Astrocyte inositol triphosphate receptor type 2 and cytosolic phospholipase A2 alpha regulate arteriole responses in mouse neocortical brain slices.
ABSTRACT: Functional hyperemia of the cerebral vascular system matches regional blood flow to the metabolic demands of the brain. One current model of neurovascular control holds that glutamate released by neurons activates group I metabotropic glutamate receptors (mGluRs) on astrocytes, resulting in the production of diffusible messengers that act to regulate smooth muscle cells surrounding cerebral arterioles. The acute mouse brain slice is an experimental system in which changes in arteriole diameter can precisely measured with light microscopy. Stimulation of the brain slice triggers specific cellular responses that can be correlated to changes in arteriole diameter. Here we used inositol trisphosphate receptor type 2 (IP(3)R2) and cytosolic phospholipase A(2) alpha (cPLA(2)?) deficient mice to determine if astrocyte mGluR activation coupled to IP(3)R2-mediated Ca(2+) release and subsequent cPLA(2)? activation is required for arteriole regulation. We measured changes in astrocyte cytosolic free Ca(2+) and arteriole diameters in response to mGluR agonist or electrical field stimulation in acute neocortical mouse brain slices maintained in 95% or 20% O(2). Astrocyte Ca(2+) and arteriole responses to mGluR activation were absent in IP(3)R2(-/-) slices. Astrocyte Ca(2+) responses to mGluR activation were unchanged by deletion of cPLA(2)? but arteriole responses to either mGluR agonist or electrical stimulation were ablated. The valence of changes in arteriole diameter (dilation/constriction) was dependent upon both stimulus and O(2) concentration. Neuron-derived NO and activation of the group I mGluRs are required for responses to electrical stimulation. These findings indicate that an mGluR/IP(3)R2/cPLA(2)? signaling cascade in astrocytes is required to transduce neuronal glutamate release into arteriole responses.
Project description:Long-term potentiation (LTP) of synaptic transmission represents the cellular basis of learning and memory. Astrocytes have been shown to regulate synaptic transmission and plasticity. However, their involvement in specific physiological processes that induce LTP in vivo remains unknown. Here we show that in vivo cholinergic activity evoked by sensory stimulation or electrical stimulation of the septal nucleus increases Ca²? in hippocampal astrocytes and induces LTP of CA3-CA1 synapses, which requires cholinergic muscarinic (mAChR) and metabotropic glutamate receptor (mGluR) activation. Stimulation of cholinergic pathways in hippocampal slices evokes astrocyte Ca²? elevations, postsynaptic depolarizations of CA1 pyramidal neurons, and LTP of transmitter release at single CA3-CA1 synapses. Like in vivo, these effects are mediated by mAChRs, and this cholinergic-induced LTP (c-LTP) also involves mGluR activation. Astrocyte Ca²? elevations and LTP are absent in IP?R2 knock-out mice. Downregulating astrocyte Ca²? signal by loading astrocytes with BAPTA or GDP?S also prevents LTP, which is restored by simultaneous astrocyte Ca²? uncaging and postsynaptic depolarization. Therefore, cholinergic-induced LTP requires astrocyte Ca²? elevations, which stimulate astrocyte glutamate release that activates mGluRs. The cholinergic-induced LTP results from the temporal coincidence of the postsynaptic activity and the astrocyte Ca²? signal simultaneously evoked by cholinergic activity. Therefore, the astrocyte Ca²? signal is necessary for cholinergic-induced synaptic plasticity, indicating that astrocytes are directly involved in brain storage information.
Project description:Activation of metabotropic glutamate receptors (mGluRs) causes membrane hyperpolarization in midbrain dopamine neurons. This hyperpolarization results from the opening of Ca(2+)-sensitive K(+) channels, which is mediated by the release of Ca(2+) from intracellular stores. Neurotransmitter-induced mobilization of Ca(2+) is generally ascribed to the action of inositol 1,4,5-triphosphate (IP(3)) in neurons. Here we show that the mGluR-mediated Ca(2+) mobilization in dopamine neurons is caused by two intracellular second messengers: IP(3) and cyclic ADP-ribose (cADPR). Focal activation of mGluRs, attained by synaptic release of glutamate or iontophoretic application of aspartate, induced a wave of Ca(2+) that spread over a distance of approximately 50 microm through dendrites and the soma. Simultaneous inhibition of both IP(3)- and cADPR-dependent pathways with heparin and 8-NH(2)-cADPR was required to block the mGluR-induced Ca(2+) release, indicating a redundancy in the signaling mechanism. Activation of ryanodine receptors was suggested to mediate the cADPR-dependent pathway, because ruthenium red, an antagonist of ryanodine receptors, inhibited the mGluR response only when the cADPR-dependent pathway was isolated by blocking the IP(3)-dependent pathway with heparin. Finally, the mGluR-mediated hyperpolarization was shown to induce a transient pause in the spontaneous firing of dopamine neurons. These results demonstrate that an excitatory neurotransmitter glutamate uses multiple intracellular pathways to exert an inhibitory control on the excitability of dopamine neurons.
Project description:It has been common experimentally to use high frequency, tetanic, stimulation to activate metabotropic glutamate receptors (mGluRs) in cortex and thalamus. To determine what type of stimulation is actually necessary to activate mGluRs we examined the effects of varying stimulation duration and intensity on activating mGluR responses. We used a thalamocortical and an intracortical slice preparation from mice and performed whole cell recordings from neurons in the ventral posterior medial nucleus or in layer 4 of primary somatosensory cortex (S1) while electrically stimulating in layer 6 of S1. Extracellular ionotropic glutamate receptor antagonists and GABAA receptor antagonists were used to isolate Group I or Group II mGluR responses. We observed that high frequency stimulation is not necessary for the activation of either Group I or Group II mGluRs. Either could be activated with as few as 2-3 pulses at stimulation frequencies around 15-20Hz. Additionally, increasing the number of pulses, intensity of stimulation, or stimulation frequency increased amplitude and duration of the mGluR response.
Project description:Activation of Group I metabotropic glutamate receptors (mGluRs) activates signaling cascades, resulting in calcium release from intracellular stores, ERK1/2 activation, and long term changes in synaptic activity that are implicated in learning, memory, and neurodegenerative diseases. As such, elucidating the molecular mechanisms underlying Group I mGluR signaling is important for understanding physiological responses initiated by the activation of these receptors. In the current study, we identify the multifunctional scaffolding protein spinophilin as a novel Group I mGluR-interacting protein. We demonstrate that spinophilin interacts with the C-terminal tail and second intracellular loop of Group I mGluRs. Furthermore, we show that interaction of spinophilin with Group I mGluRs attenuates receptor endocytosis and phosphorylation of ERK1/2, an effect that is dependent upon the interaction of spinophilin with the C-terminal PDZ binding motif encoded by Group I mGluRs. Spinophilin knock-out results in enhanced mGluR5 endocytosis as well as increased ERK1/2, AKT, and Ca(2+) signaling in primary cortical neurons. In addition, the loss of spinophilin expression results in impaired mGluR5-stimulated LTD. Our results indicate that spinophilin plays an important role in regulating the activity of Group I mGluRs as well as their influence on synaptic activity.
Project description: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.
Project description:Inositol 1,4,5-trisphosphate (IP(3)) is a second messenger that regulates intracellular Ca(2+) release through IP(3) receptors located in the sarco(endo)plasmic reticulum of cardiac myocytes. Many prohypertrophic G protein-coupled receptor (GPCR) signaling events lead to IP(3) liberation, although its importance in transducing the hypertrophic response has not been established in vivo.Here, we generated conditional, heart-specific transgenic mice with both gain- and loss-of-function for IP(3) receptor signaling to examine its hypertrophic growth effects following pathological and physiological stimulation.Overexpression of the mouse type-2 IP(3) receptor (IP(3)R2) in the heart generated mild baseline cardiac hypertrophy at 3 months of age. Isolated myocytes from overexpressing lines showed increased Ca(2+) transients and arrhythmias in response to endothelin-1 stimulation. Although low levels of IP(3)R2 overexpression failed to augment/synergize cardiac hypertrophy following 2 weeks of pressure-overload stimulation, such levels did enhance hypertrophy following 2 weeks of isoproterenol infusion, in response to Galphaq overexpression, and/or in response to exercise stimulation. To inhibit IP(3) signaling in vivo, we generated transgenic mice expressing an IP(3) chelating protein (IP(3)-sponge). IP(3)-sponge transgenic mice abrogated cardiac hypertrophy in response to isoproterenol and angiotensin II infusion but not pressure-overload stimulation. Mechanistically, IP(3)R2-enhanced cardiac hypertrophy following isoproterenol infusion was significantly reduced in the calcineurin-Abeta-null background.These results indicate that IP(3)-mediated Ca(2+) release plays a central role in regulating cardiac hypertrophy downstream of GPCR signaling, in part, through a calcineurin-dependent mechanism.
Project description:Synaptically activated calcium release from internal stores in CA1 pyramidal neurons is generated via metabotropic glutamate receptors by mobilizing IP(3). Ca(2+) release spreads as a large amplitude wave in a restricted region of the apical dendrites of these cells. These Ca(2+) waves have been shown to induce certain forms of synaptic potentiation and have been hypothesized to affect other forms of plasticity. Pairing a single backpropagating action potential (bAP) with repetitive synaptic stimulation evokes Ca(2+) release when synaptic stimulation alone is subthreshold for generating release. We examined the timing window for this synergistic effect under conditions favoring Ca(2+) release. The window, measured from the end of the train, lasted 250-500 ms depending on the duration of stimulation tetanus. The window appears to correspond to the time when both IP(3) concentration and [Ca(2+)](i) are elevated at the site of the IP(3) receptor. Detailed analysis of the mechanisms determining the duration of the window, including experiments using different forms of caged IP(3) instead of synaptic stimulation, suggest that the most significant processes are the time for IP(3) to diffuse away from the site of generation and the time course of IP(3) production initiated by activation of mGluRs. IP(3) breakdown, desensitization of the IP(3) receptor, and the kinetics of IP(3) unbinding from the receptor may affect the duration of the window but are less significant. The timing window is short but does not appear to be short enough to suggest that this form of coincidence detection contributes to conventional spike timing-dependent synaptic plasticity in these cells.
Project description:Activation of metabotropic glutamate receptors (mGluRs) often produces long-lasting effects on the excitability of cortical neurons. For example, mGluR stimulation induces long-term potentiation or depression of excitatory synaptic transmission in the hippocampus. Similarly, the effects of mGluRs on cortical epileptiform activities also are enduring. A transient application of group I mGluR agonists to hippocampal slices produces ictal-like discharges that persist for hours after the removal of the applied agonist. This action of group I mGluRs-transforming "normal" hippocampal slice into an "epileptic-like" one-may represent a form of epileptogenesis. The advent of such a model, in which epileptogenesis can be reliably induced in an in vitro preparation and the process is complete within hours, may facilitate the exploration of cellular mechanisms underlying epileptogenesis.
Project description:Functional hyperemia is the regional increase in cerebral blood flow upon increases in neuronal activity which ensures that the metabolic demands of the neurons are met. Hypertension is known to impair the hyperemic response; however, the neurovascular coupling mechanisms by which this cerebrovascular dysfunction occurs have yet to be fully elucidated. To determine whether altered cortical parenchymal arteriole function or astrocyte signaling contribute to blunted neurovascular coupling in hypertension, we measured parenchymal arteriole reactivity and vascular smooth muscle cell Ca(2+) dynamics in cortical brain slices from normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. We found that vasoconstriction in response to the thromboxane A2 receptor agonist U46619 and basal vascular smooth muscle cell Ca(2+) oscillation frequency were significantly increased in parenchymal arterioles from SHR. In perfused and pressurized parenchymal arterioles, myogenic tone was significantly increased in SHR. Although K(+)-induced parenchymal arteriole dilations were similar in WKY and SHR, metabotropic glutamate receptor activation-induced parenchymal arteriole dilations were enhanced in SHR. Further, neuronal stimulation-evoked parenchymal arteriole dilations were similar in SHR and WKY. Our data indicate that neurovascular coupling is not impaired in SHR, at least at the level of the parenchymal arterioles.
Project description:Ca(2+)-dependent pathways in neurons and astrocyte endfeet initiate changes in arteriole diameter to regulate local brain blood flow. Whether there exists a threshold of synaptic activity in which arteriole diameter is controlled independent of astrocyte endfeet Ca(2+) remains unclear. We used two-photon fluorescence microscopy to examine synaptically evoked synthetic or genetic Ca(2+) indicator signals around penetrating arterioles in acute slices of the rat neocortex. We discovered a threshold below which vasodilation occurred in the absence of endfeet Ca(2+) signals but with consistent neuronal Ca(2+) transients, suggesting endfoot Ca(2+) is not necessary for activity-dependent vasodilation under subtle degrees of brain activation.