2-AG-Mediated Control of GABAergic Signaling Is Impaired in a Model of Epilepsy.

Repeated seizures result in a persistent maladaptation of endocannabinoid (eCB) signaling, mediated part by anandamide signaling deficiency in the basolateral amygdala (BLA) that manifests as aberrant synaptic function and altered emotional behavior. Here, we determined the effect of repeated seizures (kindling) on 2-arachidonoylglycerol (2-AG) signaling on GABA transmission by directly measuring tonic and phasic eCB-mediated retrograde signaling in an in vitro BLA slice preparation from male rats. We report that both activity-dependent and muscarinic acetylcholine receptor (mAChR)-mediated depression of GABA synaptic transmission was reduced following repeated seizure activity. These effects were recapitulated in sham rats by preincubating slices with the 2-AG synthesizing enzyme inhibitor DO34. Conversely, preincubating slices with the 2-AG degrading enzyme inhibitor KML29 rescued activity-dependent 2-AG signaling, but not mAChR-mediated synaptic depression, over GABA transmission in kindled rats. These effects were not attributable to a change in cannabinoid type 1 (CB1) receptor sensitivity or altered 2-AG tonic signaling since the application of the highly selective CB1 receptor agonist CP55,940 provoked a similar reduction in GABA synaptic activity in both sham and kindled rats, while no effect of either DO34 or of the CB1 inverse agonist AM251 was observed on frequency and amplitude of spontaneous IPSCs in either sham or kindled rats. Collectively, these data provide evidence that repeated amygdala seizures persistently alter phasic 2-AG-mediated retrograde signaling at BLA GABAergic synapses, probably by impairing stimulus-dependent 2-AG synthesis/release, which contributes to the enduring aberrant synaptic plasticity associated with seizure activity.SIGNIFICANCE STATEMENT The plastic reorganization of endocannabinoid (eCB) signaling after seizures and during epileptogenesis may contribute to the negative neurobiological consequences associated with seizure activity. Therefore, a deeper understanding of the molecular basis underlying the pathologic long-term eCB signaling remodeling following seizure activity will be crucial to the development of novel therapies for epilepsy that not only target seizure activity, but, most importantly, the epileptogenesis and the comorbid conditions associated with epilepsy.

Suppression of Presynaptic Glutamate Release by Postsynaptic Metabotropic NMDA Receptor Signalling to Pannexin-1.

The impact of pannexin-1 (Panx1) channels on synaptic transmission is poorly understood. Here, we show that selective block of Panx1 in single postsynaptic hippocampal CA1 neurons from male rat or mouse brain slices causes intermittent, seconds long increases in the frequency of sEPSC following Schaffer collateral stimulation. The increase in sEPSC frequency occurred without an effect on evoked neurotransmission. Consistent with a presynaptic origin of the augmented glutamate release, the increased sEPSC frequency was prevented by bath-applied EGTA-AM or TTX. Manipulation of a previously described metabotropic NMDAR pathway (i.e., by preventing ligand binding to NMDARs with competitive antagonists or blocking downstream Src kinase) also increased sEPSC frequency similar to that seen when Panx1 was blocked. This facilitated glutamate release was absent in transient receptor potential vanilloid 1 (TRPV1) KO mice and prevented by the TRPV1 antagonist, capsazepine, suggesting it required presynaptic TRPV1. We show presynaptic expression of TRPV1 by immunoelectron microscopy and link TRPV1 to Panx1 because Panx1 block increases tissue levels of the endovanilloid, anandamide. Together, these findings demonstrate an unexpected role for metabotropic NMDARs and postsynaptic Panx1 in suppression of facilitated glutamate neurotransmission.SIGNIFICANCE STATEMENT The postsynaptic ion and metabolite channel, pannexin-1, is regulated by metabotropic NMDAR signaling through Src kinase. This pathway suppresses facilitated release of presynaptic glutamate during synaptic activity by regulating tissue levels of the transient receptor potential vanilloid 1 agonist anandamide.

Pannexin 1 activation and inhibition is permeant-selective.

KEY POINTS: The large-pore channel pannexin 1 (Panx1) is expressed in many cell types and can open upon different, yet not fully established, stimuli. Panx1 permeability is often inferred from channel permeability to fluorescent dyes, but it is currently unknown whether dye permeability translates to permeability to other molecules. Cell shrinkage and C-terminal cleavage led to a Panx1 open-state with increased permeability to atomic ions (current), but did not alter ethidium uptake. Panx1 inhibitors affected Panx1-mediated ion conduction differently from ethidium permeability, and inhibitor efficiency towards a given molecule therefore cannot be extrapolated to its effects on the permeability of another. We conclude that ethidium permeability does not reflect equal permeation of other molecules and thus is no measure of general Panx1 activity. ABSTRACT: Pannexin 1 (Panx1) is a large-pore membrane channel connecting the extracellular milieu with the cell interior. While several activation regimes activate Panx1 in a variety of cell types, the selective permeability of an open Panx1 channel remains unresolved: does a given activation paradigm increase Panx1's permeability towards all permeants equally and does fluorescent dye flux serve as a proxy for biological permeation through an open channel? To explore permeant-selectivity of Panx1 activation and inhibition, we employed Panx1-expressing Xenopus laevis oocytes and HEK293T cells. We report that different mechanisms of activation of Panx1 differentially affected ethidium and atomic ion permeation. Most notably, C-terminal truncation or cell shrinkage elevated Panx1-mediated ion conductance, but had no effect on ethidium permeability. In contrast, extracellular pH changes predominantly affected ethidium permeability but not ionic conductance. High [K+ ]o did not increase the flux of either of the two permeants. Once open, Panx1 demonstrated preference for anionic permeants, such as Cl- , lactate and glutamate, while not supporting osmotic water flow. Panx1 inhibitors displayed enhanced potency towards Panx1-mediated currents compared to that of ethidium uptake. We conclude that activation or inhibition of Panx1 display permeant-selectivity and that permeation of ethidium does not necessarily reflect an equal permeation of smaller biological molecules and atomic ions.

Constitutive SRC-mediated phosphorylation of pannexin 1 at tyrosine 198 occurs at the plasma membrane.

Pannexin 1 (PANX1)-mediated ATP release in vascular smooth muscle coordinates α1-adrenergic receptor (α1-AR) vasoconstriction and blood pressure homeostasis. We recently identified amino acids 198-200 (YLK) on the PANX1 intracellular loop that are critical for α1-AR-mediated vasoconstriction and PANX1 channel function. We report herein that the YLK motif is contained within an SRC homology 2 domain and is directly phosphorylated by SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) at Tyr198 We demonstrate that PANX1-mediated ATP release occurs independently of intracellular calcium but is sensitive to SRC family kinase (SFK) inhibition, suggestive of channel regulation by tyrosine phosphorylation. Using a PANX1 Tyr198-specific antibody, SFK inhibitors, SRC knockdown, temperature-dependent SRC cells, and kinase assays, we found that PANX1-mediated ATP release and vasoconstriction involves constitutive phosphorylation of PANX1 Tyr198 by SRC. We specifically detected SRC-mediated Tyr198 phosphorylation at the plasma membrane and observed that it is not enhanced or induced by α1-AR activation. Last, we show that PANX1 immunostaining is enriched in the smooth muscle layer of arteries from hypertensive humans and that Tyr198 phosphorylation is detectable in these samples, indicative of a role for membrane-associated PANX1 in small arteries of hypertensive humans. Our discovery adds insight into the regulation of PANX1 by post-translational modifications and connects a significant purinergic vasoconstriction pathway with a previously identified, yet unexplored, tyrosine kinase-based α1-AR constriction mechanism. This work implicates SRC-mediated PANX1 function in normal vascular hemodynamics and suggests that Tyr198-phosphorylated PANX1 is involved in hypertensive vascular pathology.

Optogenetic control with a photocleavable protein, PhoCl.

To expand the range of experiments that are accessible with optogenetics, we developed a photocleavable protein (PhoCl) that spontaneously dissociates into two fragments after violet-light-induced cleavage of a specific bond in the protein backbone. We demonstrated that PhoCl can be used to engineer light-activatable Cre recombinase, Gal4 transcription factor, and a viral protease that in turn was used to activate opening of the large-pore ion channel Pannexin-1.

Site-Specific Regulation of P2X7 Receptor Function in Microglia Gates Morphine Analgesic Tolerance.

Tolerance to the analgesic effects of opioids is a major problem in chronic pain management. Microglia are implicated in opioid tolerance, but the core mechanisms regulating their response to opioids remain obscure. By selectively ablating microglia in the spinal cord using a saporin-conjugated antibody to Mac1, we demonstrate a causal role for microglia in the development, but not maintenance, of morphine tolerance in male rats. Increased P2X7 receptor (P2X7R) activity is a cardinal feature of microglial activation, and in this study we found that morphine potentiates P2X7R-mediated Ca2+ responses in resident spinal microglia acutely isolated from morphine tolerant rats. The increased P2X7R function was blocked in cultured microglia by PP2, a Src family protein tyrosine kinase inhibitor. We identified Src family kinase activation mediated by µ-receptors as a key mechanistic step required for morphine potentiation of P2X7R function. Furthermore, we show by site-directed mutagenesis that tyrosine (Y382-384) within the P2X7R C-terminus is differentially modulated by repeated morphine treatment and has no bearing on normal P2X7R function. Intrathecal administration of a palmitoylated peptide corresponding to the Y382-384 site suppressed morphine-induced microglial reactivity and preserved the antinociceptive effects of morphine in male rats. Thus, site-specific regulation of P2X7R function mediated by Y382-384 is a novel cellular determinant of the microglial response to morphine that critically underlies the development of morphine analgesic tolerance.SIGNIFICANCE STATEMENT Controlling pain is one of the most difficult challenges in medicine and its management is a requirement of a large diversity of illnesses. Although morphine and other opioids offer dramatic and impressive relief of pain, their impact is truncated by loss of efficacy (analgesic tolerance). Understanding why this occurs and how to prevent it are of critical importance in improving pain therapies. We uncovered a novel site (Y382-384) within the P2X7 receptor that can be targeted to blunt the development of morphine analgesic tolerance, without affecting normal P2X7 receptor function. Our findings provide a critical missing mechanistic piece, site-specific modulation by Y382-384, that unifies P2X7R function to the activation of spinal microglia and the development of morphine tolerance.

Pannexin channels and ischaemia.

An ischaemic stroke occurs during loss of blood flow in the brain from the occlusion of a blood vessel. The ischaemia itself comprises a complex array of insults, including oxygen and glucose deprivation (OGD), glutamate excitotoxicity, acidification/hypercapnia, and loss of sheer forces. A substantial amount of knowledge has accumulated that define the excitotoxic cascade downstream of N-methyl-d-aspartate receptors (NMDARs). While the NMDAR can influence numerous downstream elements, one critical target during ischaemia is the ion channel, pannexin-1 (Panx1). The C-terminal region of Panx1 appears critical for its regulation under a host of physiological and pathological stimuli. We have shown using hippocampal brain slices that Panx1 is activated by NMDARs through Src family kinases. However, it is not yet certain if this involves direct phosphorylation of Panx1 or an allosteric interaction between the channel's C-terminal tail and Src. Interestingly, Panx1 opening during ischaemia and NMDAR over-activation is antagonized by an interfering peptide that comprises amino acids 305-318 of Panx1. Thus, targeting the activation of Panx1 by NMDARs and Src kinases is an attractive mechanism to reduce anoxic depolarizations and neuronal death.

Anoxia-induced NMDA receptor activation opens pannexin channels via Src family kinases.

Anoxic depolarization of pyramidal neurons results from a large inward current that is activated, in part, by excessive glutamate release during exposure to anoxia/ischemia. Pannexin-1 (Panx1) channels can be activated both by ischemia and NMDA receptors (NMDARs), but the mechanisms of Panx1 activation are unknown. We used whole-cell recordings to show that pharmacological inhibition or conditional genetic deletion of Panx1 strongly attenuates the anoxic depolarization of CA1 pyramidal neurons in acute brain slices from rats and mice. Anoxia or exogenous NMDA activated Src family kinases (SFKs), as measured by increased phosphorylation of SFKs at Y416. The SFK inhibitor PP2 prevented Src activation and Panx1 opening during anoxia. A newly developed interfering peptide that targets the SFK consensus-like sequence of Panx1 (Y308) attenuated the anoxic depolarization (AD) without affecting SFK activation. Importantly, the NMDAR antagonists, D-APV and R-CPP, attenuated AD currents carried by Panx1, and the combined application of D-APV and (10)panx (a Panx1 blocker) inhibited AD currents to the same extent as either blocker alone. We conclude that activation of NMDARs during anoxia/ischemia recruits SFKs to open Panx1, leading to sustained neuronal depolarizations.

Ionotropic receptors and ion channels in ischemic neuronal death and dysfunction.

Loss of energy supply to neurons during stroke induces a rapid loss of membrane potential that is called the anoxic depolarization. Anoxic depolarizations result in tremendous physiological stress on the neurons because of the dysregulation of ionic fluxes and the loss of ATP to drive ion pumps that maintain electrochemical gradients. In this review, we present an overview of some of the ionotropic receptors and ion channels that are thought to contribute to the anoxic depolarization of neurons and subsequently, to cell death. The ionotropic receptors for glutamate and ATP that function as ligand-gated cation channels are critical in the death and dysfunction of neurons. Interestingly, two of these receptors (P2X7 and NMDAR) have been shown to couple to the pannexin-1 (Panx1) ion channel. We also discuss the important roles of transient receptor potential (TRP) channels and acid-sensing ion channels (ASICs) in responses to ischemia. The central challenge that emerges from our current understanding of the anoxic depolarization is the need to elucidate the mechanistic and temporal interrelations of these ion channels to fully appreciate their impact on neurons during stroke.

A genetic variant of fatty acid amide hydrolase (FAAH) exacerbates hormone-mediated orexigenic feeding in mice.

Fatty acid amide hydrolase (FAAH) degrades the endocannabinoid anandamide. A polymorphism in FAAH (FAAH C385A) reduces FAAH expression, increases anandamide levels, and increases the risk of obesity. Nevertheless, some studies have found no association between FAAH C385A and obesity. We investigated whether the environmental context governs the impact of FAAH C385A on metabolic outcomes. Using a C385A knock-in mouse model, we found that FAAH A/A mice are more susceptible to glucocorticoid-induced hyperphagia, weight gain, and activation of hypothalamic AMP-activated protein kinase (AMPK). AMPK inhibition occluded the amplified hyperphagic response to glucocorticoids in FAAH A/A mice. FAAH knockdown exclusively in agouti-related protein (AgRP) neurons mimicked the exaggerated feeding response of FAAH A/A mice to glucocorticoids. FAAH A/A mice likewise presented exaggerated orexigenic responses to ghrelin, while FAAH knockdown in AgRP neurons blunted leptin anorectic responses. Together, the FAAH A/A genotype amplifies orexigenic responses and decreases anorexigenic responses, providing a putative mechanism explaining the diverging human findings.

Shadow imaging for panoptical visualization of brain tissue in vivo.

Progress in neuroscience research hinges on technical advances in visualizing living brain tissue with high fidelity and facility. Current neuroanatomical imaging approaches either require tissue fixation (electron microscopy), do not have cellular resolution (magnetic resonance imaging) or only give a fragmented view (fluorescence microscopy). Here, we show how regular light microscopy together with fluorescence labeling of the interstitial fluid in the extracellular space provide comprehensive optical access in real-time to the anatomical complexity and dynamics of living brain tissue at submicron scale. Using several common fluorescence microscopy modalities (confocal, light-sheet and 2-photon microscopy) in mouse organotypic and acute brain slices and the intact mouse brain in vivo, we demonstrate the value of this straightforward 'shadow imaging' approach by revealing neurons, microglia, tumor cells and blood capillaries together with their complete anatomical tissue contexts. In addition, we provide quantifications of perivascular spaces and the volume fraction of the extracellular space of brain tissue in vivo.

Another win for mimetic peptides in stroke: Disruption of TRPM2-NMDAR signaling.

Glutamate excitotoxicity during ischemia triggers an intracellular signaling avalanche leading to cell death, yet blocking NMDA receptors directly in human stroke trials failed. In this issue of Neuron, Zong et al. (2022) disrupt downstream NMDAR-TRPM2 coupling to improve stroke outcomes, supporting intracellular NMDAR signaling as an alternate therapeutic target.

Intrapore lipids hydrophobically gate pannexin-1 channels.

Large-pore channels such as pannexin-1 (PANX1) typically lack pore-lining constriction points, leaving only speculations on how these channels functionally "close." In this issue of Science Signaling, Kuzuya et al. found that rearrangements in the PANX1 amino-terminal helix mediate channel gating by a surprising mechanism in which lipids block the ion conduction pathway, creating a hydrophobic gate.

Astrocytes amplify neurovascular coupling to sustained activation of neocortex in awake mice.

Functional hyperemia occurs when enhanced neuronal activity signals to increase local cerebral blood flow (CBF) to satisfy regional energy demand. Ca2+ elevation in astrocytes can drive arteriole dilation to increase CBF, yet affirmative evidence for the necessity of astrocytes in functional hyperemia in vivo is lacking. In awake mice, we discovered that functional hyperemia is bimodal with a distinct early and late component whereby arteriole dilation progresses as sensory stimulation is sustained. Clamping astrocyte Ca2+ signaling in vivo by expressing a plasma membrane Ca2+ ATPase (CalEx) reduces sustained but not brief sensory-evoked arteriole dilation. Elevating astrocyte free Ca2+ using chemogenetics selectively augments sustained hyperemia. Antagonizing NMDA-receptors or epoxyeicosatrienoic acid production reduces only the late component of functional hyperemia, leaving brief increases in CBF to sensory stimulation intact. We propose that a fundamental role of astrocyte Ca2+ is to amplify functional hyperemia when neuronal activation is prolonged.

Pannexin 1 Channels as a Therapeutic Target: Structure, Inhibition, and Outlook.

Pannexin 1 (Panx1) channels are transmembrane proteins that release adenosine triphosphate and play an important role in intercellular communication. They are widely expressed in somatic and nervous system tissues, and their activity has been associated with many pathologies such as stroke, epilepsy, inflammation, and chronic pain. While there are a variety of small molecules known to inhibit Panx1, currently little is known about the mechanism of channel inhibition, and there is a dearth of sufficiently potent and selective drugs targeting Panx1. Herein we provide a review of the current literature on Panx1 structural biology and known pharmacological agents that will help provide a basis for rational development of Panx1 chemical modulators.

Stress gates an astrocytic energy reservoir to impair synaptic plasticity.

Astrocytes support the energy demands of synaptic transmission and plasticity. Enduring changes in synaptic efficacy are highly sensitive to stress, yet whether changes to astrocyte bioenergetic control of synapses contributes to stress-impaired plasticity is unclear. Here we show in mice that stress constrains the shuttling of glucose and lactate through astrocyte networks, creating a barrier for neuronal access to an astrocytic energy reservoir in the hippocampus and neocortex, compromising long-term potentiation. Impairing astrocytic delivery of energy substrates by reducing astrocyte gap junction coupling with dominant negative connexin 43 or by disrupting lactate efflux was sufficient to mimic the effects of stress on long-term potentiation. Furthermore, direct restoration of the astrocyte lactate supply alone rescued stress-impaired synaptic plasticity, which was blocked by inhibiting neural lactate uptake. This gating of synaptic plasticity in stress by astrocytic metabolic networks indicates a broader role of astrocyte bioenergetics in determining how experience-dependent information is controlled.

Author Correction: Stress gates an astrocytic energy reservoir to impair synaptic plasticity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Limiting RyR2 Open Time Prevents Alzheimer's Disease-Related Neuronal Hyperactivity and Memory Loss but Not ß-Amyloid Accumulation.

Neuronal hyperactivity is an early primary dysfunction in Alzheimer's disease (AD) in humans and animal models, but effective neuronal hyperactivity-directed anti-AD therapeutic agents are lacking. Here we define a previously unknown mode of ryanodine receptor 2 (RyR2) control of neuronal hyperactivity and AD progression. We show that a single RyR2 point mutation, E4872Q, which reduces RyR2 open time, prevents hyperexcitability, hyperactivity, memory impairment, neuronal cell death, and dendritic spine loss in a severe early-onset AD mouse model (5xFAD). The RyR2-E4872Q mutation upregulates hippocampal CA1-pyramidal cell A-type K+ current, a well-known neuronal excitability control that is downregulated in AD. Pharmacologically limiting RyR2 open time with the R-carvedilol enantiomer (but not racemic carvedilol) prevents and rescues neuronal hyperactivity, memory impairment, and neuron loss even in late stages of AD. These AD-related deficits are prevented even with continued ß-amyloid accumulation. Thus, limiting RyR2 open time may be a hyperactivity-directed, non-ß-amyloid-targeted anti-AD strategy.

Tumor-suppressive effects of pannexin 1 in C6 glioma cells.

Mammalian gap junction proteins, connexins, have long been implicated in tumor suppression. Recently, a novel family of proteins named pannexins has been identified as the mammalian counterpart of the invertebrate gap junction proteins, innexins. To date, pannexin 1 (Panx1) and pannexin 2 (Panx2) mRNAs are reported to be expressed in the brain. Most neoplastic cells, including rat C6 gliomas, exhibit reduced connexin expression, aberrant gap junctional intercellular communication (GJIC), and an increased proliferation rate. When gap junctions are up-regulated by transfecting C6 cells with connexin43, GJIC is restored and the proliferation is reduced. In this study, we examined the tumor-suppressive effects of Panx1 expression in C6 cells. Reverse transcription-PCR analysis revealed that C6 cells do not express any of the pannexin transcripts, whereas its nontumorigenic counterpart, rat primary astrocytes, exhibited mRNAs for all three pannexins. On generation of stable C6 transfectants with tagged Panx1 [myc or enhanced green fluorescent protein (EGFP)], a localization of Panx1 expression to the Golgi apparatus and plasma membrane was observed. In addition, Panx1 transfectants exhibited a flattened morphology, which differs greatly from the spindle-shaped control cells (EGFP only). Moreover, Panx1 expression increased gap junctional coupling as shown by the passage of sulforhodamine 101. Finally, we showed that stable expression of Panx1 in C6 cells significantly reduced cell proliferation in monolayers, cell motility, anchorage-independent growth, and in vivo tumor growth in athymic nude mice. Altogether, we conclude that the loss of pannexin expression may participate in the development of C6 gliomas, whereas restoration of Panx1 plays a tumor-suppressive role.