An Optical Neuron-Astrocyte Proximity Assay at Synaptic Distance Scales

Octeau, J. Christopher; Chai, Hua; Jiang, Ruotian; Bonanno, Shivan L.; Martin, Kelsey C.; Khakh, Baljit S.

doi:10.1016/j.neuron.2018.03.003

Cited by 114 publications

(144 citation statements)

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“…Moreover, NAPA can also be used to estimate the closest synaptic contacts (i.e., within 10 nm). For example, in the striatum we found there were no differences between astrocyte contacts for either D1 or D2 collateral projections (Octeau et al, 2018). Under the correct conditions and for the appropriate questions, NAPA is a powerful technique that enables interrogation of the dynamic spatial relationships between astrocyte processes and synapses in intact neural circuits.…”

Section: Of 24mentioning

confidence: 82%

Assessing Neuron–Astrocyte Spatial Interactions Using the Neuron–Astrocyte Proximity Assay

et al. 2020

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Astrocytes are morphologically complex cells with numerous close contacts with neurons at the level of their somata, branches, and branchlets. The smallest astrocyte processes make discrete contacts with synapses at scales that cannot be observed by standard light microscopy. At such contact points, astrocytes are thought to perform both homeostatic and neuromodulatory roles—functions that are proposed to be determined by their close spatial apposition. To study such spatial interactions, we previously developed a Förster resonance energy transfer (FRET)–based approach, which enables observation and tracking of the static and dynamic proximity of astrocyte processes with synapses. The approach is compatible with standard imaging techniques such as confocal microscopy and permits assessment of the most proximate contacts between astrocytes and neurons in live tissues. In this protocol article we describe the approach to analyze the contacts between striatal astrocyte processes and corticostriatal neuronal projection terminals onto medium spiny neurons. We report the required protocols in detail, including adeno‐associated virus microinjections, acute brain slice preparation, imaging, and post hoc FRET quantification. The article provides a detailed description that can be used to characterize and study astrocyte process proximity to synapses in living tissue. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Förster resonance energy transfer imaging in cultured cells Basic Protocol 2: Förster resonance energy transfer imaging with the neuron–astrocyte proximity assay in acute brain slices

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Section: Of 24mentioning

confidence: 82%

Assessing Neuron–Astrocyte Spatial Interactions Using the Neuron–Astrocyte Proximity Assay

et al. 2020

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“…The emerging combination of two‐photon calcium imaging with SBFSEM for examining neural circuits at cellular resolution may pave the way for subcellular analyses (Vishwanathan et al, ). Finally, recent multiplex Ca 2+ imaging at a single synapse‐astrocyte interface (J. P. Reynolds et al, ), application of nanotechnology to voltage recording in neurons (Jayant et al, ), and FRET‐based analysis of contacts between synapses and astrocytes (Octeau et al, ), are advances toward integrating structure and function in astrocyte mini‐circuits.…”

Section: A Roadmap To Advance the Integration Of Astrocytes Into Systmentioning

confidence: 99%

“…Estimations in the mouse hippocampus are: 1-20 neurons (Halassa, Fellin, Takano, Dong, & Haydon, 2007), 300-600 dendrites (Halassa et al, 2007), and 140,000 synapses in Bushong, Martone, Jones, andEllisman (2002), and50,700-75,200 in Chai et al (2017). Recently, a FRET-based study reports dynamic interactions of astrocytic distal processes with different types of synaptic inputs (Octeau et al, 2018). Moreover, because astrocytes are characteristically territorial, they give rise to a tiled arrangement of the brain space, which can be then seen as a patchwork of mini-circuits.…”

Section: Theoretical and Conceptual Improvementsmentioning

confidence: 99%

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A roadmap to integrate astrocytes into Systems Neuroscience

Kastanenka

Moreno-Bote

Pittà

et al. 2019

Glia

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Systems neuroscience is still mainly a neuronal field, despite the plethora of evidence supporting the fact that astrocytes modulate local neural circuits, networks, and complex behaviors. In this article, we sought to identify which types of studies are necessary to establish whether astrocytes, beyond their well‐documented homeostatic and metabolic functions, perform computations implementing mathematical algorithms that sub‐serve coding and higher‐brain functions. First, we reviewed Systems‐like studies that include astrocytes in order to identify computational operations that these cells may perform, using Ca2+ transients as their encoding language. The analysis suggests that astrocytes may carry out canonical computations in a time scale of subseconds to seconds in sensory processing, neuromodulation, brain state, memory formation, fear, and complex homeostatic reflexes. Next, we propose a list of actions to gain insight into the outstanding question of which variables are encoded by such computations. The application of statistical analyses based on machine learning, such as dimensionality reduction and decoding in the context of complex behaviors, combined with connectomics of astrocyte–neuronal circuits, is, in our view, fundamental undertakings. We also discuss technical and analytical approaches to study neuronal and astrocytic populations simultaneously, and the inclusion of astrocytes in advanced modeling of neural circuits, as well as in theories currently under exploration such as predictive coding and energy‐efficient coding. Clarifying the relationship between astrocytic Ca2+ and brain coding may represent a leap forward toward novel approaches in the study of astrocytes in health and disease.

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“…Electrophysiological methods were described previously [55,56]. Cells were visualized with infrared optics on an upright microscope (BX61WI, Olympus).…”

Section: Electrophysiology and Ex Vivo Optogeneticsmentioning

confidence: 99%

A peptidergic amygdala microcircuit modulates sexually dimorphic contextual fear

Rajbhandari

Octeau

Gonzalez

et al. 2020

Preprint

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Trauma can cause dysfunctional fear regulation leading some to develop disorders like post-traumatic stress disorder (PTSD). The amygdala regulates fear, and, PACAP and PAC1 receptors are linked to PTSD symptom severity at genetic/epigenetic levels, with a strong link in females with PTSD. We discovered a PACAPergic projection from the basomedial amygdala (BMA) to the medial intercalated cells (mICCs). In vivo optogenetic stimulation of this pathway increased cfos expression in mICCs, decreased fear retention and increased fear extinction. Selective deletion of PAC1 receptors from the mICCs in females reduced fear acquisition, but enhanced fear generalization and reduced fear extinction in males. Optogenetic stimulation of the BMA-mICCs PACAPergic pathway produced excitatory postsynaptic currents (EPSCs) in mICCs neurons, which was enhanced by PAC1 receptor antagonist, PACAP 6-38. Our findings show that mICCs modulate contextual fear in a dynamic and sexdependent manner via the microcircuit containing the BMA and mICCs, dependent on behavioral state. Results 1.PACAP-expressing neurons in the BMA innervate the medial ICCs We examined EGFP expression in the Adcyap1-EGFP mice, which restricts EGFP expression to PACAP expressing neurons [12]. Although distributed broadly in the amygdala, EGFP + cells were enriched in the lateral and basomedial nucleus (BMA) subregion with fibers in the mICCs (Fig. 1A and Fig. S1A, B). We evaluated the local afferents of BMA and found a majority of PACAPergic neurons in the BMA innervate the dorsal and ventral mICCs (Fig.1A). By comparison, we found little to no innervation of the CN by EGFP + neurons (Fig 1A. and Fig. S1B). The pattern of expression corresponds with PACAP mRNA expression, which is high in the BMA and with some expression in the mICCS (Allen Brain Atlas Mouse Brain In Situ hybridization) (Fig. S1C). To further confirm the existence of monosynaptic PACAPergic projections from BMA to mICC we used an intersectional approach as described in Fenno et. al. 2014 [13]. For this, we injected a retrogradely trafficked Cre-dependent HSV virus (hEF1α-LS1L-mCherry-IRES-flpo) into the mICCs (dorsal portion) and a Flp-dependent AAV5-EF1a-fDIO-ChR2-eYFP-WPRE into the BMA of Adcyap1-2A-Cre. Adcyap1-2A-Cre mice express Cre specifically in PACAP-containing neurons. With the intersectional approach, the HSV virus, which is Credependent and expresses FLP, retrogradely transports allowing expression specifically in PACAPergic neurons in the BMA. The AAV viruses in turn are FLP-dependent, so therefore expresses only in neurons that have FLP. This allows labeling specific projections from the BMA to mICCs. Thus, using two different approaches, we were able to confirm that PACAPergic (Adcyap1-EGFP) neurons in the BMA innervate mICCs (Fig. 1B and Fic. 1C). 2.In vivo optogenetic stimulation of BMA PACAPergic input to the mICCs decreases fear retention and increases fear extinction

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An Optical Neuron-Astrocyte Proximity Assay at Synaptic Distance Scales

Cited by 114 publications

References 69 publications

Assessing Neuron–Astrocyte Spatial Interactions Using the Neuron–Astrocyte Proximity Assay

Assessing Neuron–Astrocyte Spatial Interactions Using the Neuron–Astrocyte Proximity Assay

A roadmap to integrate astrocytes into Systems Neuroscience

A peptidergic amygdala microcircuit modulates sexually dimorphic contextual fear

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