inducing-plasticity-astrocytic-receptors-manipulation-neuronal-firing

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

University of California Riverside

The postnatal development of excitatory amino acid receptor types including kainate, N-methyl-D-aspartate (NMDA), and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) was assessed in the hippocampus, entorhinal cortex, and adjacent neocortex in normal and prenatally protein malnourished rats ages 15, 30, 90, and 220 postnatal days by quantitative autoradiography. Tritiated ligands used to measure binding site density were (3)[H]kainate, (3)[H]MK-801, and (3)[H]AMPA, respectively. Kainate receptors showed statistically significant increases in binding density in stratum lucidum of CA3 (hippocampal mossy fiber zone) in 90- and 220-day-old malnourished rats compared with age- and sex-matched controls but not in 15- or 30-day-old malnourished rats. Compared with previous anatomic studies, these results are mostly in agreement with a significantly decreased hippocampal mossy fiber plexus in 15-, 90-, and 220-day-old rats but not in 30-day-old rats. These results suggested that the increased density of postsynaptic kainate receptors located mainly on proximal apical dendrites of CA3 pyramidal cells may be compensatory to decreased glutamate release due to the reduction in mossy fiber plexus. In contrast, the density of putative NMDA and AMPA receptors quantified in prenatally malnourished rats was comparable to the density quantified in age- and sex-matched control rats, as were all three receptor types in entorhinal cortex and adjacent neocortex. Thus, the selectivity of the compensation of (3)[H]kainate-labeled mossy fiber plexus in adult but not in early postnatal developing malnourished rats may help ensure continued breeding and survival of the species under otherwise adverse environmental conditions.

Increased density of hippocampal kainate receptors but normal density of NMDA and AMPA receptors in a rat model of prenatal protein malnutrition.

Spontaneous neurotransmitter release and activation of group I metabotropic glutamate receptors (mGluRs) each play a role in the plasticity of neuronal synapses. Astrocytes may contribute to short- and long-term synaptic changes by signaling to neurons via these processes. Spontaneous whole-cell AMPA receptor (AMPAR) currents were recorded in CA1 pyramidal cells in situ while evoking Ca2+ increases in the adjacent stratum radiatum astrocytes by uncaging IP3. Whole-cell patch clamp was used to deliver caged IP3 and the Ca2+ indicator dye Oregon green BAPTA-1 to astrocytes. Neurons were patch-clamped and filled with Alexa 568 hydrazide dye to visualize their morphological relationship to the astrocyte. On uncaging of IP3, astrocyte Ca2+ responses reliably propagated as a wave into the very fine distal processes, synchronizing Ca2+ activity within astrocyte microdomains. The intracellular astrocyte Ca2+ wave coincided with a significant increase in the frequency of AMPA spontaneous EPSCs, but with no change in their kinetics. AMPAR current amplitudes were increased as well, but not significantly (p = 0.06). The increased frequency of AMPAR currents was sensitive to the group I mGluR antagonists LY367385 and 2-methyl-6-(phenylethynyl)-pyridine, suggesting that (1) astrocytes released glutamate in response to IP3 uncaging, and (2) glutamate released by astrocytes activated group I mGluRs to facilitate the release of glutamate from excitatory neuronal presynaptic boutons. These results extend previous studies, which have shown astrocyte modulation of neuronal activity in vitro and suggest that astrocyte-to-neuron signaling in intact tissue may contribute to synaptic plasticity.

Intracellular astrocyte calcium waves in situ increase the frequency of spontaneous AMPA receptor currents in CA1 pyramidal neurons.

The possibility that astrocytes are involved in brain signaling began to emerge in the late 1970s, when it was first shown that astroglia in vitro possess numerous receptors for neurotransmitters. It was later demonstrated that cultured astroglia and astrocytes in situ respond to neurotransmitters with increases in intracellular second messengers, including cyclic AMP and calcium. Astrocyte calcium responses have since been extensively studied both in culture and in intact tissue. We continue to gather information regarding the various compounds able to trigger astrocyte calcium increases, as well as the mechanisms involved in their initiation, propagation as a calcium wave within and between astrocytes, and effects on signaling within the brain. This review will focus on each of these aspects of astrocyte calcium regulation, and attempt to sort out which effects are more likely to occur in developmental, pathological, and physiological conditions. While we have come far in our understanding of the properties or potential of astrocytes' ability to signal to neurons using our array of pharmacological tools, we still understand very little regarding the level of involvement of astrocyte signaling in normal brain physiology.

Astrocyte calcium elevations: properties, propagation, and effects on brain signaling.

Astrocytes are considered the third component of the synapse, responding to neurotransmitter release from synaptic terminals and releasing gliotransmitters--including glutamate--in a Ca(2+)-dependent manner to affect neuronal synaptic activity. Many studies reporting astrocyte-driven neuronal activity have evoked astrocyte Ca(2+) increases by application of endogenous ligands that directly activate neuronal receptors, making astrocyte contribution to neuronal effect(s) difficult to determine. We have made transgenic mice that express a Gq-coupled receptor only in astrocytes to evoke astrocyte Ca(2+) increases using an agonist that does not bind endogenous receptors in brain. By recording from CA1 pyramidal cells in acute hippocampal slices from these mice, we demonstrate that widespread Ca(2+) elevations in 80%-90% of stratum radiatum astrocytes do not increase neuronal Ca(2+), produce neuronal slow inward currents, or affect excitatory synaptic activity. Our findings call into question the developing consensus that Ca(2+)-dependent glutamate release by astrocytes directly affects neuronal synaptic activity in situ.

Selective stimulation of astrocyte calcium in situ does not affect neuronal excitatory synaptic activity.

Astrocytes in the hippocampus release calcium (Ca(2+)) from intracellular stores intrinsically and in response to activation of G(q)-linked G-protein-coupled receptors (GPCRs) through the binding of inositol 1,4,5-trisphosphate (IP(3)) to its receptor (IP(3)R). Astrocyte Ca(2+) has been deemed necessary and sufficient to trigger the release of gliotransmitters, such as ATP and glutamate, from astrocytes to modulate neuronal activity. Several lines of evidence suggest that IP(3)R type 2 (IP(3)R2) is the primary IP(3)R expressed by astrocytes. To determine whether IP(3)R2 is the primary functional IP(3)R responsible for astrocytic Ca(2+) increases, we conducted experiments using an IP(3)R2 knock-out mouse model (IP(3)R2 KO). We show, for the first time, that lack of IP(3)R2 blocks both spontaneous and G(q)-linked GPCR-mediated increases in astrocyte Ca(2+). Furthermore, neuronal G(q)-linked GPCR Ca(2+) increases remain intact, suggesting that IP(3)R2 does not play a major functional role in neuronal calcium store release or may not be expressed in neurons. Additionally, we show that lack of IP(3)R2 in the hippocampus does not affect baseline excitatory neuronal synaptic activity as measured by spontaneous EPSC recordings from CA1 pyramidal neurons. Whole-cell recordings of the tonic NMDA receptor-mediated current indicates that ambient glutamate levels are also unaffected in the IP(3)R2 KO. These data show that IP(3)R2 is the key functional IP(3)R driving G(q)-linked GPCR-mediated Ca(2+) increases in hippocampal astrocytes and that removal of astrocyte Ca(2+) increases does not significantly affect excitatory neuronal synaptic activity or ambient glutamate levels.

Loss of IP3 receptor-dependent Ca2+ increases in hippocampal astrocytes does not affect baseline CA1 pyramidal neuron synaptic activity.

Astrocytes comprise approximately half of the volume of the adult mammalian brain and are the primary neuronal structural and trophic supportive elements. Astrocytes are organized into distinct nonoverlapping domains and extend elaborate and dense fine processes that interact intimately with synapses and cerebrovasculature. The recognition in the mid 1990s that astrocytes undergo elevations in intracellular calcium concentration following activation of G protein-coupled receptors by synaptically released neurotransmitters demonstrated not only that astrocytes display a form of excitability but also that astrocytes may be active participants in brain information processing. The roles that astrocytic calcium elevations play in neurophysiology and especially in modulation of neuronal activity have been intensely researched in recent years. This review will summarize the current understanding of the function of astrocytic calcium signaling in neurophysiological processes and discuss areas where the role of astrocytes remains controversial and will therefore benefit from further study.

What is the role of astrocyte calcium in neurophysiology?

A number of exciting findings have been made in astrocytes during the past 15 years that have led many researchers to redefine how the brain works. Astrocytes are now widely regarded as cells that propagate Ca(2+) over long distances in response to stimulation, and, similar to neurons, release transmitters (called gliotransmitters) in a Ca(2+)-dependent manner to modulate a host of important brain functions. Although these discoveries have been very exciting, it is essential to place them in the proper context of the approaches used to obtain them to determine their relevance to brain physiology. This review revisits the key observations made in astrocytes that greatly impact how they are thought to regulate brain function, including the existence of widespread propagating intercellular Ca(2+) waves, data suggesting that astrocytes signal to neurons through Ca(2+)-dependent release of glutamate, and evidence for the presence of vesicular machinery for the regulated exocytosis of gliotransmitters.

Sorting out astrocyte physiology from pharmacology.

The concept that astrocytes release neuroactive molecules (gliotransmitters) to affect synaptic transmission has been a paradigm shift in neuroscience research over the past decade. This concept suggests that astrocytes, together with pre- and postsynaptic neuronal elements, make up a functional synapse. Astrocyte release of gliotransmitters (for example, glutamate and adenosine triphosphate) is generally accepted to be a Ca2+-dependent process. We used two mouse lines to either selectively increase or obliterate astrocytic Gq G protein-coupled receptor Ca2+ signaling to further test the hypothesis that astrocytes release gliotransmitters in a Ca2+-dependent manner to affect synaptic transmission. Neither increasing nor obliterating astrocytic Ca2+ fluxes affects spontaneous and evoked excitatory synaptic transmission or synaptic plasticity. Our findings suggest that, at least in the hippocampus, the mechanisms of gliotransmission need to be reconsidered.

Hippocampal short- and long-term plasticity are not modulated by astrocyte Ca2+ signaling.

Dendritic spines are dynamic, actin-rich structures that form the postsynaptic sites of most excitatory synapses in the brain. The F-actin severing protein cofilin has been implicated in the remodeling of dendritic spines and synapses under normal and pathological conditions, by yet unknown mechanisms. Here we report that β-arrestin-2 plays an important role in NMDA-induced remodeling of dendritic spines and synapses via translocation of active cofilin to dendritic spines. NMDAR activation triggers cofilin activation through calcineurin and phosphatidylinositol 3-kinase (PI3K)-mediated dephosphorylation and promotes cofilin translocation to dendritic spines that is mediated by β-arrestin-2. Hippocampal neurons lacking β-arrestin-2 develop mature spines that fail to remodel in response to NMDA. β-Arrestin-2-deficient mice exhibit normal hippocampal long-term potentiation, but significantly impaired NMDA-dependent long-term depression and spatial learning deficits. Moreover, β-arrestin-2-deficient hippocampal neurons are resistant to Aβ-induced dendritic spine loss. Our studies demonstrate unique functions of β-arrestin-2 in NMDAR-mediated dendritic spine and synapse plasticity through spatial control over cofilin activation.

Cofilin under control of β-arrestin-2 in NMDA-dependent dendritic spine plasticity, long-term depression (LTD), and learning.

A prominent area of neuroscience research over the past 20 years has been the acute modulation of neuronal synaptic activity by Ca(2+)-dependent release of the transmitters ATP, D-serine, and glutamate (called gliotransmitters) by astrocytes. Although the physiological relevance of this mechanism is under debate, emerging evidence suggests that there are critical factors in addition to Ca(2+) that are required for gliotransmitters to be released from astrocytes. Interestingly, these factors include activated microglia and the proinflammatory cytokine Tumor Necrosis Factor α (TNFα), chemotactic cytokine Stromal cell-Derived Factor-1α (SDF-1α), and inflammatory mediator prostaglandin E2 (PGE(2)). Of note, microglial activation and release of inflammatory molecules from activated microglia and reactive astrocytes can occur within minutes of a triggering stimulus. Therefore, activation of astrocytes by inflammatory molecules combined with Ca(2+) elevations may lead to gliotransmitter release, and be an important step in the early sequence of events contributing to hyperexcitability, excitotoxicity, and neurodegeneration in the damaged or diseased brain. In this review, we will first examine evidence questioning Ca(2+)-dependent gliotransmitter release from astrocytes in healthy brain tissue, followed by a close examination of recent work suggesting that Ca(2+)-dependent gliotransmitter release occurs as an early event in the development of neurological disorders and neuroinflammatory and neurodegenerative diseases.

Calcium Signaling and Gliotransmission in Normal vs. Reactive Astrocytes.

Very little is known about the ability of astrocytic receptors to exhibit plasticity as a result of changes in neuronal activity. Here we provide evidence for bidirectional scaling of astrocytic group I metabotropic glutamate receptor signaling in acute mouse hippocampal slices following long-term changes in neuronal firing rates. Plasticity of astrocytic mGluRs was measured by recording spontaneous and evoked Ca²⁺ elevations in both astrocytic somata and processes. An exogenous astrocytic Gq G protein-coupled receptor was resistant to scaling, suggesting that the alterations in astrocyte Ca²⁺ signaling result from changes in activity of the surface mGluRs rather than a change in intracellular G protein signaling molecules. These findings suggest that astrocytes actively detect shifts in neuronal firing rates and adjust their receptor signaling accordingly. This type of long-term plasticity in astrocytes resembles neuronal homeostatic plasticity and might be important to ensure an optimal or expected level of input from neurons.

Bidirectional scaling of astrocytic metabotropic glutamate receptor signaling following long-term changes in neuronal firing rates.

It is estimated that one in 26 people will develop epilepsy in their lifetime, amounting to almost 12 million people in the United States alone (Hesdorffer et al., 2011). Epilepsy is a group of conditions characterized by sporadic occurrence of seizures and unconsciousness. This severely limits the ability to perform everyday tasks and leads to increased difficulty with learning and memory, maintenance of steady employment, driving, and overall socioeconomic integration. A greater understanding of the cellular and molecular mechanisms underlying seizures and epilepsy is necessary, as it may lead to novel antiepileptic treatments. In this chapter, we will review the current literature surrounding the involvement of glial cells in epilepsy with particular emphasis on review of human tissue studies and some possible underlying mechanisms. Based on the current evidence and hypotheses of glial mechanisms in epilepsy, novel therapeutic opportunities for the treatment of epilepsy will also be presented.

Glial cell changes in epilepsy: overview of the clinical problem and therapeutic opportunities.

One of the most important functions of astrocytes is removal of glutamate released during synaptic transmission. Surprisingly, the mechanisms by which astrocyte glutamate uptake is acutely modulated remain to be clarified. Astrocytes express metabotropic glutamate receptors (mGluRs) and other G protein-coupled receptors (GPCRs), which are activated during neuronal activity. Here, we test the hypothesis that astrocytic group I mGluRs acutely regulate glutamate uptake by astrocytes in situ. This hypothesis was tested in acute mouse hippocampal slices. Activation of astrocytic mGluRs, using a tetanic high-frequency stimulus (HFS) applied to Schaffer collaterals, led to potentiation of the amplitude of the synaptically evoked glutamate transporter currents (STCs) and associated charge transfer without changes in kinetics. Similar potentiation of STCs was not observed in the presence of group I mGluR antagonists or the PKC inhibitor, PKC 19-36, suggesting that HFS-induced potentiation of astrocyte glutamate uptake is astrocytic group I mGluR and PKC dependent. Pharmacological stimulation of a transgenic GPCR (MrgA1R), expressed exclusively in astrocytes, also potentiated STC amplitude and charge transfer, albeit quicker and shorter lasting compared with HFS-induced potentiation. The amplitude of the slow, inward astrocytic current due to potassium (K(+)) influx was also enhanced following activation of the endogenous mGluRs or the astrocyte-specific MrgA1 Gq GPCRs. Taken together, these findings suggest that astrocytic group I mGluR activation has a synergistic, modulatory effect on the uptake of glutamate and K(+).

Astrocytic group I mGluR-dependent potentiation of astrocytic glutamate and potassium uptake.

Astrocyte Gq GPCR and IP3 receptor-dependent Ca(2+) elevations occur spontaneously in situ and in vivo. These events vary considerably in size, often remaining confined to small territories of astrocyte processes called "microdomains" and sometimes propagating over longer distances that can include the soma. It has remained unclear whether these events are driven by constitutive (basal) GPCR signaling activity, neuronal action potential-dependent or quantal vesicular release, or some combination of these mechanisms. Here, we applied manipulations to increase or inhibit neuronal vesicular neurotransmitter release together with low-level stimulation of Schaffer collaterals in acute mouse hippocampal slices in an effort to determine the mechanisms underlying spontaneous astrocyte Ca(2+) events. We found no significant change in spontaneous microdomain astrocyte Ca(2+) elevations when neuronal action potentials were significantly enhanced or blocked. The astrocyte Ca(2+) activity was also not affected by inhibitors of group I mGluRs. However, blockade of miniature neurotransmitter release using Bafilomycin A1 significantly reduced the frequency of microdomain astrocyte Ca(2+) elevations. We then tested whether astrocyte Ca(2+) microdomains can be evoked by low intensity SC stimulation. Importantly, microdomains could not be reproduced even using single, low intensity pulses to the SCs at a minimum distance from the astrocyte. Evoked astrocyte Ca(2+) responses most often included the cell soma, were reduced by group I mGluR antagonists, and were larger in size compared to spontaneous Ca(2+) microdomains. Overall, our findings suggest that spontaneous microdomain astrocyte Ca(2+) elevations are not driven by neuronal action potentials but require quantal release of neurotransmitter which cannot be replicated by stimulation of Schaffer collaterals.

Astrocyte calcium microdomains are inhibited by Bafilomycin A1 and cannot be replicated by low-level Schaffer collateral stimulation in situ.

Astrocytes are a predominant glial cell type in the nervous systems, and are becoming recognized as important mediators of normal brain function as well as neurodevelopmental, neurological, and neurodegenerative brain diseases. Although numerous potential mechanisms have been proposed to explain the role of astrocytes in the normal and diseased brain, research into the physiological relevance of these mechanisms in vivo is just beginning. In this review, we will summarize recent developments in innovative and powerful molecular approaches, including knockout mouse models, transgenic mouse models, and astrocyte-targeted gene transfer/expression, which have led to advances in understanding astrocyte biology in vivo that were heretofore inaccessible to experimentation. We will examine the recently improved understanding of the roles of astrocytes - with an emphasis on astrocyte signaling - in the context of both the healthy and diseased brain, discuss areas where the role of astrocytes remains debated, and suggest new research directions.

Todd A. Fiacco

Department of Cell Biology and Neuroscience,
Center for Glial-Neuronal Interactions

University of California Riverside

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Todd A. Fiacco

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cell Biology and Neuroscience,Center for Glial-Neuronal Interactions

University of California Riverside

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Department of Cell Biology and Neuroscience,
Center for Glial-Neuronal Interactions