Protocol to downregulate GABAergic-astrocyte signaling via astrocyte-selective ablation of GABAB receptor in adult mice.

Here, we present a protocol to selectively downregulate GABAB receptor (GABABR) expression in astrocytes of mouse medial prefrontal cortex (mPFC). We first describe the procedure of surgeries and viral injections. We then detail genetic, histological, and functional characterizations of astrocytic GABABR ablation using RT-PCR, imaging, and behavioral assays. The use of GABAB flox mice can be easily adapted to generate astrocyte-selective GABABR ablation in different brain areas and postnatal stages, leading to local downregulation of GABAergic-astrocyte signaling without developmental issues. For complete details on the use and execution of this protocol, please refer to Mederos et al. (2021).

Sleep down state-active ID2/Nkx2.1 interneurons in the neocortex.

Pyramidal cells and GABAergic interneurons fire together in balanced cortical networks. In contrast to this general rule, we describe a distinct neuron type in mice and rats whose spiking activity is anti-correlated with all principal cells and interneurons in all brain states but, most prevalently, during the down state of non-REM (NREM) sleep. We identify these down state-active (DSA) neurons as deep-layer neocortical neurogliaform cells that express ID2 and Nkx2.1 and are weakly immunoreactive to neuronal nitric oxide synthase. DSA neurons are weakly excited by deep-layer pyramidal cells and strongly inhibited by several other GABAergic cell types. Spiking of DSA neurons modified the sequential firing order of other neurons at down-up transitions. Optogenetic activation of ID2+Nkx2.1+ interneurons in the posterior parietal cortex during NREM sleep, but not during waking, interfered with consolidation of cue discrimination memory. Despite their sparsity, DSA neurons perform critical physiological functions.

GABAergic signaling to astrocytes in the prefrontal cortex sustains goal-directed behaviors.

GABA interneurons play a critical role in higher brain functions. Astrocytic glial cells interact with synapses throughout the whole brain and are recognized as regulatory elements of excitatory synaptic transmission. However, it is largely unknown how GABAergic interneurons and astrocytes interact and contribute to stable performance of complex behaviors. Here, we found that genetic ablation of GABAB receptors in medial prefrontal cortex astrocytes altered low-gamma oscillations and firing properties of cortical neurons, which affected goal-directed behaviors. Remarkably, working memory deficits were restored by optogenetic stimulation of astrocytes with melanopsin. Furthermore, melanopsin-activated astrocytes in wild-type mice enhanced the firing rate of cortical neurons and gamma oscillations, as well as improved cognition. Therefore, our work identifies astrocytes as a hub for controlling inhibition in cortical circuits, providing a novel pathway for the behaviorally relevant midrange time-scale regulation of cortical information processing and consistent goal-directed behaviors.

Melanopsin for Time-Controlling Activation of Astrocyte -Neuron Networks.

Melanopsin, a mammalian G-protein-coupled photopigment, is a novel optical tool which enables studying astrocyte-neuron networks. Here, we describe the required guidelines to take advantage of this promising optical tool for functional neuron-glia studies. The selective expression of melanopsin in astrocytes allows triggering astrocytic Ca2+ signaling, changes in synaptic transmission, and modifying behavioral responses.

Prefrontal - subthalamic pathway supports action selection in a spatial working memory task.

Subthalamic nucleus (STN) is the main source of feed-forward excitation in the basal ganglia and a main target of therapeutic deep brain stimulation in movement disorders. Alleviation of motor symptoms during STN stimulation can be accompanied by deterioration of abilities to quickly choose between conflicting alternatives. Cortical afferents to the subthalamic region (ST), comprising STN and zona incerta (ZI), include projections from the medial prefrontal cortex (mPFC), yet little is known about prefrontal-subthalamic coordination and its relevance for decision-making. Here we combined electrophysiological recordings with optogenetic manipulations of projections from mPFC to ST in mice as they performed a spatial working memory task (T-maze) or explored an elevated plus maze (anxiety test). We found that gamma oscillations (30-70 Hz) are coordinated between mPFC and ST at theta (5-10 Hz) and, less efficiently, at sub-theta (2-5 Hz) frequencies. An optogenetic detuning of the theta/gamma cross-frequency coupling between the regions into sub-theta range impaired performance in the T-maze, yet did not affect anxiety-related behaviors in the elevated plus maze. Both detuning and inhibition of the mPFC-ST pathway led to repeated incorrect choices in the T-maze. These effects were not associated with changes of anxiety and motor activity measures. Our findings suggest that action selection in a cognitively demanding task crucially involves theta rhythmic coordination of gamma oscillatory signaling in the prefrontal-subthalamic pathway.

GABAergic-astrocyte signaling: A refinement of inhibitory brain networks.

Interneurons play a critical role in precise control of network operation. Indeed, higher brain capabilities such as working memory, cognitive flexibility, attention, or social interaction rely on the action of GABAergic interneurons. Evidence from excitatory neurons and synapses has revealed astrocytes as integral elements of synaptic transmission. However, GABAergic interneurons can also engage astrocyte signaling; therefore, it is tempting to speculate about different scenarios where, based on particular interneuron cell type, GABAergic-astrocyte interplay would be involved in diverse outcomes of brain function. In this review, we will highlight current data supporting the existence of dynamic GABAergic-astrocyte communication and its impact on the inhibitory-regulated brain responses, bringing new perspectives on the ways astrocytes might contribute to efficient neuronal coding.

Monitoring Interneuron-Astrocyte Signaling and Its Consequences on Synaptic Transmission.

Whole-cell patch clamp allows the characterization of synaptic transmission in neurons. It is possible to manipulate astrocytic activity and record how these glial cells affect neuronal networks. Here we describe the methodology to monitor the endogenously activation of astrocytes by inhibitory synaptic activity. Afterward, such glial activation will let us study the consequences of interneuron-astrocyte signaling on excitatory neurotransmission at hippocampal synapses.

Melanopsin for precise optogenetic activation of astrocyte-neuron networks.

Optogenetics has been widely expanded to enhance or suppress neuronal activity and it has been recently applied to glial cells. Here, we have used a new approach based on selective expression of melanopsin, a G-protein-coupled photopigment, in astrocytes to trigger Ca2+ signaling. Using the genetically encoded Ca2+ indicator GCaMP6f and two-photon imaging, we show that melanopsin is both competent to stimulate robust IP3-dependent Ca2+ signals in astrocyte fine processes, and to evoke an ATP/Adenosine-dependent transient boost of hippocampal excitatory synaptic transmission. Additionally, under low-frequency light stimulation conditions, melanopsin-transfected astrocytes can trigger long-term synaptic changes. In vivo, melanopsin-astrocyte activation enhances episodic-like memory, suggesting melanopsin as an optical tool that could recapitulate the wide range of regulatory actions of astrocytes on neuronal networks in behaving animals. These results describe a novel approach using melanopsin as a precise trigger for astrocytes that mimics their endogenous G-protein signaling pathways, and present melanopsin as a valuable optical tool for neuron-glia studies.

Astrocyte-Neuron Networks: A Multilane Highway of Signaling for Homeostatic Brain Function.

Research on glial cells over the past 30 years has confirmed the critical role of astrocytes in pathophysiological brain states. However, most of our knowledge about astrocyte physiology and of the interactions between astrocytes and neurons is based on the premises that astrocytes constitute a homogeneous cell type, without considering the particular properties of the circuits or brain nuclei in which the astrocytes are located. Therefore, we argue that more-sophisticated experiments are required to elucidate the specific features of astrocytes in different brain regions, and even within different layers of a particular circuit. Thus, in addition to considering the diverse mechanisms used by astrocytes to communicate with neurons and synaptic partners, it is necessary to take into account the cellular heterogeneity that likely contributes to the outcomes of astrocyte-neuron signaling. In this review article, we briefly summarize the current data regarding the anatomical, molecular and functional properties of astrocyte-neuron communication, as well as the heterogeneity within this communication.

Neuron-astrocyte signaling is preserved in the aging brain.

Astrocytes play crucial roles in brain homeostasis and are emerging as regulatory elements of neuronal and synaptic physiology by responding to neurotransmitters with Ca2+ elevations and releasing gliotransmitters that activate neuronal receptors. Aging involves neuronal and astrocytic alterations, being considered risk factor for neurodegenerative diseases. Most evidence of the astrocyte-neuron signaling is derived from studies with young animals; however, the features of astrocyte-neuron signaling in adult and aging brain remain largely unknown. We have investigated the existence and properties of astrocyte-neuron signaling in physiologically and pathologically aging mouse hippocampal and cortical slices at different lifetime points (0.5 to 20 month-old animals). We found that astrocytes preserved their ability to express spontaneous and neurotransmitter-dependent intracellular Ca2+ signals from juvenile to aging brains. Likewise, resting levels of gliotransmission, assessed by neuronal NMDAR activation by glutamate released from astrocytes, were largely preserved with similar properties in all tested age groups, but DHPG-induced gliotransmission was reduced in aged mice. In contrast, gliotransmission was enhanced in the APP/PS1 mouse model of Alzheimer's disease, indicating a dysregulation of astrocyte-neuron signaling in pathological conditions. Disruption of the astrocytic IP3 R2 mediated-signaling, which is required for neurotransmitter-induced astrocyte Ca2+ signals and gliotransmission, boosted the progression of amyloid plaque deposits and synaptic plasticity impairments in APP/PS1 mice at early stages of the disease. Therefore, astrocyte-neuron interaction is a fundamental signaling, largely conserved in the adult and aging brain of healthy animals, but it is altered in Alzheimer's disease, suggesting that dysfunctions of astrocyte Ca2+ physiology may contribute to this neurodegenerative disease. GLIA 2017 GLIA 2017;65:569-580.

A First-in-Class Small-Molecule that Acts as a Dual Inhibitor of HDAC and PDE5 and that Rescues Hippocampal Synaptic Impairment in Alzheimer's Disease Mice.

The targeting of two independent but synergistic enzymatic activities, histone deacetylases (HDACs, class I and HDAC6) and phosphodiesterase 5 (PDE5), has recently been validated as a potentially novel therapeutic approach for Alzheimer's disease (AD). Here we report the discovery of a new first-in-class small-molecule (CM-414) that acts as a dual inhibitor of PDE5 and HDACs. We have used this compound as a chemical probe to validate this systems therapeutics strategy, where an increase in the activation of cAMP/cGMP-responsive element-binding protein (CREB) induced by PDE5 inhibition, combined with moderate HDAC class I inhibition, leads to efficient histone acetylation. This molecule rescued the impaired long-term potentiation evident in hippocampal slices from APP/PS1 mice. Chronic treatment of Tg2576 mice with CM-414 diminished brain Aß and tau phosphorylation (pTau) levels, increased the inactive form of GSK3ß, reverted the decrease in dendritic spine density on hippocampal neurons, and reversed their cognitive deficits, at least in part by inducing the expression of genes related to synaptic transmission. Thus, CM-414 may serve as the starting point to discover balanced dual inhibitors with an optimal efficacy and safety profile for clinical testing on AD patients.

Insulin Regulates Astrocytic Glucose Handling Through Cooperation With IGF-I.

Brain activity requires a flux of glucose to active regions to sustain increased metabolic demands. Insulin, the main regulator of glucose handling in the body, has been traditionally considered not to intervene in this process. However, we now report that insulin modulates brain glucose metabolism by acting on astrocytes in concert with IGF-I. The cooperation of insulin and IGF-I is needed to recover neuronal activity after hypoglycemia. Analysis of underlying mechanisms show that the combined action of IGF-I and insulin synergistically stimulates a mitogen-activated protein kinase/protein kinase D pathway resulting in translocation of GLUT1 to the cell membrane through multiple protein-protein interactions involving the scaffolding protein GAIP-interacting protein C terminus and the GTPase RAC1. Our observations identify insulin-like peptides as physiological modulators of brain glucose handling, providing further support to consider the brain as a target organ in diabetes.

Activity-dependent switch of GABAergic inhibition into glutamatergic excitation in astrocyte-neuron networks.

Interneurons are critical for proper neural network function and can activate Ca2+ signaling in astrocytes. However, the impact of the interneuron-astrocyte signaling into neuronal network operation remains unknown. Using the simplest hippocampal Astrocyte-Neuron network, i.e., GABAergic interneuron, pyramidal neuron, single CA3-CA1 glutamatergic synapse, and astrocytes, we found that interneuron-astrocyte signaling dynamically affected excitatory neurotransmission in an activity- and time-dependent manner, and determined the sign (inhibition vs potentiation) of the GABA-mediated effects. While synaptic inhibition was mediated by GABAA receptors, potentiation involved astrocyte GABAB receptors, astrocytic glutamate release, and presynaptic metabotropic glutamate receptors. Using conditional astrocyte-specific GABAB receptor (Gabbr1) knockout mice, we confirmed the glial source of the interneuron-induced potentiation, and demonstrated the involvement of astrocytes in hippocampal theta and gamma oscillations in vivo. Therefore, astrocytes decode interneuron activity and transform inhibitory into excitatory signals, contributing to the emergence of novel network properties resulting from the interneuron-astrocyte interplay.