Molecular approaches for manipulating astrocytic signaling in vivo

Astrocytes are the predominant glial type in the central nervous system and play important roles in assisting neuronal function and network activity. Astrocytes exhibit complex signaling systems that are essential for their normal function and the homeostasis of the neural network. Altered signaling in astrocytes is closely associated with neurological and psychiatric diseases, suggesting tremendous therapeutic potential of these cells. To further understand astrocyte function in health and disease, it is important to study astrocytic signaling in vivo. In this review, we discuss molecular tools that enable the selective manipulation of astrocytic signaling, including the tools to selectively activate and inactivate astrocyte signaling in vivo. Lastly, we highlight a few tools in development that present strong potential for advancing our understanding of the role of astrocytes in physiology, behavior, and pathology

Modulation of the autonomic nervous system and behaviour by acute glial cell G q protein-coupled receptor activation in vivo: Glial GPCR signalling in physiology and behaviourin vivo

Glial fibrillary acidic protein (GFAP)-expressing cells (GFAP+ glial cells) are the predominant cell type in the central and peripheral nervous systems. Our understanding of the role of GFAP+ glial cells and their signalling systems in vivo is limited due to our inability to manipulate these cells and their receptors in a cell type-specific and non-invasive manner. To circumvent this limitation, we developed a transgenic mouse line (GFAP-hM3Dq mice) that expresses an engineered Gq protein-coupled receptor (Gq-GPCR) known as hM3Dq DREADD (designer receptor exclusively activated by designer drug) selectively in GFAP+ glial cells. The hM3Dq receptor is activated solely by a pharmacologically inert, but bioavailable, ligand (clozapine-N-oxide; CNO), while being non-responsive to endogenous GPCR ligands. In GFAP-hM3Dq mice, CNO administration increased heart rate, blood pressure and saliva formation, as well as decreased body temperature, parameters that are controlled by the autonomic nervous system (ANS). Additionally, changes in activity-related behaviour and motor coordination were observed following CNO administration. Genetically blocking inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ increases in astrocytes failed to interfere with CNO-mediated changes in ANS function, locomotor activity or motor coordination. Our findings reveal an unexpectedly broad role of GFAP+ glial cells in modulating complex physiology and behaviour in vivo and suggest that these effects are not dependent on IP3-dependent increases in astrocytic Ca2+

Intracellular Astrocyte Calcium Waves In Situ Increase the Frequency of Spontaneous AMPA Receptor Currents in CA1 Pyramidal Neurons

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 cell

Astrocyte Calcium Signaling: From Observations to Functions and the Challenges Therein

We provide an overview of recent progress on the study of astrocyte intracellular Ca2+ signaling. We consider the methods that have been used to monitor astrocyte Ca2+ signals, the various types of Ca2+ signals that have been discovered (waves, microdomains, and intrinsic fluctuations), the approaches used to broadly trigger and block Ca2+ signals, and, where possible, the proposed and demonstrated physiological roles for astrocyte Ca2+ signals within neuronal microcircuits. Although important progress has been made, we suggest that further detailed work is needed to explore the biophysics and molecular mechanisms of Ca2+ signaling within entire astrocytes, including their fine distal extensions, such as processes that interact spatially with neurons and blood vessels. Improved methods are also needed to mimic and block molecularly defined types of Ca2+ signals within genetically specified populations of astrocytes. Moreover, it will be essential to study astrocyte Ca2+ activity in vivo to distinguish between pharmacological and physiological activity, and to study Ca2+ activity in situ to rigorously explore mechanisms. Once methods to reliably measure, mimic, and block specific astrocyte Ca2+ signals with high temporal and spatial precision are available, researchers will be able to carefully explore the correlative and causative roles that Ca2+ signals may play in the functions of astrocytes, blood vessels, neurons, and microcircuits in the healthy and diseased brain

Hippocampal Astrocytes In Situ Respond to Glutamate Released from Synaptic Terminals

A long-standing question in neurobiology is whether astrocytes respond to the neuronal release of neurotransmitter

Retroviral Inhibition of cAMP-Dependent Protein Kinase Inhibits Myelination But Not Schwann Cell Mitosis Stimulated by Interaction with Neurons

Schwann cells are the myelinating glia of the peripheral nervous system. Neuron-Schwann cell contact profoundly affects several aspects of Schwann cell phenotype, including stimulation of mitosis and myelin formation. Many reports suggest that neuronal contact exerts this influence on Schwann cells by elevating Schwann cell cAMP and activating cAMP-dependent protein kinase A (PKA). To elucidate the importance of Schwann cell PKA in neuronal stimulation of Schwann cell mitosis and myelination, the gene encoding the PKA inhibitory protein RIalphaAB or PKIEGFP was delivered to Schwann cells using retroviral vectors. PKA inhibitory retroviral vectors effectively blocked forskolin-stimulated Schwann cell mitosis and morphological change, demonstrating the ability of the vectors to inhibit PKA in infected Schwann cells. Treatment of dorsal root ganglia neuron-Schwann cell cocultures with H-89 (10 microm) or KT5720 (1-10 microm), chemical inhibitors selective for PKA, significantly inhibited neuronal stimulation of Schwann cell mitosis. In contrast, retrovirus-mediated inhibition of Schwann cell PKA had no effect on the ability of neurons to stimulate Schwann cell mitosis. However, markedly fewer myelin segments were formed by Schwann cells expressing PKA inhibitory proteins compared with controls. These results suggest that activation of Schwann cell PKA is required for myelin formation but not for Schwann cell mitosis stimulated by interaction with neurons

Nuclear Factor κB-COX2 Pathway Activation in Non-myelinating Schwann Cells Is Necessary for the Maintenance of Neuropathic Pain in vivo

Chronic neuropathic pain leads to long-term changes in the sensitivity of both peripheral and central nociceptive neurons. Glial fibrillary acidic protein (GFAP)-positive glial cells are closely associated with the nociceptive neurons including astrocytes in the central nervous system (CNS), satellite glial cells (SGCs) in the sensory ganglia, and non-myelinating Schwann cells (NMSCs) in the peripheral nerves. Central and peripheral GFAP-positive cells are involved in the maintenance of chronic pain through a host of inflammatory cytokines, many of which are under control of the transcription factor nuclear factor κB (NFκB) and the enzyme cyclooxygenase 2 (COX2). To test the hypothesis that inhibiting GFAP-positive glial signaling alleviates chronic pain, we used (1) a conditional knockout (cKO) mouse expressing Cre recombinase under the hGFAP promoter and a floxed COX2 gene to inactivate the COX2 gene specifically in GFAP-positive cells; and (2) a tet-Off tetracycline transactivator system to suppress NFκB activation in GFAP-positive cells. We found that neuropathic pain behavior following spared nerve injury (SNI) significantly decreased in COX2 cKO mice as well as in mice with decreased glial NFκB signaling. Additionally, experiments were performed to determine whether central or peripheral glial NFκB signaling contributes to the maintenance of chronic pain behavior following nerve injury. Oxytetracycline (Oxy), a blood-brain barrier impermeable analog of doxycycline was employed to restrict transgene expression to CNS glia only, leaving peripheral glial signaling intact. Signaling inactivation in central GFAP-positive glia alone failed to exhibit the same analgesic effects as previously observed in animals with both central and peripheral glial signaling inhibition. These data suggest that the NFκB-COX2 signaling pathway in NMSCs is necessary for the maintenance of neuropathic pain in vivo

A Literature Review on Care at the End-of-Life in the Emergency Department

The hospitalisation and management of patients at the end-of-life by emergency medical services is presenting a challenge to our society as the majority of people approaching death explicitly state that they want to die at home and the transition from acute care to palliation is difficult. In addition, the escalating costs of providing care at the end-of-life in acute hospitals are unsustainable. Hospitals in general and emergency departments in particular cannot always provide the best care for patients approaching end-of-life. The main objectives of this paper are to review the existing literature in order to assess the evidence for managing patients dying in the emergency department, and to identify areas of improvement such as supporting different models of care and evaluating those models with health services research. The paper identified six main areas where there is lack of research and/or suboptimal policy implementation. These include uncertainty of treatment in the emergency department; quality of life issues, costs, ethical and social issues, interaction between ED and other health services, and strategies for out of hospital care. The paper concludes with some areas for policy development and future research