogenic seizure foci in Lgi1-mutant rats, where astrocytic Kir4.1 channels were significantly down-regulated during the development of epileptogenesis [16,17]. Astrocytic Kir4.1 channels mediate spatial K + buffering and glutamate uptake into astrocytes via EAAT1 and EAAT2 [18][19][20][21]29,30], playing a crucial role in the development of epilepsy [21]. Thus, down-regulation of Kir4.1 channels might at least partly be involved in the elevation of hippocampal glutamate level in Lgi1-mutant rats. ...
Leucine-rich glioma-inactivated 1 (LGI1) was identified as a causative gene of autosomal dominant lateral temporal lobe epilepsy. We previously reported that Lgi1-mutant rats carrying a missense mutation (L385R) showed audiogenic seizure-susceptibility. To explore the pathophysiological mechanisms underlying Lgi1-related epilepsy, we evaluated changes in glutamate and GABA release in Lgi1-mutant rats. Acoustic priming (AP) for audiogenic seizure-susceptibility was performed by applying intense sound stimulation (130 dB, 10 kHz, 5 min) on postnatal day 16. Extracellular glutamate and GABA levels in the hippocampus CA1 region were evaluated at 8 weeks of age, using in vivo microdialysis techniques. Under naïve conditions without AP, glutamate and GABA release evoked by high-K+ depolarization was more prominent in Lgi1-mutant than in wild-type (WT) rats. The AP treatment on day 16 significantly increased basal glutamate levels and depolarization-induced glutamate release both in Lgi1-mutant and WT rats, yielding greater depolarization-induced glutamate release in Lgi1-mutant rats. On the other hand, the AP treatment enhanced depolarization-induced GABA release only in WT rats, and not in Lgi1-mutant rats, illustrating reduced GABAergic neurotransmission in primed Lgi1-mutant rats. The present results suggest that enhanced glutamatergic and reduced GABAergic neurotransmission are involved in the audiogenic seizure-susceptibility associated with Lgi1-mutation.
... Aβ also inhibits glutamate uptake (Lauderback et al., 1999) and interrupts K+ homeostasis (Cheung et al., 2015;Price et al., 2021). These changes not only increase [glut] o by inhibiting the activity of glutamate transporters in astrocytes, but also elevate [K + ] o by disrupting the astrocytic K + spatial buffering (Olsen and Sontheimer, 2008;Rimmele et al., 2017) and K + siphoning (Kofuji and Newman, 2004;Butt and Kalsi, 2006); both are mediated by K ir 4.1 channels that are highly expressed in astrocytes, but not in neurons (Butt and Kalsi, 2006;Djukic et al., 2007;Seifert et al., 2009 Figure 7). However, this unique astrocyte dysfunction is understudied in the field; and therefore, requires more attention and investigation in the future. ...
The normal function of the medial prefrontal cortex (mPFC) is essential for regulating neurocognition, but it is disrupted in the early stages of Alzheimer’s disease (AD) before the accumulation of Aβ and the appearance of symptoms. Despite this, little is known about how the functional activity of medial prefrontal cortex pyramidal neurons changes as Alzheimer’s disease progresses during aging. We used electrophysiological techniques (patch-clamping) to assess the functional activity of medial prefrontal cortex pyramidal neurons in the brain of 3xTg-Alzheimer’s disease mice modeling early-stage Alzheimer’s disease without Aβ accumulation. Our results indicate that firing rate and the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) were significantly increased in medial prefrontal cortex neurons from young Alzheimer’s disease mice (4–5-month, equivalent of <30-year-old humans) compared to agematched control mice. Blocking ionotropic glutamatergic NMDA receptors, which regulate neuronal excitability and Ca2+ homeostasis, abolished this neuronal hyperactivity. There were no changes in Ca2+ influx through the voltage-gated Ca2+ channels (VGCCs) or inhibitory postsynaptic activity in medial prefrontal cortex neurons from young Alzheimer’s disease mice compared to controls. Additionally, acute exposure to Aβ42 potentiated medial prefrontal cortex neuronal hyperactivity in young Alzheimer’s disease mice but hadnoeffects oncontrols. These findings indicate that the hyperactivity of medial prefrontal cortex pyramidal neurons at early-stage Alzheimer’s disease is induced byanabnormalincreaseinpresynapticglutamate releaseandpostsynaptic NMDA receptor activity, which initiates neuronal Ca2+ dyshomeostasis. Additionally, because accumulated Aβ forms unconventional but functional Ca2+ channels in medialprefrontal cortex neuronsinthelatestageofAlzheimer’sdisease,ourstudy also suggests an exacerbated Ca2+ dyshomeostasis in medial prefrontal cortex pyramidal neurons following overactivation of such VGCCs.
... According to the literature, there are two possible theories: GJIC is thought to be critical for maintaining tissue homeostasis and propagating electrical and metabolic signals in cell populations [60]. Consistent with this, GJs can suppress seizure activity by redistributing K + and glutamate (Glu) [61][62][63][64][65]. Astrocytes are electrically and metabolically connected to each other through gap junctions mainly composed of Cx43 and Cx30, forming a functional network [66,67]. ...
Connexin 43 (Cx43) is most widely distributed in mammals, especially in the cardiovascular and nervous systems. Its phosphorylation state has been found to be regulated by the action of more than ten kinases and phosphatases, including mitogen-activated protein kinase/extracellular signaling and regulating kinase signaling. In addition, the phosphorylation status of different phosphorylation sites affects its own synthesis and assembly and the function of the gap junctions (GJs) to varying degrees. The phosphorylation of Cx43 can affect the permeability, electrical conductivity, and gating properties of GJs, thereby having various effects on intercellular communication and affecting physiological or pathological processes in vitro and in vivo. Therefore, clarifying the relationship between Cx43 phosphorylation and specific disease processes will help us better understand the disease. Based on the above clinical and preclinical findings, we present in this review the functional significance of Cx43 phosphorylation in multiple diseases and discuss the potential of Cx43 as a drug target in Cx43-related disease pathophysiology, with an emphasis on the importance of connexin 43 as an emerging therapeutic target in cardiac and neuroprotection.
... In the process known as spatial K + buffering, the excess neuronally released [K + ] o is passively taken up by astrocytes via Kir4.1 channels, distributed in the GJ-coupled network and released at sites of lower [K + ] o [26]. This process, which is driven by the difference between the local K + equilibrium potential and the more negative membrane potential of the glial syncytium, cannot be sustained in the absence of coupling, resulting in stronger local [K + ] o increases and consequently in a more pronounced neuronal depolarisation and enhanced neuronal excitability [26][27][28][29][30]. In addition to K + , the redistribution of Na + ions through the GJ-coupled astrocytic network might also play an important role in preventing neuronal hyperexcitability. ...
The gap-junction-coupled astroglial network plays a central role in the regulation of neuronal activity and synchronisation, but its involvement in the pathogenesis of neuronal diseases is not yet understood. Here, we present the current state of knowledge about the impact of impaired glial coupling in the development and progression of epilepsy and discuss whether astrocytes represent alternative therapeutic targets. We focus mainly on temporal lobe epilepsy (TLE), which is the most common form of epilepsy in adults and is characterised by high therapy resistance. Functional data from TLE patients and corresponding experimental models point to a complete loss of astrocytic coupling, but preservation of the gap junction forming proteins connexin43 and connexin30 in hippocampal sclerosis. Several studies further indicate that astrocyte uncoupling is a causal event in the initiation of TLE, as it occurs very early in epileptogenesis, clearly preceding dysfunctional changes in neurons. However, more research is needed to fully understand the role of gap junction channels in epilepsy and to develop safe and effective therapeutic strategies targeting astrocytes.
... The existence of gap junctions between astrocytes and oligodendrocytes leads to the formation of a functional syncytium that supports the movement of intracellular metabolic substances from astrocytes to oligodendrocytes [1]. A further important function of this coupling is the dilution of increased K + concentration in oligodendrocytes through direct passage into the astrocytic cytoplasm [17]. Finally, astrocytes release gliotransmitters such as glutamate, purine, and gamma-aminobutyric acid (GABA) (in response to neurotransmitters released from nearby synapses) into the synaptic cleft and thus can regulate neuronal excitability [18]. ...
Neuroglial cells, and especially astrocytes, constitute the most varied group of central nervous system (CNS) cells, displaying substantial diversity and plasticity during development and in disease states. The morphological changes exhibited by astrocytes during the acute and chronic stages following CNS injury can be characterized more precisely as a dynamic continuum of astrocytic reactivity. Different subpopulations of reactive astrocytes may be ascribed to stages of degenerative progression through their direct pathogenic influence upon neurons, neuroglia, the blood-brain barrier, and infiltrating immune cells. Multiple sclerosis (MS) constitutes an autoimmune demyelinating disease of the CNS. Despite the previously held notion that reactive astrocytes purely form the structured glial scar in MS plaques, their continued multifaceted participation in neuroinflammatory outcomes and oligodendrocyte and neuronal function during chronicity, suggest that they may be an integral cell type that can govern the pathophysiology of MS. From a therapeutic-oriented perspective, astrocytes could serve as key players to limit MS progression, once the integral astrocyte–MS relationship is accurately identified. This review aims toward delineating the current knowledge, which is mainly focused on immunomodulatory therapies of the relapsing–remitting form, while shedding light on uncharted approaches of astrocyte-specific therapies that could constitute novel, innovative applications once the role of specific subgroups in disease pathogenesis is clarified.
... Membrane potential is essential for maintaining the function of various protein machinery and transmitting signals between cells [2]. If the membrane potential is not properly maintained, cells can enter an overexcited state, leading to diseases such as epilepsy [3]. The cell membrane potential is highly sensitive to the extracellular potassium ion concentration, and the membrane potential changes sensitively depending on the potassium concentration [4]. ...
K2P channels, also known as two-pore domain K+ channels, play a crucial role in maintaining the cell membrane potential and contributing to potassium homeostasis due to their leaky nature. The TREK, or tandem of pore domains in a weak inward rectifying K+ channel (TWIK)-related K+ channel, subfamily within the K2P family consists of mechanical channels regulated by various stimuli and binding proteins. Although TREK1 and TREK2 within the TREK subfamily share many similarities, β-COP, which was previously known to bind to TREK1, exhibits a distinct binding pattern to other members of the TREK subfamily, including TREK2 and the TRAAK (TWIK-related acid-arachidonic activated K+ channel). In contrast to TREK1, β-COP binds to the C-terminus of TREK2 and reduces its cell surface expression but does not bind to TRAAK. Furthermore, β-COP cannot bind to TREK2 mutants with deletions or point mutations in the C-terminus and does not affect the surface expression of these TREK2 mutants. These results emphasize the unique role of β-COP in regulating the surface expression of the TREK family.
... This redistribution of K þ in space was initially described in Müller glial cells of the retina. 126,127 Perisynaptic processes of Müller glia cover synapses in the inner plexiform layer of the retina. Neuronal activity triggers substantial rises in [K þ ] o ; excess K þ enters glial cells through K ir 4.1 channels, while K þ subsequently is equilibrated throughout the cell and is released (again through K ir 4.1 channels) to the subretinal space, vitreous humour, or perivascular space. ...
... During intense neuronal activity, the extracellular concentration of K + ([K + ] o ) increases locally and is thought to be cleared by unidirectional redistribution through the astrocyte network (syncytium) toward regions of low K + such as blood vessels. Astroglial K + spatial buffering thus regulates the extracellular concentration of K + ([K + ] o ) and controls neuronal excitability in the CNS [7][8][9]. ...
A single sub-anesthetic dose of ketamine evokes rapid and long-lasting beneficial effects in patients with a major depressive disorder. However, the mechanisms underlying this effect are unknown. It has been proposed that astrocyte dysregulation of extracellular K+ concentration ([K+]o) alters neuronal excitability, thus contributing to depression. We examined how ketamine affects inwardly rectifying K+ channel Kir4.1, the principal regulator of K+ buffering and neuronal excitability in the brain. Cultured rat cortical astrocytes were transfected with plasmid-encoding fluorescently tagged Kir4.1 (Kir4.1-EGFP) to monitor the mobility of Kir4.1-EGFP vesicles at rest and after ketamine treatment (2.5 or 25 µM). Short-term (30 min) ketamine treatment reduced the mobility of Kir4.1-EGFP vesicles compared with the vehicle-treated controls (p < 0.05). Astrocyte treatment (24 h) with dbcAMP (dibutyryl cyclic adenosine 5′-monophosphate, 1 mM) or [K+]o (15 mM), which increases intracellular cAMP, mimicked the ketamine-evoked reduction of mobility. Live cell immunolabelling and patch-clamp measurements in cultured mouse astrocytes revealed that short-term ketamine treatment reduced the surface density of Kir4.1 and inhibited voltage-activated currents similar to Ba2+ (300 µM), a Kir4.1 blocker. Thus, ketamine attenuates Kir4.1 vesicle mobility, likely via a cAMP-dependent mechanism, reduces Kir4.1 surface density, and inhibits voltage-activated currents similar to Ba2+, known to block Kir4.1 channels.
... AQP4 expression has also been reported to be colocalized with Kir4.1, the inwardly rectifying potassium channel, on the endfeet processes of astrocytes in the brain and retinal Müller cells [7,20]. Typically, these retinal regions of co-localized Kir4.1 and AQP4 channels act as potassium [K + ] sinks for regulating high concentrations of [K + ] in the extracellular space around active neurons [21,22]. This physical coincidence led to early suggestions of the coupling of water transport and K + regulation by Müller cells [7,20,23], though more recent studies have failed to demonstrate changes in Kir4.1 expression or K + currents in AQP4 knockout mice [24]. ...
Purpose: Spatial co-localization of aquaporin water channels (AQP4) and inwardly rectifying potassium ion channels (Kir4.1) on the endfeet regions of glial cells has been suggested as the basis of functionally interrelated mechanisms of osmoregulation in brain edema. The aim of this study was to investigate the spatial and temporal changes in the expression of AQP4 and Kir4.1 channels in an avascular retina during the first week of the optical induction of refractive errors. Methods: Three-day-old hatchling chicks were randomly assigned to three groups and either did not wear lenses or were monocularly goggled with ±10D lenses for varying times up to 7 days before biometric assessment. Retinal tissue was prepared either for western blot analysis to show the presence of the AQP4 and Kir4.1 protein in the chick retina or for immunolocalization using AQP4 and Kir4.1 antibodies to determine the regional distribution and intensity of labeling during the induction of refractive errors. Results: As expected, ultrasonography demonstrated that all eyes showed rapid elongation post hatching. Negative lens-wearing eyes elongated faster than fellow eyes or normal non goggled eyes and became progressively more myopic with time post lensing. Positive lens-wearing eyes showed reduced ocular growth compared to normal controls and developed a hyperopic refraction. Quantitative immunohistochemistry revealed the upregulation of AQP4 channel expression on Müller cells in the retinal nerve fiber layer during the first 2 days of negative lens wear. Kir4.1 channel upregulation in the inner plexiform layer was only found on day 4 of positive lens wear during the development of refractive hyperopia. Conclusions: These results indicate that the expression of AQP4 and Kir4.1 channels on Müller cells is associated with the changes in ocular volume seen during the induction of refractive errors. However, the sites of greatest expression and the temporal pattern of the upregulation of AQP4 and Kir4.1 were dissimilar, indicating a dissociation of AQP4 and Kir4.1 function during refractive error development. Increased AQP4 expression in the nerve fiber layer is suggested to contribute to the rapid axial elongation and movement of fluid into the vitreous cavity in the presence of minus lenses; whereas, upregulation of Kir4.1 channels appears to play a role in limiting axial elongation in the presence of plus lenses. The processes by which osmoregulation is maintained during rapid growth of organs such as the young eye are unknown. Indeed, fluid dynamics in the eye are not well understood [1-3], but it is generally accepted that osmoregulation of the retina is primarily controlled by solute-linked transport through the ion channels and transporter mechanisms of the retinal pigment epithelium (RPE) and the Müller glial cells that span the retina from the vitreal border to the sub-retinal space [4-9]. The importance of the Müller glial cells in retinal osmoregulation began to emerge after the discovery of specialized transmembrane water channels known as aquaporins (AQPs) [10]. AQP0, AQP1, AQP4, and AQP9 proteins have all been found in mammalian retina [11-14], but
... Taken together, these data indicate K + conductance through Kir channels is important for KCC function and maintenance of [Cl − ] i , presumably through Kir mediated membrane polarization or K + recycling, but additional studies are needed to ascertain the specific ionic requirements for KCC function and functional relationship between Kir channels, KCC, and GGCC. Interestingly, pharmacological or genetic inhibition of Drosophila Kir2 has been reported lead to increased central nerve firing rates was speculated to be due to alterations in K + clearance during neurotransmission (Chen and Swale, 2018), which has been described for mammals (Djukic et al., 2007;Kofuji and Newman, 2004). While it is likely that inhibition of K + clearance is partly responsible for increased neural firing rates, it is also conceivable that inhibition of Kir channels leads to hyperexcitability through reduced KCC activity by direct (i.e. ...
The K+/Cl- cotransporter (KCC) is the primary mechanism by which mature neurons maintain low intracellular chloride (Cl-) concentration and has been shown to be functionally coupled to the GABA-gated chloride channels (GGCC) in Drosophila central neurons. Further, pharmacological inhibition of KCC has been shown to lead to acute toxicity of mosquitoes that highlights the toxicological relevance of insect KCC. Yet, gaps in knowledge remain regarding physiological drivers of KCC function and interactions of ion flux mechanisms upstream of GGCC in insects. Considering this, we employed electrophysiological and fluorescent microscopy techniques to further characterize KCC in the insect nervous system. Fluorescent microscopy indicated insect KCC2 is expressed in rdl neurons, which is the neuron type responsible for GABA-mediated neurotransmission, and are coexpressed with inward rectifier potassium (Kir) 2 channels. Coexpression of Kir2 and KCC2 suggested the possibility of functional coupling between these two K+ flux pathways. Indeed, extracellular recordings of Drosophila CNS showed pre-block of Kir channels prior to block of KCC led to a significant (P < 0.001) increase in CNS firing rates over baseline that when taken together, supports functional coupling of Kir to KCC function. Additionally, we documented a synergistic increase to toxicity of VU0463271, an established KCC inhibitor, above the expected additive toxicity after co-treatment with the Kir inhibitor, VU041. These data expand current knowledge regarding the physiological roles of KCC and Kir channels in the insect nervous system by defining additional pathways that facilitate inhibitory neurotransmission through GGCC.
... In the normal brain, Kir4.1 is expressed in astrocytes, where it functions to buffer extracellular potassium through passive uptake following neuronal firing. [40][41][42] Disruption of Kir4.1 function results in elevated extracellular K + levels, leading to subsequent neuronal excitability that may culminate in seizures. 26,43,44 Despite these links, our studies are the first to describe a role for Kir4.1 in GRE, which brings into focus the role of epilepsy-associated genes in GRE and glioma progression. ...
Seizures are a frequent pathophysiological feature of malignant glioma. Recent studies implicate peritumoral synaptic dysregulation as a driver of brain hyperactivity and tumor progression; however, the molecular mechanisms that govern these phenomena remain elusive. Using scRNA-seq and intraoperative patient ECoG recordings, we show that tumors from seizure patients are enriched for gene signatures regulating synapse formation. Employing a human-to-mouse in vivo functionalization pipeline to screen these genes, we identify IGSF3 as a mediator of glioma progression and dysregulated neural circuitry that manifests as spreading depolarization (SD). Mechanistically, we discover that IGSF3 interacts with Kir4.1 to suppress potassium buffering and found that seizure patients exhibit reduced expression of potassium handlers in proliferating tumor cells. In vivo imaging reveals that dysregulated synaptic activity emanates from the tumor-neuron interface, which we confirm in patients. Our studies reveal that tumor progression and seizures are enabled by ion dyshomeostasis and identify SD as a driver of disease.
... Specifically, AQP4 promotes the clearance of Aβ, and the dysregulation of AQP4 leads to the accumulation of Aβ in the brain [141,142]. Astrocytes of the NVU also maintain extracellular potassium concentrations in the brain via a process called "potassium siphoning" [143,144]. It is reasonable to predict that changes in potassium channel function may regulate brain function. ...
The cerebral vascular system stringently regulates cerebral blood flow (CBF). The components of the blood–brain barrier (BBB) protect the brain from pathogenic infections and harmful substances, efflux waste, and exchange substances; however, diseases develop in cases of blood vessel injuries and BBB dysregulation. Vascular pathology is concurrent with the mechanisms underlying aging, Alzheimer’s disease (AD), and vascular dementia (VaD), which suggests its involvement in these mechanisms. Therefore, in the present study, we reviewed the role of vascular dysfunction in aging and neurodegenerative diseases, particularly AD and VaD. During the development of the aforementioned diseases, changes occur in the cerebral blood vessel morphology and local cells, which, in turn, alter CBF, fluid dynamics, and vascular integrity. Chronic vascular inflammation and blood vessel dysregulation further exacerbate vascular dysfunction. Multitudinous pathogenic processes affect the cerebrovascular system, whose dysfunction causes cognitive impairment. Knowledge regarding the pathophysiology of vascular dysfunction in neurodegenerative diseases and the underlying molecular mechanisms may lead to the discovery of clinically relevant vascular biomarkers, which may facilitate vascular imaging for disease prevention and treatment.
... Potassium ion homeostasis is also involved in multiple feedback pathways mediated by glia (Fig. 2f). Neuronal activity results in local accumulations of extracellular K + ([K + ] e ) that may promptly be abated by spatial buffering by glia (Kofuji and Newman 2004). Significantly, synaptically evoked Ca 2+ signaling in astrocytes (yellow pathway) can also regulate [K + ] e to modulate both spontaneous synaptic release at excitatory synapses and neuronal firing (Wang et al. 2006). ...
Three decades after A. P. de Silva’s seminal paper introduced the concept of logic gates at the molecular level, the field of molecular logic gates (MLGs) has witnessed significant advancements. MLGs are devices designed to perform logical operations, utilizing one or more physical or chemical stimulus signals (inputs) to generate an output response. Notably, MLGs have found diverse applications, with optical detection of analytes emerging as a notable evolution of traditional chemosensors. Organic synthesis methods are pivotal in crafting molecular architectures tailored as optical devices capable of analyte detection through logical functions. This review delves into the fundamental aspects and physical–chemical properties of MLGs, with a particular emphasis on synthetic strategies driving their design.
Mature hippocampal astrocytes exhibit a linear current-to-voltage (I-V) K + membrane conductance, which is called passive conductance. It is estimated to enable astrocytes to keep potassium homeostasis in the brain. We previously reported that the TWIK-1/TREK-1 heterodimeric channels are crucial for astrocytic passive conductance. However, the regulatory mechanism of these channels by other binding proteins still remains elusive. Here, we identified Na+/H + exchange regulator-1 (NHERF-1), a protein highly expressed in astrocytes, as a candidate interaction partner for these channels. NHERF-1 endogenously bound to TWIK-1/TREK-1 in hippocampal cultured astrocytes. When NHERF-1 is overexpressed or silenced, surface expression and activity of TWIK-1/TREK-1 heterodimeric channels were inhibited or enhanced, respectively. Furthermore, we confirmed that reduced astrocytic passive conductance by NHERF-1 overexpressing in the hippocampus increases kainic acid (KA)-induced seizure sensitivity. Taken together, these results suggest that NHERF-1 is a key regulator of TWIK-1/TREK-1 heterodimeric channels in astrocytes and suppression of TREK-1 surface expression by NHERF-1 increases KA-induced seizure susceptibility via reduction of astrocytic passive conductance.
The extracellular potassium ion concentration in the brain exerts a significant influence on cellular excitability and intercellular communication. Perturbations in the extracellular potassium ion level are closely correlated with various chronic neuropsychiatric disorders including depression. However, a critical gap persists in performing real‐time and long‐term monitoring of extracellular potassium ions, which is necessary for comprehensive profiling of chronic neuropsychiatric diseases. Here, a fiber potassium ion sensor (FKS) that consists of a soft conductive fiber with a rough surface and a hydrophobic‐treated transduction layer interfaced with a potassium ion‐selective membrane is found to solve this problem. The FKS demonstrates stable interfaces between its distinct functional layers in an aqueous environment, conferring an exceptional stability of 6 months in vivo, in stark contrast to previous reports with working durations from hours to days. Upon implantation into the mouse brain, the FKS enables effective monitoring of extracellular potassium ion dynamics under diverse physiological states including anesthesia, forced swimming, and tail suspension. Using this FKS, tracking of extracellular potassium ion fluctuations that align with behaviors associated with the progression of depression over months is achieved, demonstrating its usability in studying chronic neuropsychiatric disorders from a new biochemical perspective.
Spreading depolarizations (SDs) are an enigmatic and ubiquitous co-morbidity of neural dysfunction. SDs are propagating waves of local field depolarization and increased extracellular potassium. They increase the metabolic demand on brain tissue, resulting in changes in tissue blood flow, and are associated with adverse neurological consequences including stroke, epilepsy, neurotrauma, and migraine. Their occurrence is associated with poor patient prognosis through mechanisms which are only partially understood. Here we show in vivo that two (structurally dissimilar) drugs, which suppress astroglial gap junctional communication, can acutely suppress SDs. We found that mefloquine hydrochloride (MQH), administered IP, slowed the propagation of the SD potassium waveform and intermittently led to its suppression. The hemodynamic response was similarly delayed and intermittently suppressed. Furthermore, in instances where SD led to transient tissue swelling, MQH reduced observable tissue displacement. Administration of meclofenamic acid (MFA) IP was found to reduce blood flow, both proximal and distal, to the site of SD induction, preceding a large reduction in the amplitude of the SD-associated potassium wave. We introduce a novel image processing scheme for SD wavefront localization under low-contrast imaging conditions permitting full-field wavefront velocity mapping and wavefront parametrization. We found that MQH administration delayed SD wavefront's optical correlates. These two clinically used drugs, both gap junctional blockers found to distinctly suppress SDs, may be of therapeutic benefit in the various brain disorders associated with recurrent SDs.
Glial cells were once thought of as simple support players in the central nervous system (CNS). However, the latest studies of glial cells have shown that they are actually of significance and play a variety of functions. Once thought to be passive supportive cells, astrocytes are now important in maintaining neurotransmitter balance, controlling synaptic activity, and regulating blood flow in the brain. Oligodendrocytes and Schwann cells, besides making myelin, are closely involved in controlling how fast nerve signals travel and maintaining the health of axons. Microglia had been thought to be only immune observers, but they also help to control the immune response and control synapses. Ependymal cells, which are sometimes ignored, are important in regulating cerebrospinal fluid circulation, directing the location of neural stem cells, and enabling cell communication. This chapter explores distinct glial cell types -astrocytes, oligodendrocytes, Schwann cells, microglia, and ependymal cells- highlighting their newfound, intricate functionalities and interactions with neurons.
Citation: Everaerts, K.; Thapaliya, P.; Pape, N.; Durry, S.; Eitelmann, S.; Roussa, E.; Ullah, G.; Rose, C.R. Inward Operation of Sodium-Bicarbonate Cotransporter 1 Promotes Astrocytic Na + Loading and Loss of ATP in Mouse Neocortex during Brief Chemical Ischemia. Cells 2023, 12, 2675. https://doi. Abstract: Ischemic conditions cause an increase in the sodium concentration of astrocytes, driving the breakdown of ionic homeostasis and exacerbating cellular damage. Astrocytes express high levels of the electrogenic sodium-bicarbonate cotransporter1 (NBCe1), which couples intracellular Na + homeostasis to regulation of pH and operates close to its reversal potential under physiological conditions. Here, we analyzed its mode of operation during transient energy deprivation via imaging astrocytic pH, Na + , and ATP in organotypic slice cultures of the mouse neocortex, complemented with patch-clamp and ion-selective microelectrode recordings and computational modeling. We found that a 2 min period of metabolic failure resulted in a transient acidosis accompanied by a Na + increase in astrocytes. Inhibition of NBCe1 increased the acidosis while decreasing the Na + load. Similar results were obtained when comparing ion changes in wild-type and Nbce1-deficient mice. Mathematical modeling replicated these findings and further predicted that NBCe1 activation contributes to the loss of cellular ATP under ischemic conditions, a result confirmed experimentally using FRET-based imaging of ATP. Altogether, our data demonstrate that transient energy failure stimulates the inward operation of NBCe1 in astrocytes. This causes a significant amelioration of ischemia-induced astrocytic acidification, albeit at the expense of increased Na + influx and a decline in cellular ATP.
In the last two decades, there has been increasing evidence supporting non-neuronal cells as active contributors to neurodegenerative disorders. Among glial cells, astrocytes play a pivotal role in driving amyotrophic lateral sclerosis (ALS) progression, leading the scientific community to focus on the “astrocytic signature” in ALS. Here, we summarized the main pathological mechanisms characterizing astrocyte contribution to MN damage and ALS progression, such as neuroinflammation, mitochondrial dysfunction, oxidative stress, energy metabolism impairment, miRNAs and extracellular vesicles contribution, autophagy dysfunction, protein misfolding, and altered neurotrophic factor release. Since glutamate excitotoxicity is one of the most relevant ALS features, we focused on the specific contribution of ALS astrocytes in this aspect, highlighting the known or potential molecular mechanisms by which astrocytes participate in increasing the extracellular glutamate level in ALS and, conversely, undergo the toxic effect of the excessive glutamate. In this scenario, astrocytes can behave as “producers” and “targets” of the high extracellular glutamate levels, going through changes that can affect themselves and, in turn, the neuronal and non-neuronal surrounding cells, thus actively impacting the ALS course. Moreover, this review aims to point out knowledge gaps that deserve further investigation.
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the loss of both upper and lower motor neurons, resulting in muscle weakness, atrophy, paralysis, and eventually death. Motor cortical hyperexcitability is a common phenomenon observed at the presymptomatic stage of ALS. Both cell-autonomous (the intrinsic properties of motor neurons) and non-cell-autonomous mechanisms (cells other than motor neurons) are believed to contribute to cortical hyperexcitability. Decoding the pathological relevance of these dynamic changes in motor neurons and glial cells has remained a major challenge. This review summarizes the evidence of cortical hyperexcitability from both clinical and preclinical research, as well as the underlying mechanisms. We discuss the potential role of glial cells, particularly microglia, in regulating abnormal neuronal activity during the disease progression. Identifying early changes such as neuronal hyperexcitability in the motor system may provide new insights for earlier diagnosis of ALS and reveal novel targets to halt the disease progression.
The human brain is composed of nearly one hundred billion neurons and an equal number of glial cells, including macroglia, i.e., astrocytes and oligodendrocytes, and microglia, the resident immune cells of the brain. In the last few decades, compelling evidence has revealed that glial cells are far more active and complex than previously thought. In particular, astrocytes, the most abundant glial cell population, not only take part in brain development, metabolism, and defense against pathogens and insults, but they also affect sensory, motor, and cognitive functions by constantly modulating synaptic activity. Not surprisingly, astrocytes are actively involved in neurodegenerative diseases (NDs) and other neurological disorders like brain tumors, in which they rapidly become reactive and mediate neuroinflammation. Reactive astrocytes acquire or lose specific functions that differently modulate disease progression and symptoms, including cognitive impairments. Astrocytes express several types of ion channels, including K+, Na+, and Ca2+ channels, transient receptor potential channels (TRP), aquaporins, mechanoreceptors, and anion channels, whose properties and functions are only partially understood, particularly in small processes that contact synapses. In addition, astrocytes express ionotropic receptors for several neurotransmitters. Here, we provide an extensive and up-to-date review of the roles of ion channels and ionotropic receptors in astrocyte physiology and pathology. As examples of two different brain pathologies, we focus on Alzheimer’s disease (AD), one of the most diffuse neurodegenerative disorders, and glioblastoma (GBM), the most common brain tumor. Understanding how ion channels and ionotropic receptors in astrocytes participate in NDs and tumors is necessary for developing new therapeutic tools for these increasingly common neurological conditions.
Epilepsy is a group of neurological diseases which requires significant economic costs for the treatment and care of patients. The central point of epileptogenesis stems from the failure of synaptic signal transmission mechanisms, leading to excessive synchronous excitation of neurons and characteristic epileptic electroencephalogram activity, in typical cases being manifested as seizures and loss of consciousness. The causes of epilepsy are extremely diverse, which is one of the reasons for the complexity of selecting a treatment regimen for each individual case and the high frequency of pharmacoresistant cases. Therefore, the search for new drugs and methods of epilepsy treatment requires an advanced study of the molecular mechanisms of epileptogenesis. In this regard, the investigation of molecular chaperones as potential mediators of epileptogenesis seems promising because the chaperones are involved in the processing and regulation of the activity of many key proteins directly responsible for the generation of abnormal neuronal excitation in epilepsy. In this review, we try to systematize current data on the role of molecular chaperones in epileptogenesis and discuss the prospects for the use of chemical modulators of various chaperone groups’ activity as promising antiepileptic drugs.
Background
Paroxysmal kinesigenic dyskinesia is the representative form of paroxysmal dyskinesia, and its mechanism is unclear. Although paroxysmal kinesigenic dyskinesia is mostly attributed to genetic factors, more than 60% of paroxysmal kinesigenic dyskinesia cases are of uncertain mutations. We searched for novel genetic causes of paroxysmal kinesigenic dyskinesia and explored the corresponding pathophysiology.
Methods
A cohort of 476 probands with primary paroxysmal kinesigenic dyskinesia of uncertain genetic causes were enrolled for whole exome sequencing. Gene Ranking, Identification and Prediction Tool, a method of case-control analysis,was applied to identify the candidate genes. Another 46 probands were subsequently screened with Sanger sequencing. Whole-cell patch-clamp recording was applied to verify the electrophysiological impact of the identified variants. Amouse model with cerebellar heterozygous knockout of the candidate gene was generated via adeno-associated virus injection, and dyskinesia-like phenotype inducement and rotarod tests were performed. In vivo multiunit electrical recording was applied to investigate the change in neural excitability in knockout mice.
Results
Heterozygous variants of potassium channel inwardly rectifying subfamily J member 10 (KCNJ10) mainly clustered in patients withparoxysmal kinesigenic dyskinesia compared with the control groups. Fifteenvariants were detected in 16 out of 522 probands (frequency = 3.07%). Patients with KCNJ10 variants tended to have a later onset age and shorter duration of attacks than patients with proline-rich transmembrane protein 2 mutations. Inwardly rectifying potassium channel 4.1 (Kir4.1) is highly expressed in the cerebellum of mice,and its expression pattern is consistent with the natural course of paroxysmal kinesigenic dyskinesia. Further electrophysiological recordings revealed that all the variants identified in patients led to different degrees of reduction in Kir4.1 currents, and mice with heterozygous conditional knockout of Kcnj10 in the cerebellum presented dystonic posture with epidural KCl stimulation in cerebellum, as well as poor motor coordination and motor learning ability in rotarod tests. The firing rate of deep cerebellar nuclei was significantly elevated in Kcnj10-cKO mice, indicating abnormal hyperexcitability in the Kir4.1-deficient mouse model.
Conclusion
We identified heterozygous mutations of KCNJ10 as a novel genetic cause of paroxysmal kinesigenic dyskinesia. Based on the findings in the present study, we suppose that the impaired function of Kir4.1 might lead to defective homeostatic maintenance of extracellular potassium and glutamate levels and thus cause abnormal neuronal excitability. The findings elucidated the pathogenesis of paroxysmal kinesigenic dyskinesia, thoughadditional efforts are needed to reveal the role of Kir4.1 in movement disorders.
The role of glia, particularly astrocytes, in mediating the central nervous system’s response to injury and neurodegenerative disease is an increasingly well studied topic. These cells perform myriad support functions under physiological conditions but undergo behavioral changes – collectively referred to as ‘reactivity’ – in response to the disruption of neuronal homeostasis from insults, including glaucoma. However, much remains unknown about how reactivity alters disease progression – both beneficially and detrimentally – and whether these changes can be therapeutically modulated to improve outcomes. Historically, the heterogeneity of astrocyte behavior has been insufficiently addressed under both physiological and pathological conditions, resulting in a fragmented and often contradictory understanding of their contributions to health and disease. Thanks to increased focus in recent years, we now know this heterogeneity encompasses both intrinsic variation in physiological function and insult-specific changes that vary between pathologies. Although previous studies demonstrate astrocytic alterations in glaucoma, both in human disease and animal models, generally these findings do not conclusively link astrocytes to causative roles in neuroprotection or degeneration, rather than a subsequent response. Efforts to bolster our understanding by drawing on knowledge of brain astrocytes has been constrained by the primacy in the literature of findings from peri-synaptic ‘gray matter’ astrocytes, whereas much early degeneration in glaucoma occurs in axonal regions populated by fibrous ‘white matter’ astrocytes. However, by focusing on findings from astrocytes of the anterior visual pathway – those of the retina, unmyelinated optic nerve head, and myelinated optic nerve regions – we aim to highlight aspects of their behavior that may contribute to axonal vulnerability and glaucoma progression, including roles in mitochondrial turnover and energy provisioning. Furthermore, we posit that astrocytes of the retina, optic nerve head and myelinated optic nerve, although sharing developmental origins and linked by a network of gap junctions, may be best understood as distinct populations residing in markedly different niches with accompanying functional specializations. A closer investigation of their behavioral repertoires may elucidate not only their role in glaucoma, but also mechanisms to induce protective behaviors that can impede the progressive axonal damage and retinal ganglion cell death that drive vision loss in this devastating condition.
This review focuses on Receptor for Advanced Glycation Endproducts/Diaphonous related formin1 (RAGE/Diaph1) interaction as a modulator of actin cytoskeleton dynamics in peripheral nervous system (PNS) in diabetes. Deciphering the complex molecular interactions between RAGE and Diaph1 is crucial in expanding our understanding of diabetic length dependent neuropathy (DLDN). DLDN is a common neurological disorder in patients with diabetes. It is well known that actin cytoskeletal homeostasis is disturbed during DLDN. Thus, we review the current status of knowledge about RAGE/Diaph1 impact on actin cytoskeletal malfunctions in PNS and DLDN progression in diabetes. We also survey studies about small molecules that may block RAGE/Diaph1 axis and thus inhibit the progression of DLDN. Finally, we explore examples of cytoskeletal long-non coding RNAs (lncRNAs) currently unrelated to DLDN, to discuss their potential role in this disease. Most recent studies indicated that lncRNAs have a great potential in many research areas, including RAGE/Diaph1 axis as well as DLDN. Altogether, this review is aimed at giving us an insight into the involvement of cytoskeletal lncRNAs in DLDN.
Astrocytes and neurons extensively interact in the brain. Identifying astrocyte and neuron proteomes is essential for elucidating the protein networks that dictate their respective contributions to physiology and disease. Here we used cell-specific and subcompartment-specific proximity-dependent biotinylation¹ to study the proteomes of striatal astrocytes and neurons in vivo. We evaluated cytosolic and plasma membrane compartments for astrocytes and neurons to discover how these cells differ at the protein level in their signalling machinery. We also assessed subcellular compartments of astrocytes, including end feet and fine processes, to reveal their subproteomes and the molecular basis of essential astrocyte signalling and homeostatic functions. Notably, SAPAP3 (encoded by Dlgap3), which is associated with obsessive–compulsive disorder (OCD) and repetitive behaviours2–8, was detected at high levels in striatal astrocytes and was enriched within specific astrocyte subcompartments where it regulated actin cytoskeleton organization. Furthermore, genetic rescue experiments combined with behavioural analyses and molecular assessments in a mouse model of OCD⁴ lacking SAPAP3 revealed distinct contributions of astrocytic and neuronal SAPAP3 to repetitive and anxiety-related OCD-like phenotypes. Our data define how astrocytes and neurons differ at the protein level and in their major signalling pathways. Moreover, they reveal how astrocyte subproteomes vary between physiological subcompartments and how both astrocyte and neuronal SAPAP3 mechanisms contribute to OCD phenotypes in mice. Our data indicate that therapeutic strategies that target both astrocytes and neurons may be useful to explore in OCD and potentially other brain disorders.
Inorganic ions are indispensable substances in living systems, and are widely involved in many essential biological processes. Increasing evidence has shown that the disruption of ion homeostasis is closely related to health problems; thus, the in situ evaluation of ion levels and monitoring their dynamic changes in the living body are critical for precise diagnosis and therapy of diseases. Currently, along with the development of advanced imaging probes, optical imaging and magnetic resonance imaging (MRI) are becoming two major imaging approaches for the identification of ion dynamics. In this review, the design and fabrication of ion-sensitive fluorescent/MRI probes are introduced from the perspective of imaging principles. Furthermore, the recent advances in dynamic imaging of ion levels in living organisms, as well as understanding of ion dyshomeostasis related progression and early diagnosis of diseases, are summarized. Finally, the future perspectives of state-of-the-art ion-sensitive probes for biomedical applications are briefly discussed.
Introduction
Identification of early adaptive and maladaptive neuronal stress responses is an important step in developing targeted neuroprotective therapies for degenerative disease. In glaucoma, retinal ganglion cells (RGCs) and their axons undergo progressive degeneration resulting from stress driven by sensitivity to intraocular pressure (IOP). Despite therapies that can effectively manage IOP many patients progress to vision loss, necessitating development of neuronal-based therapies. Evidence from experimental models of glaucoma indicates that early in the disease RGCs experience altered excitability and are challenged with dysregulated potassium (K⁺) homeostasis. Previously we demonstrated that certain RGC types have distinct excitability profiles and thresholds for depolarization block, which are associated with sensitivity to extracellular K⁺.
Methods
Here, we used our inducible mouse model of glaucoma to investigate how RGC sensitivity to K⁺ changes with exposure to elevated IOP.
Results
In controls, conditions of increased K⁺ enhanced membrane depolarization, reduced action potential generation, and widened action potentials. Consistent with our previous work, 4 weeks of IOP elevation diminished RGC light-and current-evoked responses. Compared to controls, we found that IOP elevation reduced the effects of increased K⁺ on depolarization block threshold, with IOP-exposed cells maintaining greater excitability. Finally, IOP elevation did not alter axon initial segment dimensions, suggesting that structural plasticity alone cannot explain decreased K⁺ sensitivity.
Discussion
Thus, in response to prolonged IOP elevation RGCs undergo an adaptive process that reduces sensitivity to changes in K⁺ while diminishing excitability. These experiments give insight into the RGC response to IOP stress and lay the groundwork for mechanistic investigation into targets for neuroprotective therapy.
Objective:
To determine the role of ion concentrations and ion pump activity in conduction block of myelinated axon induced by a long-duration direct current (DC).
Methods:
A new axonal conduction model for myelinated axons based on the classical Frankenhaeuser-Huxley (FH) equations is developed that includes ion pump activity and allows the intracellular and extracellular Na+ and K+ concentrations to change with axonal activity.
Results:
Action potential generation, propagation, and acute DC block occurring within a short period (milliseconds) that do not significantly change the ion concentrations or trigger ion pump activity are successfully simulated by the new model in a similar way as the classical FH model. Different from the classical model, the new model also successfully simulates the post-stimulation block phenomenon, i.e., the axonal conduction block occurring after terminating a long-duration (30 seconds) DC stimulation as observed recently in animal studies. The model reveals a significant K+ accumulation outside the axonal node as the possible mechanism underlying the post-DC block that is slowly reversed by ion pump activity during the post-stimulation period.
Conclusion:
Changes in ion concentrations and ion pump activity play an important role in post-stimulation block induced by long-duration DC stimulation.
Significance:
Long-duration stimulation is used clinically for many neuromodulation therapies, but the effects on axonal conduction/block are poorly understood. This new model will be useful for better understanding of the mechanisms underlying long-duration stimulation that changes ion concentrations and triggers ion pump activity.
Elevation of intraocular pressure (IOP) is a major risk factor for neurodegeneration in glaucoma. Glial cells, which play an important role in normal functioning of retinal neurons, are well involved into retinal ganglion cell (RGC) degeneration in experimental glaucoma animal models generated by elevated IOP. In response to elevated IOP, mGluR I is first activated and Kir4.1 channels are subsequently inhibited, which leads to the activation of Müller cells. Müller cell activation is followed by a complex process, including proliferation, release of inflammatory and growth factors (gliosis). Gliosis is further regulated by several factors. Activated Müller cells contribute to RGC degeneration through generating glutamate receptor-mediated excitotoxicity, releasing cytotoxic factors and inducing microglia activation. Elevated IOP activates microglia, and following morphological and functional changes, these cells, as resident immune cells in the retina, show adaptive immune responses, including an enhanced release of pro-inflammatory factors (tumor neurosis factor-α, interleukins, etc.). These ATP and Toll-like receptor-mediated responses are further regulated by heat shock proteins, CD200R, chemokine receptors, and metabotropic purinergic receptors, may aggravate RGC loss. In the optic nerve head, astrogliosis is initiated and regulated by a complex reaction process, including purines, transmitters, chemokines, growth factors and cytokines, which contributes to RGC axon injury through releasing pro-inflammatory factors and changing extracellular matrix in glaucoma. The effects of activated glial cells on RGCs are further modified by the interplay among different types of glial cells. This review is concluded by presenting an in-depth discussion of possible research directions in this field in the future.
Membrane water transport is critically involved in brain volume homeostasis and in the pathogenesis of brain edema. The cDNA encoding aquaporin-4 (AQP4) water channel protein was recently isolated from rat brain. We used immunocytochemistry and high-resolution immunogold electron microscopy to identify the cells and membrane domains that mediate water flux through AQP4. The AQP4 protein is abundant in glial cells bordering the subarachnoidal space, ventricles, and blood vessels. AQP4 is also abundant in osmosensory areas, including the supraoptic nucleus and subfornical organ. Immunogold analysis demonstrated that AQP4 is restricted to glial membranes and to subpopulations of ependymal cells. AQP4 is particularly strongly expressed in glial membranes that are in direct contact with capillaries and pia. The highly polarized AQP4 expression indicates that these cells are equipped with specific membrane domains that are specialized for water transport, thereby mediating the flow of water between glial cells and the cavities filled with CSF and the intravascular space.
Postembedding immunogold labeling was used to examine the subcellular distribution of the inwardly rectifying K⁺ channel Kir4.1 in rat retinal Müller cells and to compare this with the distribution of the water channel aquaporin-4 (AQP4). The quantitative analysis suggested that both molecules are enriched in those plasma membrane domains that face the vitreous body and blood vessels. In addition, Kir4.1, but not AQP4, was concentrated in the basal ∼300–400 nm of the Müller cell microvilli. These data indicate that AQP4 may mediate the water flux known to be associated with K⁺ siphoning in the retina. By its highly differentiated distribution of AQP4, the Müller cell may be able to direct the water flux to select extracellular compartments while protecting others (the subretinal space) from inappropriate volume changes. The identification of specialized membrane domains with high Kir4.1 expression provides a morphological correlate for the heterogeneous K⁺ conductance along the Müller cell surface. GLIA 26:47–54, 1999. © 1999 Wiley-Liss, Inc.
An inwardly rectifying potassium channel predominantly expressed in glial cells, Kir4.1/KAB-2, has a sequence of Ser-Asn-Val in its carboxyl-terminal end, suggesting a possible interaction with an anchoring protein
of the PSD-95 family. We examined the effects of PSD-95 on the distribution and function of Kir4.1 in a mammalian cell line.
When Kir4.1 was expressed alone, the channel immunoreactivity was distributed homogeneously. In contrast, when co-expressed
with PSD-95, prominent clustering of Kir4.1 in the cell membrane occurred. Kir4.1 was co-immunoprecipitated with PSD-95 in
the co-expressed cells. Glutathione S-transferase-fusion protein of COOH terminus of Kir4.1 bound to PSD-95. These interactions disappeared when the Ser-Asn-Val
motif was deleted. The magnitude of whole-cell Kir4.1 current was increased by 2-fold in cells co-expressing Kir4.1 and PSD-95
compared with cells expressing Kir4.1 alone. SAP97, another member of the PSD-95 family, showed similar effects on Kir4.1.
Furthermore, we found that Kir4.1 as well as SAP97 distributed not diffusely but clustered in retinal glial cells. Therefore,
PSD-95 family proteins may be a physiological regulator of the distribution and function of Kir4.1 in glial cells.
Electrophysiological techniques were used to determine the ion selectivity properties and the spatial distribution of the membrane conductance of amphibian Müller cells. Membrane potential changes recorded during ion substitution experiments in frog (Rana pipiens) retinal slices demonstrated that the Müller cell K+:Na+ membrane permeability ratio is approximately 490:1 and that cell Cl- permeability is extremely low. In frog retinal slices, Müller cell input resistance was 8.5 megohms when measured in the inner plexiform layer and 4.8 megohms when measured in the optic fiber layer. Intact, enzymatically dissociated salamander (Ambystoma tigrinum) cells had an input resistance of 7.9 megohms, whereas cells lacking their endfoot process (removed by surgical microdissection or by shearing force) had a resistance of 152 megohms. Pressure ejection of a 100 mM K+ solution near the proximal surface of the endfeet of dissociated salamander cells produced depolarizations 7 times greater than did ejections near the lateral face of the endfoot and 24 to 50 times greater than did ejections near other cell regions. Similar K+ ejection results were obtained from Müller cells in salamander and frog retinal slices. Taken together, these results demonstrate that in both the frog and the salamander, approximately 95% of the total membrane conductance of Müller cells is localized in the cell's endfoot process. In salamander, the specific membrane resistance of the endfoot membrane was estimated to be 32 ohm X cm2 whereas the specific resistance of the remainder of the cell was 7300 ohm X cm2. This remarkably nonuniform conductance distribution has important consequences for theories concerning K+ regulation in the retina and for mechanisms underlying electroretinogram generation.
Recordings of light-evoked changes in extracellular K+ concentration (delta[K+]o) were obtained in the retinas of frog and mudpuppy. In eyecup preparations, various recording approaches were used and provided evidence for a K increase near the outer plexiform layer (distal K increase). This distal K increase could be pharmacologically dissociated from the well-known, large K increase in the proximal retina by the application of ethanol and gamma-aminobutyric acid. The distal K increase also often showed surround antagonism. A retinal slice preparation was used to permit electrode placement into the desired retinal layers under direct visual control and without the risk of electrode damage to adjacent layers. In the slice, a distinct distal K increase was found in the outer plexiform layer, in addition to the prominent K increase in the inner plexiform layer. Compared with eyecups, only weak K increases were found in the nuclear layers of the slice. This suggests that the K responses observed in the nuclear layers of eyecups may be generated by K+ diffusing along the electrode track from the plexiform layers. In the context of current models of ERG b-wave generation, the magnitude of the recorded distal K increase, compared with the proximal K increase, seems too small to give rise to the b-wave. However, the distal K increase may be differentially depressed by electrode dead space. It is also possible that if certain aspects of the models of b-wave generation were modified, then the observed distal K increase could give rise to the b-wave.
Efflux of K+ from dissociated salamander Müller cells was measured with ion-selective microelectrodes. When the distal end of an isolated cell was exposed to high concentrations of extracellular K+, efflux occurred primarily from the endfoot, a cell process previously shown to contain most of the K+ conductance of the cell membrane. Computer simulations of K+ dynamics in the retina indicate that shunting ions through the Müller cell endfoot process is more effective in clearing local increases in extracellular K+ from the retina than is diffusion through extracellular space.
Spreading depression (SD) and the related hypoxic SD-like depolarization (HSD) are characterized by rapid and nearly complete depolarization of a sizable population of brain cells with massive redistribution of ions between intracellular and extracellular compartments, that evolves as a regenerative, “all-or-none” type process, and propagates slowly as a wave in brain tissue. This article reviews the characteristics of SD and HSD and the main hypotheses that have been proposed to explain them. Both SD and HSD are composites of concurrent processes. Antagonists of N-methyl-d-aspartate (NMDA) channels or voltage-gated Na ⁺ or certain types of Ca ²⁺ channels can postpone or mitigate SD or HSD, but it takes a combination of drugs blocking all known major inward currents to effectively prevent HSD. Recent computer simulation confirmed that SD can be produced by positive feedback achieved by increase of extracellular K ⁺ concentration that activates persistent inward currents which then activate K ⁺ channels and release more K ⁺ . Any slowly inactivating voltage and/or K ⁺ -dependent inward current could generate SD-like depolarization, but ordinarily, it is brought about by the cooperative action of the persistent Na ⁺ current I Na,P plus NMDA receptor-controlled current. SD is ignited when the sum of persistent inward currents exceeds persistent outward currents so that total membrane current turns inward. The degree of depolarization is not determined by the number of channels available, but by the feedback that governs the SD process. Short bouts of SD and HSD are well tolerated, but prolonged depolarization results in lasting loss of neuron function. Irreversible damage can, however, be avoided if Ca ²⁺ influx into neurons is prevented.
This chapter presents certain arguments in favor of an active uptake of K+ in exchange for Na+ mediated by the glial Na+, K+-ATPase by comparing results obtained with cerebral cortex slices, fractions enriched in neuronal and glial structures, and cultured astrocytes. It is well known that brain extracellular K+ is maintained at a resting level of 3 mM but can increase up to 10 or 20 mM during intense nerve activity. The K+ activation of the glial enzyme is maintained through a partial purification procedure, demonstrating that this glial property does not depend upon the integrity of the cells or the membranes but is related to the structure of the protein itself. Nearly homogeneous populations of living astrocytes can be obtained in culture using mechanically or enzymatically dissociated newborn rat cerebral cortex. Brain cortex of adult mammalian brain is able to concentrate K+ ions within the intracellular space.
Recordings of light-evoked changes in extracellular K+ concentration (delta[K+]o) were obtained in the retinas of frog and mudpuppy. In eyecup preparations, various recording approaches were used and provided evidence for a K increase near the outer plexiform layer (distal K increase). This distal K increase could be pharmacologically dissociated from the well-known, large K increase in the proximal retina by the application of ethanol and gamma-aminobutyric acid. The distal K increase also often showed surround antagonism. A retinal slice preparation was used to permit electrode placement into the desired retinal layers under direct visual control and without the risk of electrode damage to adjacent layers. In the slice, a distinct distal K increase was found in the outer plexiform layer, in addition to the prominent K increase in the inner plexiform layer. Compared with eyecups, only weak K increases were found in the nuclear layers of the slice. This suggests that the K responses observed in the nuclear layers of eyecups may be generated by K+ diffusing along the electrode track from the plexiform layers. In the context of current models of ERG b-wave generation, the magnitude of the recorded distal K increase, compared with the proximal K increase, seems too small to give rise to the b-wave. However, the distal K increase may be differentially depressed by electrode dead space. It is also possible that if certain aspects of the models of b-wave generation were modified, then the observed distal K increase could give rise to the b-wave.
Müller cells, the principal glial cells of the retina, exhibit a high degree of functional and morphological polarization. An inward rectifying K+ channel, the dominant ion channel in Müller cells, is localized preferentially to cell endfeet, which terminate on the vitreal surface of the retina and on blood vessels. Two acid/ base transport systems, an Na+/HCO 3- cotransporter and a CI-/HCO3 - anion exchanger, also are localized preferentially to the endfeet. These functional specializations facilitate the ability of Müller cells to regulate extracellular ion levels in the retina. Müller cells regulate extracellular K+ levels by transporting K+ away from the neural retina to the vitreous humor and the subretinal space. Müller cells may also regulate retinal CO2 and pH by the combined action of cell carbonic anhydrase and acid/base transporters localized to the endfeet, and they may control blood flow by the depolarization-induced release of potassium and protons from cell endfeet onto blood vessels. The physiology of ion transport in CNS astrocytes is not understood as well as it is in Müller cells. The presence of inward rectifying K+ channels and acid/base transporters in astrocytes, however, suggests that these cells may utilize mechanisms similar to those of Müller cells in regulating the extracellular microenvironment and in controlling blood flow. The Neuroscientist 2:109-117, 1996
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.
The water channel AQP4 is concentrated in perivascular and subpial membrane domains of brain astrocytes. These membranes form
the interface between the neuropil and extracerebral liquid spaces. AQP4 is anchored at these membranes by its carboxyl terminus
to α-syntrophin, an adapter protein associated with dystrophin. To test functions of the perivascular AQP4 pool, we studied
mice homozygous for targeted disruption of the gene encoding α-syntrophin (α-Syn−/−). These animals show a marked loss of AQP4 from perivascular and subpial membranes but no decrease in other membrane domains,
as judged by quantitative immunogold electron microscopy. In the basal state, perivascular and subpial astroglial end-feet
were swollen in brains of α-Syn−/− mice compared to WT mice, suggesting reduced clearance of water generated by brain metabolism. When stressed by transient
cerebral ischemia, brain edema was attenuated in α-Syn−/− mice, indicative of reduced water influx. Surprisingly, AQP4 was strongly reduced but α-syntrophin was retained in perivascular
astroglial end-feet in WT mice examined 23 h after transient cerebral ischemia. Thus α-syntrophin-dependent anchoring of AQP4
is sensitive to ischemia, and loss of AQP4 from this site may retard the dissipation of postischemic brain edema. These studies
identify a specific, syntrophin-dependent AQP4 pool that is expressed at distinct membrane domains and which mediates bidirectional
transport of water across the brain–blood interface. The anchoring of AQP4 to α-syntrophin may be a target for treatment of
brain edema, but therapeutic manipulations of AQP4 must consider the bidirectional water flux through this molecule.
We measured activity‐dependent changes in [K ⁺ ] o with K ⁺ ‐selective microelectrodes in adult rat optic nerve, a CNS white matter tract, to investigate the factors responsible for post‐stimulus recovery of [K ⁺ ] o .
Post‐stimulus recovery of [K ⁺ ] o followed a double‐exponential time course with an initial, fast time constant, τ fast , of 0.9 ± 0.2 s (mean ± s.d. ) and a later, slow time constant, τ slow , of 4.2 ± 1 s following a 1 s, 100 Hz stimulus. τ fast , but not τ slow , decreased with increasing activity‐dependent rises in [K ⁺ ] o . τ slow , but not τ fast , increased with increasing stimulus duration.
Post‐stimulus recovery of [K ⁺ ] o was temperature sensitive. The apparent temperature coefficients ( Q 10 , 27–37°C) for the fast and slow components following a 1 s, 100 Hz stimulus were 1.7 and 2.6, respectively.
Post‐stimulus recovery of [K ⁺ ] o was sensitive to Na ⁺ pump inhibition with 50 μM strophanthidin. Following a 1 s, 100 Hz stimulus, 50 μM strophanthidin increased τ fast and τ slow by 81 and 464%, respectively. Strophanthidin reduced the temperature sensitivity of post‐stimulus recovery of [K ⁺ ] o .
Post‐stimulus recovery of [K ⁺ ] o was minimally affected by the K ⁺ channel blocker Ba ²⁺ (0.2 m m ). Following a 10 s, 100 Hz stimulus, 0.2 m m Ba ²⁺ increased τ fast and τ slow by 24 and 18%, respectively.
Stimulated increases in [K ⁺ ] o were followed by undershoots of [K ⁺ ] o . Post‐stimulus undershoot amplitude increased with stimulus duration but was independent of the peak preceding [K ⁺ ] o increase.
These observations imply that two distinct processes contribute to post‐stimulus recovery of [K ⁺ ] o in central white matter. The results are compatible with a model of K ⁺ removal that attributes the fast, initial phase of K ⁺ removal to K ⁺ uptake by glial Na ⁺ pumps and the slower, sustained decline to K ⁺ uptake via axonal Na ⁺ pumps.
Previous electrophysiological evidence has indicated that astrocytes and oligodendrocytes express inwardly rectifying K+ channels both in vitro and in vivo. Here, for the first time, we have undertaken light microscopic immunohistochemical studies demonstrating the location of one such channel, Kir4.1, in both cell types in regions of the rat CNS. Some astrocytes such as those in the deep cerebellar nuclei, Bergmann glia, retinal Müller cells, and a subset in hippocampus express Kir4.1 immunoreactivity, but not others including those in white matter. Oligodendrocytes also express this protein, strongly in perikarya and to a lesser extent in their processes. Expression of Kir4.1 in astrocytes and oligodendrocytes would enable these cells to clear extracellular K+ through this channel, whereas nonexpressors might use other mechanisms. GLIA 30:362–372, 2000. © 2000 Wiley-Liss, Inc.
Extracellular potassium activity (ak) and field potentials (fp) were measured in the nucleus ventro-postero-lateralis (VPL) thalami in order to assess the extent of thalamic participation in cortical seizure activity. Small increases (up to 0.7 mmole/l) or decreases (up to 0.2 mmole/1) in ak were induced by electrical stimulation of the contralateral forepaw. These changes in ak were spatially more limited than the simultaneously recorded fp. Similar observations were made during weak electrical stimulation of the somatosensory cortex and during interictal spikes in a cortical penicillin focus. Large and widespread increases in ak to levels of 11.6 mmoles/l and slow negative fps of 8 mV accompanied seizure generation either in a cortical penicillin focus or during intense repetitive electrical stimulation of the cortical surface. Subsequent to such increases ak fell to subnormal levels. The amplitudes and durations of such undershoots were correlated with the amplitudes of the preceding increases in ak. Sometimes thalamic seizures ceased before cortical epileptic episodes. This resulted in a decrease of cortical EEG amplitudes. After ablation of the sensorimotor cortex seizures in forepaw-VPL could be induced by stimulation of the cortical white matter underlying either forepaw or hind paw representation areas of the somatosensory cortex. These results further support the conclusion that specific thalamic nuclei participate in seizure generation and may serve as a subcortical route of seizure spread.
Recent cloning of a family of genes encoding inwardly rectifying K+ channels has provided the opportunity to explain some venerable problems in membrane biology. An expanding number of novel inwardly rectifying K+ channel clones has revealed multiple channel subfamilies that have specialized roles in cell function. The molecular determinants of inward rectification have been largely elucidated with the discovery of endogenous polyamines that act as voltage-dependent intracellular channel blockers, and with the identification of a critical site in the channel that mediates high-affinity block by both polyamines and Mg2+.
Inwardly rectifying K+ (Kir) channels are considered to play the major role in the spatial buffering of glial cells. We have examined the electrophysiological properties of Kir channels in isolated rabbit Müller cells (retinal glial cells). Although a previous study reported that three kinds of Kir channels with different conductance and rectification properties were expressed in distinct regions of rabbit Müller cell membrane, we could record only a single population of Kir channels from the distal end to the endfoot in 205 successful cell-attached patches. The identified Müller cell Kir channel had a unitary conductance of 25 pS in the inward direction with symmetrical 153 mM K+ condition. The conductance and gating properties of the Müller cell Kir channels were identical to those of the KAB-2/Kir4.1 heterologously expressed in a mammalian cultured cell line, HEK293T cells. Thus KAB-2/Kir4.1 was the predominant glial Kir channel not only in the brain, but also in the retina. Because its rectification is intermediate, this Kir channel may contribute to both the intrusion and the extrusion of K+ ions across glial cell membrane and may be the major pathway for redistribution of extracellular K+ ions in the central nervous system.
The effects of systemic LSD (10--25 micrograms/kg) on visually evoked [K+]o responses from the striate cortex have been investigated in cats. Elevation of [K+]o was dependent on the orientation, and direction of stimulus movement and showed ocular dominance. LSD most commonly produced a depression of [K+]o transients and a deterioration in their directional selectivity. These observations suggest that the effect of LSD is to produce a net depression in visually evoked neuronal firing.
The development of potassium specific ion exchanger microelectrodes has enabled investigators to measure directly brain extracellular potassium ion activity. Although serum potassium in various species ranges between 3.5 and 6 mEq/l, brain extracellular potassium is maintained at a level close to 3 mEq/l independent of fluctuations in serum values. Despite this buffering of the internal brain environment by extracerebral changes, local variations in extracellular potassium occur in response to evoked neuronal activity, seizures, and spreading depression. Mechanisms involved in the maintenance of this ionic homeostasis in the brain include mediated transport at the level of the cerebral capillary and the choroid plexus epithelium. In addition, there are ouabain-sensitive clearance mechanisms presumably involving Na,K-ATPase that participate in the removal of excess potassium. The relative roles of simple diffusion, high glial cell conductance of potassium, and active ionic pumps in restoring basal potassium levels after activity are still controversial.
Levels of extracellular potassium activity (aK) during repetitive electrical stimulation were measured with ion sensitive microelectrodes in the somatosensory cortex of the cat to determine maximum values ("ceiling" levels) under different experimental conditions. The maximal values of aK were 10.2 mequiv./1 during stimulation of the cortical surface (CS) or of the nucleus ventroposterolateralis thalami (VPL) and during selfsustained afterdischarges (SAD). Similarly, peak values were 6.5 mequiv./1 for the nucleus ventrolateralis anterior and 4 mequiv./1 for the nucleus centromedianus as well as for the nucleus cuneatus. The rise in aK during a test stimulus with constant intensity and frequency was inversely related to the level of aK produced by a preceding stimulation. Also rise in aK during SAD was smaller when it started from an enhanced level of aK. During repetitive stimulation of CS or VPL a rise in aK was not observed when aK was increased to levels above 10 mequiv./1 by superfusion with potassium enriched solutions. An electrophoretically evoked K+ test signal was reduced between 10 and 48% when applied during stimulus induced increased levels of aK. Stimulus induced potassium changes could become negative when aK was increased to levels above 7 mequiv./1 by local electrophoresis, while the stimulus induced increase in neuronal discharge rate did not disappear or reverse. Amplitudes of ECoG and local evoked potentials were reduced as aK increased during stimulation or superfusion. It is suggested that the ceiling in its steady state is maintained by an active K+ uptake mechanism which balances extra releases of K+. Decreased release of K+ at increased levels of aK may in addition limit the rise in aK.
The kinetic characterization of the Na/K/Cl cotransport of cultured astrocytes and evidence for its involvement in volume regulation and K+ net uptake during K+ clearance are reviewed. Emphasis is put on experimental evidence for a proposed sodium cycle in astrocytes; this cycle involves a Na(+)-K+ ATPase that is stimulated by both a high external K+ and intracellular Na+. Elevated external K+ also stimulates the Na/K/Cl carrier, transporting these ions inward. As a result Na+ is cycled across the membrane, carried inward by the Na/K/Cl carrier, and returned by the Na(+)-K+ ATPase. Both functionally coupled mechanisms lead to intracellular KCl accumulation and inward movements of water to compensate for increased osmolarity. The combined cycle is expected to play a major role in the regulation of physiological K+ levels in the brain.
During onset and offset of illumination, considerable changes in extracellular K+ concentration ([K+]c) occur within particular retinal layers. There are two ways in which glial cells may control [K+]c: (1) by space-independent processes, for example, by K+ uptake due to the Na(+)-K+ ATPase, and (2) by space-dependent processes, that is, by spatial buffering currents flowing through K+ channels. Rabbit retinal Müller (glial) cells were studied for expression of mechanisms supporting both kinds of processes. This review demonstrates that rabbit Müller cells have Na-K pumps whose distribution and properties are highly adapted to meet the needs of efficient K+ clearance. Furthermore, spatial buffering currents through specialized K+ channels of Müller cells greatly accelerate retinal K+ clearance during and after stimulation.
1. The M-wave is a light-adapted response of proximal retina consisting of phasic negative field potentials at light onset and offset that are spatially tuned for small stimuli. We measured light-dependent changes in extracellular K+ concentration ([K+]o) in proximal retina to investigate the hypothesis that the M-wave originates from Müller cell responses to changes in [K+]o. 2. Extracellular field potentials, and changes in [K+]o evoked in response to circular spots of light flashed on steady backgrounds, were recorded with double-barreled K(+)-sensitive electrodes placed in the retina at different depths. 3. Increases in [K+]o during illumination and at light offset were maximal in proximal retina, with the On [K+]o increase located more proximally than the Off increase. The [K+]o increase during illumination consisted of a phasic and sustained response, whereas the Off [K+]o increase was predominantly phasic. The spatial tuning of the [K+]o increases was similar to the tuning of the field potentials. 4. The Off-field potential was larger than the On potential; it tended to be maximal more distally and was more sharply localized in retinal depth. Stimulus-response characteristics of the field potentials were not altered by intravitreal tetrodotoxin (TTX; 3.8 microM) sufficient to block retinal ganglion cell action potentials. 5. There were no rod contributions to the proximal [K+]o increases and field potentials recorded at the background illuminations used in this study (9.5-11.5 log q.deg-2.s-1). 6. An intravitreal injection of L- or DL-2-amino-4-phosphonobutyric acid (APB; 1 mM) was used to block On-system neuronal responses in proximal retina and isolate Off-system responses. After APB the [K+]o response consisted of a sustained decrease in [K+]o during illumination followed by an overshoot at light offset, while the field potential was a sustained positive response at light onset followed by an initially phasic negative response at light onset followed by an initially phasic negative response at light offset. These responses retained spatial tuning. To isolate the On-system components, the APB-isolated responses were subtracted from the controls. The [K+]o response now consisted of a sustained increase during illumination followed by an undershoot at light offset. The field potential was a sustained negative potential with an initial phasic peak that decayed at Off. Results with kynurenate (KYN; 5 mM) and (+/-)cis-2,3-piperidine (PDA; 5 mM) confirmed the sustained nature of the On component.(ABSTRACT TRUNCATED AT 400 WORDS)
We examined the role of Müller (glial) cells in buffering light-evoked changes in extracellular K+ concentration, [K+]o, in the isolated retina of the toad, Bufo marinus. We found evidence for two opposing Müller cell current loops that are generated by a light-evoked increase in [K+]o in the inner plexiform layer. These current loops, which are involved in the generation of the M-wave of the electroretinogram (ERG), prevent the accumulation of K+ in the inner plexiform layer by transporting K+ both to vitreous and to distal retina. In addition, under dark-adapted conditions, we found evidence for a Müller cell current loop that is generated by a light-evoked decrease in [K+]o in the receptor layer. This current loop, which is involved in the generation of the slow PIII component of the ERG, helps to buffer the light-evoked decrease in [K+]o throughout distal retina by transporting K+ from vitreous. The spatial buffering fluxes of K+ can be abolished by blocking Müller cell K+ conductance with 200 microM Ba2+. The separate contributions of the M-wave and slow PIII currents to Müller cell spatial buffering were isolated by various pharmacological treatments that were designed to enhance or suppress light-evoked activity in specific retinal neurons. Our results show that Müller cell K+ currents not only buffer light-evoked increases in [K+]o, but also buffer light-evoked decreases in [K+]o, and thereby diminish any deleterious effects upon neuronal function that could arise in response to large changes in [K+]o in the plexiform layers. Moreover, our results emphasize that spatial buffering currents generate many components of the electroretinogram.
We describe the electrophysiological properties of acutely isolated type-1 astrocytes using a new "tissue print" dissociation procedure. Because the enzymes used did not destroy or modify the ion channels, and the cells retained many processes, the properties may reflect those in vivo. The types of ion channels in type-1 astrocytes changed rapidly during the first 10 postnatal days, when they attained their adult phenotype. This change was dependent on the presence of neurons. In culture, most of these channel types were not expressed, but a phenotype more typical of that in vivo could be induced by co-culture with neurons. The electrophysiological properties of astrocytes make some existing hypotheses of astrocyte function less likely.
In 20 cats anaesthetized with alpha-chloralose and spinalized at the thoracolumbar junction we investigated the role of stimulation induced accumulation of extracellular potassium in the spinal cord in the processing of nociceptive discharges from the knee joint. For that we electrically stimulated the posterior articular nerve of the knee. We further performed innocuous and noxious stimulation of the knee and of other parts of the leg and studied the effect of an acute inflammation of the knee on [K+]0 in the spinal cord. Innocuous stimulation of the skin (brushing or touching) and innocuous movements in the knee joint all induced rises in [K+]0 which were maximal at recording depths of 1500 to 2200 microns below the surface of the cord dorsum. Peak increases were 0.4 mM for touching the leg and 1.7 mM during rhythmic flexion/extension of the knee joint. Noxious stimulation of the skin, the paw, the tendon and noxious movements of the knee joint also produced rises in [K+]0, which were somewhat larger for the individual types of stimuli than those produced by innocuous intensities. Electrical stimulation of the posterior articular nerve induced rises in [K+]0 by up to 0.6 mM. Stimulus intensities sufficient to activate unmyelinated group IV fibers were only slightly effective in raising [K+]0 above the levels reached during stimulation of myelinated group II and III fibers. During development of an acute inflammation of the knee joint (induced by kaolin and carrageenan), increases in [K+]0 and associated field potentials became larger by about 25%. We assume that this reflects an increase in neuronal responses. In conclusion, changes in [K+]0 in the spinal cord are somewhat larger during noxious stimulation than during innocuous stimulation. The absolute level reached depended more on the site and type of stimulation than on the actual stimulus intensity itself. Hence a critical role of spinal K+ accumulation for nociception is unlikely.
A major function of glial cells in the central nervous system is to buffer the extracellular potassium concentration, [K+]o. A local rise in [K+]o causes potassium ions to enter glial cells, which have membranes that are highly permeable to K+; potassium then leaves the glial cells at other locations where [K+]o has not risen. We report here the first study of the individual ion channels mediating potassium buffering by glial cells. The patch-clamp technique was employed to record single channel currents in Müller cells, the radial glia of the vertebrate retina. Those cells have 94% of their potassium conductance in an endfoot apposed to the vitreous humour, causing K+ released from active retinal neurones to be buffered preferentially to the vitreous. Recordings from patches of endfoot and cell body membrane show that a single type of inward-rectifying K+ channel mediates potassium buffering at both cell locations. The non-uniform density of K+ conductance is due to a non-uniform distribution of one type of K+ channel, rather than to the cell expressing high conductance channels at the endfoot and low conductance channels elsewhere on the cell.
The distribution of K+ conductance across the surface of retinal Müller cells was determined in 5 mammalian species--rabbit, guinea pig, mouse, owl monkey, and cat--and in tiger salamander. Potassium conductance was measured by monitoring cell depolarizations evoked by focal ejections of a high-K+ solution onto the surface of freshly dissociated cells. This technique measured the total K+ conductance of a given cell region (regional conductance), i.e., the specific K+ conductance times the total surface area in that region. In mammalian species with avascular retinas (rabbit, guinea pig), the regional K+ conductance within the middle portion of the cell was only a fraction (10.6-28.9%) of the endfoot conductance, while the conductance of the distal (photo-receptor) end of the cell was approximately half (41.2-49.8%) the endfoot conductance. In 2 species with vascularized retinas (mouse and owl monkey), by contrast, the regional K+ conductance within the middle portion of the cell was as large as 125.5-129.8% of the endfoot conductance. In these cells the K+ conductance of the distal end was 68.3-82.9% of the endfoot value. In cat, a third vascularized species, the K+ conductance was highest (187.1% of the endfoot value) at the distal end of the cell. In tiger salamander, which has an avascular retina, the regional K+ conductance of all regions distal to the endfoot was only 2.4-15.7% of the endfoot value. Differences in the distributions of regional K+ conductance observed in the 6 species raise the possibility that in vascularized mammalian retinas, the high-K+ conductance of the middle portion of Müller cells is associated with retinal blood vessels. The results are consistent with the hypothesis that, in avascular species, Müller cells aid in regulating extracellular K+ levels by transferring (siphoning) excess K+ principally into the vitreous humor, while in at least some vascularized species (mouse, monkey), excess K+ is transferred by Müller cells into retinal capillaries, as well as into the vitreous.
1. Double-barrelled ion-sensitive micro-electrodes were used to measure changes in the intracellular activities of K+, Na+ and Cl- (aiK, aiNa, aiCl) in glial cells of slices from guinea-pig olfactory cortex during repetitive stimulation of the lateral olfactory tract. 2. Base-line levels of aiK, aiNa and aiCl were about 66, 25 and 6 mM, respectively, for cells with resting potentials higher than -80 mV. During stimulation, intraglial aiK and aiCl increased, whereas aiNa decreased. Within about 2 min after stimulation the ion activities returned to their base-line levels. 3. The Cl- equilibrium potential was found to be close to the membrane potential (Em). There was also a strong correlation between changes of Em and aiCl. These observations indicate a high Cl- conductance of the glial cell membrane. 4. In the presence of Ba2+, the usual depolarizing response of the glial cells to a rise of the extracellular K+ activity (aeK) reversed into a membrane hyperpolarization. Furthermore, Ba2+ strongly reduced the stimulus-related rise of intraglial aiK. An additional application of ouabain blocked both the membrane hyperpolarization as well as the remaining rise of aiK. 5. In conclusion, our data show that glial cells in guinea-pig olfactory cortex slices possess at least two mechanisms of K+ accumulation. One mechanism is sensitive to the K+ channel blocker Ba2+ and might be a passive KCl influx. The other appears to be the electrogenic Na+/K+ pump, which can be activated by excess extracellular K+.
The membrane properties of Müller cells, the principal glial cells of the vertebrate retina, have been characterized in a series of physiological experiments on freshly dissociated cells. In species lacking a retinal circulation (tiger salamander, rabbit, guinea pig), the end-foot of the Müller cell has a much higher K+ conductance than do other cell regions. In species with retinal circulation (mouse, cat, owl monkey) the K+ conductance of the end-foot is greater than the conductance of the proximal process of the cell. In these species, however, the K+ conductance of the soma and distal process is equal to, or greater than, the end-foot conductance. Müller cells also possess four voltage-dependent ion channels, including an inward rectifying K+ channel. These membrane specializations may aid in the regulation of extracellular K+ levels by Müller cells in the retina. High end-foot conductance shunts excess K+ out through the end-foot, where it diffuses into the vitreous humor. In vascularized retinae, excess K+ may also be transferred to the ablumenal wall of capillaries, where it could be transported into the blood.
White matter is a compact structure consisting primarily of neuronal axons and glial cells. As in other parts of the nervous system, the function of glial cells in white matter is poorly understood. We have explored the electrophysiological properties of two types of glial cells found predominantly in white matter: type 2 astrocytes and oligodendrocytes. Whole-cells and single-channel patch-clamp techniques were used to study these cell types in postnatal rat optic nerve cultures prepared according to the procedures of Raff et al. (Nature, 303:390-396, 1983b). Type 2 astrocytes in culture exhibit a "neuronal" channel phenotype, expressing at least six distinct ion channel types. With whole-cell recording we observed three inward currents: a voltage-sensitive sodium current qualitatively similar to that found in neurons and both transient and sustained calcium currents. In addition, type 2 astrocytes had two components of outward current: a delayed potassium current which activated at 0 mV and an inactivating calcium-dependent potassium current which activated at -30 mV. Type 2 astrocytes in culture could be induced to fire single regenerative potentials in response to injections of depolarizing current. Single-channel recording demonstrated the presence of an outwardly rectifying chloride channel in both type 2 astrocytes and oligodendrocytes, but this channel could only be observed in excised patches. Oligodendrocytes expressed only one other current: an inwardly rectifying potassium current that is mediated by 30- and 120-pS channels. Because these channels preferentially conduct potassium from outside to inside the cell, and because they are open at the resting potential of the cell, they would be appropriate for removing potassium from the extracellular space; thus it is proposed that oligodendrocytes, besides myelinating axons, play an important role in potassium regulation in white matter. The conductances present in oligodendrocytes suggest a "modulated Boyle and Conway mechanism" of potassium accumulation.
The aim of this investigation is to estimate the contribution of spatial glial K+ buffer currents to extracellular K+ homeostasis during enhanced neuronal activity. Neuronal hyperactivity was induced by electrical stimulation of the cortical surface or the ventrobasal thalamic nuclei of cats (5-50 Hz, 0.1-0.2 ms, two to three times threshold stimulation intensity, 5-20 s). The accompanying slow field potential changes were recorded simultaneously across the grey matter with vertical assemblies of eight micropipettes glued 300 microns apart. Using the Poisson equation, the amplitudes of the underlying current sources and sinks were calculated. The current source densities depended on the depth of recording, frequency, strength, and duration of the stimulation. Current sinks, corresponding to a removal of 0.1-0.5 mmoles of monovalent cations per liter of brain tissue and second from the extracellular space, were observed in middle cortical layers, whereas sources appeared at superficial and deeper sites. These sinks and sources might represent K+ moved across glial membranes by spatial buffer currents. The consequences of glial buffer currents of this magnitude were investigated with model calculations. It turned out that measurements of electrolyte and volume changes of the extracellular space (Dietzel et al. Exp. Brain Res. 40:432-439, 1980; Exp. Brain Res. 46:73-84, 1982) could only partially be explained by spatial buffer currents of this magnitude. Comparison of the calculated values with intracellular measurements in neurons and glial cells (Coles et al. Ann. N.Y. Acad. Sci. 481:303-317, 1986; Ballanyi et al. J. Physiol. 382:159-174, 1987) suggests that spatial buffering combines with an approximately equimolar KCl transport and, depending on the preparation, also K+/Na+-exchange across glial membranes.
Activity-dependent variations in extracellular potassium concentration in the central nervous system may be regulated, in
part, by potassium spatial buffering currents in glial cells. The role of spatial buffering in the retina was assessed by
measuring light-evoked potassium changes in amphibian eyecups. The amplitude of potassium increases in the vitreous humor
was reduced to approximately 10 percent by 50 micromolar barium, while potassium increases in the inner plexiform layer were
largely unchanged. The decrease in the vitreal potassium response was accurately simulated with a numerical model of potassium
current flow through Muller cells, the principal glial cells of the retina. Barium also substantially increased the input
resistance of Muller cells and blocked the Muller cell-generated M-wave, indicating that barium blocks the potassium channels
of Muller cells. Thus, after a light-evoked potassium increase within the retina, there is a substantial transfer of potassium
from the retina to the vitreous humor by potassium current flow through Muller cells.
Analysis of neural activity-dependent fluctuations in K+, H+, and ECS dimensions in the developing RON has revealed major changes during the first two to three postnatal weeks. The emergence of the adult ceiling level for evoked extracellular K+ (10 to 12 mM) and significant ECS shrinkage are roughly correlated in time with the proliferation and maturation of glial cells in this structure. This observation and others have led to the hypothesis that ECS shrinkage depends upon electrolyte and water transport into glial cells with subsequent swelling. Development of the adult K+ ceiling level may also depend upon glial cells, but it is likely that other factors contribute to this homeostatic mechanism. Marked alterations in activity-dependent pHo shifts were seen with development and may be related to changes in the activity of carbonic anhydrase in this structure. The technological means are at hand to pursue these questions vigorously in an effort to provide further insight into the mechanisms of ionic and fluid homeostasis of brain ECS, and the developing RON appears to be a useful model system in this regard.
The distribution of potassium conductance over the surface of freshly dissociated salamander astrocytes was determined by
monitoring cell depolarizations evoked by focal increases in the extracellular potassium concentration. The specific potassium
conductance of the endfoot processes of these cells was approximately tenfold higher than the conductance of other cell regions.
This dramatically nonuniform conductance distribution may play an important role in the regulation of extracellular potassium
levels by glia in the brain.
K+ and Cl- transport using 42K+ and 36Cl- was studied in primary astrocyte cultures prepared from neonatal rat brains. A component of 42K+ uptake was sensitive to both furosemide and bumetanide with maximum inhibition being obtained at 1 and 0.01 mM concentrations of the inhibitors, respectively. Furosemide and bumetanide also markedly inhibited uptake of 36Cl-. 42K+ uptake in the presence of ouabain was also sensitive to the omission of medium Na+ and Cl-. These results suggest the existence of a K+ + Na+ + Cl- cotransport system in astrocyte cultures which in many cells has been shown to be involved in volume regulation. We studied volume changes using uptake of [14C]3-O- methyl-D-glucose ([14C]3-OMG), and also ion transport, in attached cells in response to exposure to hyper- or hypotonic medium. Exposure to medium made hypertonic with mannitol resulted in shrinkage of the [14C]3-OMG space of the cells, but did not affect 36Cl- content, expressed as nmol/mg protein. Exposure to hypotonic medium led to a marked increase in the [14C]3-OMG space, rapidly followed by a decrease towards control values. After the cells were then exposed to isotonic medium there was an immediate decrease followed by a slower increase in the [14C]3-OMG space. The increase in the [14C]3-OMG space was partially inhibited by 1 mM furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)
Mammalian glial cells were identified and studied in the optic nerves of anaesthetized rats.
Cells with membrane potentials of 77–85 mV were located in the optic nerve with capillary micropipettes. These were shown to be neuroglia by iontophoretic injection of a fluorescent dye through the recording electrode, followed by histological verification of the location of the dye. No distinction was made between astroglia and oligodendroglia.
Neuroglial cells gave no impulse activity. Their membrane potential was studied in isolated optic nerves by varying the ionic composition of the bathing fluid. The glial membrane potential depends predominantly on a transmembrane gradient of potassium ions.
The time course of local changes of the extracellular space (ES) was investigated by measuring concentration changes of repeatedly injected tetramethylammonium (TMA+) and choline (Ch+) ions for which cell membranes are largely impermeable. After stimulus-induced extracellular [K+] elevations the δ[TMA+] and δ[Ch+] signals recorded with nominally K+-selective liquid ion-exchanger microelectrodes increased by up to 100%, thus indicating a reduction of the ES down to one half of its initial size. The shrinkage was maximal at sites where the K+ release into the ES was also largest. At very superficial and deep layers, however, considerable increases in extracellular K+ concentration were not accompanied by significant reductions in the ES. These findings can be explained as a consequence of K+ movement through spatially extended cell structures. Calculations based on a model combining the spatial buffer mechanism of Kuffler and Nicholls (1966) to osmolarity changes caused by selective K+ transport through primarily K+ permeable membranes support this concept.
Following stimulation additional iontophoretically induced [K+]o rises were reduced in amplitude by up to 35%, even at sites where maximal decreases of the ES were observed. This emphasizes the importance of active uptake for K+ clearance out of the ES.
Work with ion-selective microelectrodes on the retina of the honeybee drone has shown that potassium is released from photoreceptors during activity and enters glial cells. Measurements of the extracellular voltage gradients indicate that, in this preparation, currents flowing through the glial cells in the 'spatial buffer' pattern account for a large fraction of the glial K+ entry in the active region of the tissue.