Astrocytic GPCR-Induced Ca2+ Signaling Is Not Causally Related to Local Cerebral Blood Flow Changes.

Activation of Gq-type G protein-coupled receptors (GPCRs) gives rise to large cytosolic Ca2+ elevations in astrocytes. Previous in vitro and in vivo studies have indicated that astrocytic Ca2+ elevations are closely associated with diameter changes in the nearby blood vessels, which astrocytes enwrap with their endfeet. However, the causal relationship between astrocytic Ca2+ elevations and blood vessel diameter changes has been questioned, as mice with diminished astrocytic Ca2+ signaling show normal sensory hyperemia. We addressed this controversy by imaging cortical vasculature while optogenetically elevating astrocyte Ca2+ in a novel transgenic mouse line, expressing Opto-Gq-type GPCR Optoα1AR (Astro-Optoα1AR) in astrocytes. Blue light illumination on the surface of the somatosensory cortex induced Ca2+ elevations in cortical astrocytes and their endfeet in mice under anesthesia. Blood vessel diameter did not change significantly with Optoα1AR-induced Ca2+ elevations in astrocytes, while it was increased by forelimb stimulation. Next, we labeled blood plasma with red fluorescence using AAV8-P3-Alb-mScarlet in Astro-Optoα1AR mice. We were able to identify arterioles that display diameter changes in superficial areas of the somatosensory cortex through the thinned skull. Photo-stimulation of astrocytes in the cortical area did not result in noticeable changes in the arteriole diameters compared with their background strain C57BL/6. Together, compelling evidence for astrocytic Gq pathway-induced vasodiameter changes was not observed. Our results support the notion that short-term (<10 s) hyperemia is not mediated by GPCR-induced astrocytic Ca2+ signaling.

Astrocyte calcium signaling transforms cholinergic modulation to cortical plasticity in vivo.

Global brain state dynamics regulate plasticity in local cortical circuits, but the underlying cellular and molecular mechanisms are unclear. Here, we demonstrate that astrocyte Ca(2+) signaling provides a critical bridge between cholinergic activation, associated with attention and vigilance states, and somatosensory plasticity in mouse barrel cortex in vivo. We investigated first whether a combined stimulation of mouse whiskers and the nucleus basalis of Meynert (NBM), the principal source of cholinergic innervation to the cortex, leads to enhanced whisker-evoked local field potential. This plasticity is dependent on muscarinic acetylcholine receptors (mAChR) and N-methyl-d-aspartic acid receptors (NMDARs). During the induction of this synaptic plasticity, we find that astrocytic [Ca(2+)](i) is pronouncedly elevated, which is blocked by mAChR antagonists. The elevation of astrocytic [Ca(2+)](i) is crucial in this type of synaptic plasticity, as the plasticity could not be induced in inositol-1,4,5-trisphosphate receptor type 2 knock-out (IP(3)R2-KO) mice, in which astrocytic [Ca(2+)](i) surges are diminished. Moreover, NBM stimulation led to a significant increase in the extracellular concentration of the NMDAR coagonist d-serine in wild-type mice when compared to IP(3)R2-KO mice. Finally, plasticity in IP(3)R2-KO mice could be rescued by externally supplying d-serine. Our data present coherent lines of in vivo evidence for astrocytic involvement in cortical plasticity. These findings suggest an unexpected role of astrocytes as a gate for cholinergic plasticity in the cortex.

In vivo intracellular recording suggests that gray matter astrocytes in mature cerebral cortex and hippocampus are electrophysiologically homogeneous.

Previous anatomical and in vitro electrophysiology studies suggest that astrocytes are heterogeneous in physiology, morphology, and biochemical content. However, the extent to which this diversity applies to in vivo conditions is largely unknown. To characterize and classify the physiological and morphological properties of cerebral cortical and hippocampal astrocytes in the intact brain, we performed in vivo intracellular recordings from single astrocytes using anesthetized mature rats. Astrocytes were classified based on their glial fibrillary acidic protein (GFAP) immunoreactivity and cell body locations. We analyzed morphometric measures such as the occupied volume and polarity, as well as physiological characteristics such as the mean membrane potential. These measurements did not show obvious segregation into subpopulations, suggesting that gray matter astrocytes in the cortex and hippocampus are composed of a homogeneous population in mature animals. The membrane potential of astrocytes in both cortex and hippocampus fluctuated within a few millivolts in the presence of spontaneous network activity. These membrane potential fluctuations of an astrocyte showed a significant variability that depended on the local field potential state and cell body location. We attribute the variability of the membrane potential fluctuations to local potassium concentration changes due to neuronal activity.

Transient Astrocytic Gq Signaling Underlies Remote Memory Enhancement.

Astrocytes elicit transient Ca2+ elevations induced by G protein-coupled receptors (GPCRs), yet their role in vivo remains unknown. To address this, transgenic mice with astrocytic expression of the optogenetic Gq-type GPCR, Optoα1AR, were established, in which transient Ca2+ elevations similar to those in wild type mice were induced by brief blue light illumination. Activation of cortical astrocytes resulted in an adenosine A1 receptor-dependent inhibition of neuronal activity. Moreover, sensory stimulation with astrocytic activation induced long-term depression of sensory evoked response. At the behavioral level, repeated astrocytic activation in the anterior cortex gradually affected novel open field exploratory behavior, and remote memory was enhanced in a novel object recognition task. These effects were blocked by A1 receptor antagonism. Together, we demonstrate that GPCR-triggered Ca2+ elevation in cortical astrocytes has causal impacts on neuronal activity and behavior.

Transcranial Direct Current Stimulation (tDCS) Induces Adrenergic Receptor-Dependent Microglial Morphological Changes in Mice.

Transcranial direct current stimulation (tDCS) has been reported for its beneficial effects on memory formation and various brain disorders. While the electrophysiological readout of tDCS effects is subtle, astrocytes have been demonstrated to elicit Ca2+ elevations during tDCS in a rodent model. This study aimed to elucidate the effects of tDCS on another major glial cell type, microglia, by histology and in vivo imaging. tDCS performed in awake conditions induced a significant change in the pixel intensity distribution of Iba-1 immunohistochemistry, and microglial somata were enlarged when examined 3 h after tDCS. These effects were blocked by adrenergic receptor antagonists or in IP3R2 (inositol trisphosphate receptor type 2)-deficient mice, which lack large cytosolic Ca2+ elevations in astrocytes. No obvious changes were observed in isoflurane-anesthetized mice. Furthermore, in vivo two-photon imaging of microglia showed a reduction of motility that was blocked by a ß2-adrenergic receptor antagonist. Our observations add support for the influence of noradrenaline in tDCS and suggest possible interactions between microglia and astrocytes to express functional changes associated with tDCS.

Intracellular labeling of single cortical astrocytes in vivo.

Glial cells have traditionally been considered to play supportive roles in the central nervous system. As recent experimental evidence suggests glial cells' participation in neural information processing, there has been a need to monitor the physiology of glial cells in vivo in the matured brain. Concurrently, identification and classification of the recorded glial cells is essential as there are at least several different kinds of glial cells. Past studies have achieved in vivo intracellular electrophysiological recording of glial cells using sharp glass microelectrodes, however, morphological recovery and identification of the recorded cells have hardly been done, due to technical difficulties. We demonstrate that use of large fragment biotinylated dextran amine (BDA) is an effective way to label a single glial cell recorded with a sharp microelectrode in vivo. Furthermore, the tracer signal amplification was achieved by a combination of avidin biotinylated horseradish peroxidase macromolecular complex (ABC) and tyramide-based methods, making multiple immunohistochemistry feasible. Using the method described in this study, we have successfully recorded and labeled cortical glial cells including astrocytes, oligodendrocytes, and microglia.

Volume transmission signalling via astrocytes.

The influence of astrocytes on synaptic function has been increasingly studied, owing to the discovery of both gliotransmission and morphological ensheathment of synapses. While astrocytes exhibit at best modest membrane potential fluctuations, activation of G-protein coupled receptors (GPCRs) leads to a prominent elevation of intracellular calcium which has been reported to correlate with gliotransmission. In this review, the possible role of astrocytic GPCR activation is discussed as a trigger to promote synaptic plasticity, by affecting synaptic receptors through gliotransmitters. Moreover, we suggest that volume transmission of neuromodulators could be a biological mechanism to activate astrocytic GPCRs and thereby to switch synaptic networks to the plastic mode during states of attention in cerebral cortical structures.

Neurons associated with the flip-flop activity in the lateral accessory lobe and ventral protocerebrum of the silkworm moth brain.

The lateral accessory lobe (LAL) and the ventral protocerebrum (VPC) are a pair of symmetrical neural structures in the insect brain. The LAL-VPC is regarded as the major target of olfactory responding neurons as well as the control center for olfactory-evoked sequential zigzag turns. Previous studies of the silkworm moth Bombyx mori showed that these turns are controlled by long-lasting anti-phasic activities of the flip-flopping descending neurons with dendrites in the LAL-VPC. To elucidate the neural mechanisms underlying the generation of this alternating activity between the LAL-VPC units of both hemispheres, we first analyzed the detailed neural architecture of the LAL-VPC and identified five subregions. We then investigated the morphology and physiological responses of the LAL-VPC neurons by intracellular recording and staining and morphologically identified three types of bilateral neurons and three types of unilateral neurons. Bilateral neurons showed either brief or cyclic long-lasting responses. At least some neurons of the latter type produced gamma-aminobutyric acid (GABA). Unilateral neurons linking the LAL and VPC, in contrast, showed long-lasting or quick alternating activity. Timing analysis of the activity onset of each neural type suggests that quick reciprocal neural transmission between unilateral neurons would be responsible for the generation of long-lasting activity in one LAL-VPC unit, which lasts for up to a few seconds. Reciprocal inhibition and excitation by the bilateral neurons with long-lasting activities would mediate the alternating long-lasting activity between both LAL-VPC units, which might last for up to 20 seconds.