Ca2+ activity maps of astrocytes tagged by axo-astrocytic AAV transfer
Creators: name: Georgiou, Leonidas
name: Echeverría, Anaí
name: Georgiou, Achilleas
name: Kuhn, Bernd
Publisher: name: Zenodo
Year: 2022
Subjects: astrocyte
in vivo calcium imaging
AAV
two-photon microscopy

description: Astrocytes exhibit localized Ca<sup>2+</sup> microdomain (MD) activity thought to be actively involved in information processing in the brain. However, functional organization of Ca<sup>2+</sup> MDs in space and time in relationship to behavior and neuronal activity is poorly understood. Here, we first show that Adeno-Associated Virus (AAV) particles transfer anterogradely from axons to astrocytes. Then we use this axo-astrocytic AAV transfer to express genetically encoded Ca<sup>2+</sup> indicators at high contrast circuit-specifically. In combination with two-photon microscopy and unbiased, event-based analysis we investigated cortical astrocytes embedded in the vibrissal thalamocortical circuit. We found a wide range of Ca<sup>2+</sup> MD signals, some of which were ultrafast (≤300 ms). Frequency and size of signals were extensively increased by locomotion but only subtly with sensory stimulation. The overlay of these signals resulted in behavior dependent maps with characteristic Ca<sup>2+</sup> activity hotspots, maybe representing memory engrams. These functional subdomains are stable over days, suggesting subcellular specialization.
description: The analysis code and guide on how to use the data can be found here: https://github.com/Achilleas/aqua-py-analysis Readme files are included in the Github repository above. Also Readme files for each figure can be found in supplemental information (Data_curation_SA) uploaded on Zenodo. Funding provided by: Okinawa Institute of Science and Technology School Corporation<br>Crossref Funder Registry ID: http://dx.doi.org/10.13039/100007715<br>Award Number:
description: All experimental procedures were approved by the OIST Institutional Animal Care and Use Committee (IACUC) in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC International) accredited facility. <b>Animals </b> Male, 1 to 3-month-old C57/BL6-J mice (Japan Clea) were used. Experiments were performed during the dark period of the 12/12-h dark/light cycle. <b>Surgeries </b> Mice were anesthetized by an intraperitoneal (ip) injection of a mixture of Medetomidine (0.3 mg/kg), Midazolam (4 mg/kg), and Butorphanol (5 mg/kg) in 0.9% saline solution. Carprofen (5 μg/g; ip), Dexamethasone (2 μg/g; intramuscular) and Buprenorphine (0.1 μg/g; subcutaneous) were administered to reduce inflammation, immune response, and pain, respectively. Following anesthesia induction, the eyes of the mouse were covered with mycochlorin eye ointment (Sato Pharmaceutical Co., Ltd). The mouse head hair was removed with a hair trimmer and hair removal cream (Veet®). The head of the animal was fixed on a stereotactic apparatus and its skin was sterilized with iodine. If further anesthesia was needed, 1% isoflurane was administered. In preparations without chronic window implantation, a single midline sagittal incision of the scalp was performed. The exposed skull was cleaned with the local anesthetic lidocaine. The injection coordinates were based on the Paxinos' Mouse Brain Atlas <i>(53)</i>, and previous calibration injections. The skull was thinned at the desired coordinates using a dental drill (OS-40, Osada) with a diamond drill bit (TRUSCO, T2-532M). A sterile glass pipette with a broken tip was used to puncture the thinned skull at the coordinates established to minimize the exposure of the brain. Using beveled glass pipettes (10-15μm tip diameter), AAVs were injected into ventral posteromedial nucleus (VPM; 1.8 mm posterior, 1.7 mm lateral, 3.5 mm below dura) and for some experiments additionally in barrel cortex (BX) (1.8 mm posterior, 3.2 mm lateral, 0.5 mm below dura) for intersectional strategies. AAVs were injected at a rate of ~70 nl/ 5 min by air pressure application. The pipette was left in place for at least 5 min before and after injection. After injections, the cut skin was closed using superglue. Animals were housed individually and allowed to recover for at least one week, unless otherwise stated. For chronic window implantation surgeries (54), the skin was sterilized with iodine before cutting it to expose the skull. The skull was cleaned with lidocaine solution (2% in saline). The region above the injection site in BX was marked, and a circle of ~2 mm radius was drawn around the marking. The skull surrounding the marked circle was thinned with the drill. The bone above the intended craniotomy was gently lifted after gluing a wooden cotton swap with superglue gel to the bone, to expose the dura without damaging it <i>(55)</i>. Any bleeding was cleaned carefully with Carprofen soaked gelfoam (Pfizer). AAVs were injected into VPM only or both VPM and BX (intersectional strategy) using beveled glass pipettes (10-15 μm tip diameter), at VPM: 1.8 mm posterior, 1.7 mm lateral, 3.5 mm below dura, and BX: 1.8 mm posterior, 3.2 mm lateral, 0.5 mm below dura. In all intersectional strategies, AAVs were first injected in the VPM followed immediately be a second injection in BX. AAVs were injected at a rate of ~70 nl/ 5 min by air pressure application. The pipette was left in place for at least 5 min before and after injection. A 5-mm diameter glass coverslip was placed on the craniotomy and fixed to the bone with super glue. Additionally, an aluminum head plate was mounted and fixed with dental cement (Super-Bond). Super-Bond was also used to cover any exposed skull. The animals were individually housed and allowed to recover for at least one week. <b>Adeno-associated viruses </b> <b>Intersectional strategy – astrocytes: </b>To assess whether AAV1 tags astrocytes anterograde the injection site, 140 nl (1:1 ratio) AAV2/1-CMV-Pl-Cre-rBG (1.2x10<sup>13</sup> GC/mL) and AAV2/1-hSyn-TurboRFP-WPRE-rBG (3.9x10<sup>13</sup> GC/mL) (University of Pennsylvania Viral Vector Core) were co-injected in VPM and 100 nl AAV2/5-GFaABC1D-Flex-Lck-GCaMP6f-WPRE-SV40 (1.0x10<sup>13</sup> GC/mL) (Sirion Biotech) was injected in BX. For sparse labelling of BX astrocytes with membrane tagged GCaMP6f for <i>in vivo </i>imaging we injected: 140nl AAV2/1-CMV-Pl-Cre-rBG (1.2x10<sup>13</sup> GC/mL; University of Pennsylvania Viral Vector Core) in VPM and 140nl AAV2/5-GFaABC1D-Flex-Lck-GCaMP6f-WPRE-SV40 (1.0x10<sup>13</sup> GC/mL; Sirion Biotech) in BX. <b>Intersectional strategy – neurons: </b>To assess if a similar intersectional strategy can be used to label neurons, 140 nl (1:1 ratio) AAV2/1-CMV-Pl-Cre-rBG (1.2x10<sup>13</sup> GC/mL) and AAV2/1-hSyn-TurboRFP-WPRE-rBG (3.9x10<sup>13</sup> GC/mL) (University of Pennsylvania Viral Vector Core) were co-injected in VPM and 100nl AAV9-Syn-Flex-GCaMP6f-WPRE-SV40 (2.8x10<sup>13</sup> GC/mL) (University of Pennsylvania Viral Vector Core) was injected in BX. <b>Single VPM injection strategies: </b>To test whether injection in VPM alone is sufficient for inducing cell labelling in BX and what elements of the AAV vector are needed to do so, 140 nl (1:1 ratio) of different AAV combinations were injected only in VPM. <b>Simple strategy: </b>AAV2/1-hSyn-TurboRFP-WPRE-rBG and AAV2/1-CAG-GCaMP6f-WPRE-SV40 (1.33x10<sup>13</sup> GC/mL) (University of Pennsylvania Viral Vector Core). <b>Combinatorial strategy: </b>AAV2/1-CMV-Pl-Cre-rBG and AAV2/1-CAG-Flex-eGFP-WPRE-bGH (9.16x10<sup>13 </sup>GC/mL) (University of Pennsylvania Viral Vector Core). Or:<b> </b>AAV2/1-CMV-Pl-Cre-rBG and AAV2/1-CAG-Flex-tdTomato-WPRE-rBG (4.65x10<sup>13 </sup>GC/mL) (University of Pennsylvania Viral Vector Core). For sparse labelling of BX astrocytes and neurons along with thalamocortical axons for <i>in vivo </i>imaging, we injected 120nl (1:1:1 ratio) AAV2/1-CMV-Pl-Cre-rBG (1.2x10<sup>13</sup> GC/mL), AAV2/1-CAG-Flex-GCaMP6f-WPRE-SV40 (1.33x10<sup>13</sup> GC/mL) and AAV2/1.hSyn-TurboRFP-WPRE-rBG (3.9x10<sup>13</sup> GC/mL) (University of Pennsylvania Viral Vector Core) in VPM. <b>AAV capsid tracking experiment</b> To track AAV capsids we used two groups of mice (n=3 each). Each mouse was injected twice, i.e. in each VPM (left and right) once: The first group got the first injection 12 days before the perfusion and the second injection into the contralateral VPM 1 h before the perfusion. The second group received its first injection 24 h before the perfusion and the second the injection into the contralateral VPM 1 h before the perfusion. Injections 1 h before perfusion were used as positive controls for the Anti-VP1 antibody to label AAV capsids at the injection site, within 1 h post injection. One injection of 140 nl, AAV2/1-CMV-Pl-Cre-rBG and AAV2/1-CAG-Flex-tdTomato-WPRE-rBG (1:1 ratio) was injected per VPM of each mouse. <b>Brain slice preparation. </b>Mice were deeply anesthetized with a mixture of Medetomidine (0.3mg/kg), Midazolam (4 mg/kg), and Butorphanol (5 mg/kg) in 0.9% saline solution and transcardially perfused with PBS followed by PLP (4% paraformaldehyde, 1.2% lysine, and 0.2% periodate in 0.1 M phosphate buffer) fixation. The brains were extracted and stored in PLP at 4ºC for a minimum of 48 h. For extended storage PLP was replaced with PBS. A vibratome (VT1000S, Leica) was used to cut 100 μm thick coronal slices. The slices were mounted on glass slides with Mowiol and stored at 4º C. For immunohistochemistry preparation, PLP fixed brains were cryoprotected using 20% sucrose solution in PBS overnight. The brains were then trimmed and embedded in optimal cutting temperature (OCT) compound (Tissue-Tek). The samples were frozen at -80º C for 1 h. A cryotome (Leica, CM3050 S) was used to cut 50 μm thick coronal slices at -15º C which were immediately transferred into wells filled with PBS. <b>Immunohistochemistry - AAV capsid tracking. </b>We used immunohistochemistry to tag AAV capsids with anti-VP1 antibody (Antibodies-online, ABIN933221). Brain sections cut with the cryotome were washed in PBS and then incubated in 20% normal goat serum in permeabilization solution (0.3% Triton-X-100, 0.05% sodium azide, PBS) for 1 h. The serum was replaced with 1:20 mouse Anti-AAV1 monoclonal antibody diluted in permeabilization solution. The slices were incubated for 24 h at 4º C. The samples were washed with PBS and incubated in goat anti-mouse polyclonal secondary antibody Alexa Fluor 488 (abcam, ab150113) 1:200 (diluted in permeabilization solution) for 2-3 h, in the dark, at room temperature. The brain slices were washed with PBS, mounted on glass slides with DAPI Antifade Mounting Medium (VECTASHIELD), and stored at 4ºC. <b>Immunohistochemistry – S100β and NeuN staining. </b>We used anti-S100β antibody (rabbit monoclonal, EP1576Y, abcam, ab52642) as an astrocyte specific marker. 50 μm thick coronal slices (see brain slice preparation) were prepared from mice sacrificed 3 weeks after intersectional injection of 140 nl (1:1 ratio) AAV1-CMV-Cre and AAV1-hSyn-TurboRFP in VPM and 100 nl AAV5-GFaABC1D-FLEx-Lck-GCaMP6f in BX (see intersectional strategy – astrocytes). The brain sections were washed 4 times in PBS and then incubated in 20% normal donkey serum in permeabilization solution (0.3% Triton X-100, 0.02% sodium azide, PBS) for 1 h. The serum was replaced with 1:500 anti-S100β antibody diluted in permeabilization solution. The slices were incubated for 24 h at 4º C. The samples were washed 4 times with PBS and incubated with the secondary antibody (donkey anti-rabbit polyclonal antibody, Alexa Fluor 647, abcam ab150075) 1:200 (diluted in permeabilization solution) for 2-3 h, in the dark, at room temperature. The brain slices were washed 4 times with PBS, mounted on glass slides with DAPI Antifade Mounting Medium (VECTASHIELD), and stored at 4º C. We used anti-NeuN antibody (rabbit monoclonal, EPR12763, abcam ab177487) as a neuron specific marker. 50 μm thick coronal slices (see brain slice preparation) were prepared from mice sacrificed 3 weeks after intersectional injection of 140 nl (1:1 ratio) AAV1-CMV-Cre and AAV1-hSyn-TurboRFP in VPM and 100nl AAV9-Syn-FLEx-GCaMP6f in BX (see intersectional strategy – neurons). The brain sections were washed 4 times in PBS and then incubated in 20% normal donkey serum in permeabilization solution (0.3% Triton X-100, 0.02% sodium azide, PBS) for 1 h. The serum was replaced with 1:1000 anti-NeuN antibody diluted in permeabilization solution. The slices were incubated for 24 h at 4º C. The samples were washed 4 times with PBS and incubated with secondary antibody (donkey anti-rabbit, polyclonal antibody, Alexa Fluor 647, abcam ab150075) 1:200 (diluted in permeabilization solution) for 2-3 h, in the dark, at room temperature. The brain slices were washed with PBS, mounted on glass slides with DAPI Antifade Mounting Medium (VECTASHIELD), and stored at 4º C. <b>Confocal imaging and analysis in fixed slices. </b>LSM 510 META ConfoCor3 (Carl Zeiss) and LSM 710 (Carl Zeiss) confocal microscopes were used to image fixed brain slices. LSM 510 was used for all <i>ex vivo </i>imaging, except for antibody-labelled capsid samples in the cortex. For LSM 510, a 488 nm argon laser and a 561 nm Diode-Pumped Solid-State (DPSS) laser were used to excite green fluorophores (eGFP, GCaMP6f, Alexa Fluor 488) and red fluorophores (tdTomato, TurboRFP) respectively. A 405 nm diode was used to excite DAPI. Immuno-stained AAV capsids in BX were imaged with LSM 710 using a 63x /N.A. 1.46 a-Plan-Apochromat oil objective. <b>Quantification of capsid puncta density in BX.</b> tdTomato<sup>+</sup> astrocyte and neuron cell bodies (n =15) were randomly selected from 3 brain slices mainly in layer 2/3 and 4 (n = 3 mice) ~1700 μm lateral of the injection tract. We captured 3D image stacks of 67.5 x 67.5 μm<sup>2</sup> field of view (16 bit, 1024 x 1024 pixel), 0.7 μm interspaced from each other at 0.84 AU and averaged 8 times. Alexa-Fluor 488 and tdTomato were excited using a 488 nm Argon laser and 543 nm Helium-Neon laser, respectively. For control, 3D stacks with the same parameters were acquired on contralateral hemisphere BX (~1700 μm lateral; ipsilateral the control VPM injection). To quantify the number of capsid puncta, images were first converted to 8 bit and a 1-1-1-pixel 3D Gaussian filter was applied. Brightness, contrast, and threshold parameters were established empirically and kept constant. Fluorescent puncta of 0.1 - 0.5 μm<sup>2</sup> in size were automatically counted using Fiji <i>(49)</i> plugin Analyze Particles. The size-range parameter was based on empirical observations of puncta sizes since they could be easily identified by eye. Unpaired two tailed t-tests were used to compare the puncta density between a) BX cell bodies (n = 15) vs their local background (n=12) and b) BX background vs background of contralateral BX, that was devoid of tdTomato<sup>+</sup> cell bodies (1 h after injection of its ipsilateral, control VPM; n = 10). The sampling volume was calculated to be equal to the image area x 0.7 μm x number of Z sections. Puncta density was calculated to be the number of puncta / sampling volume. <b>Quantification of cell types. </b>A high-resolution confocal microscope LSM 880 Airyscan (Carl Zeiss) was used to image fixed brain slices. A 488 nm argon, 561 nm Diode-Pumped Solid-State (DPSS) and 633 nm helium-neon lasers were used to excite GCaMP6f, TurboRFP and Alexa Fluor 647 respectively. A 405 nm diode was used to excite DAPI. Images were taken with a 20x objective. Astrocyte test: we randomly selected the nuclei (DAPI<sup>+</sup>) of BX cells expressing GCaMP6f with a cloud-like morphology in anti-S100β stained brain slices (n=3/mouse, 3 mice). For each cell, we captured a 3D image stack of 142 x 142 x 7 μm (1024 x1024 pixel, averaged 4 times, 1 μm interspaced). We recorded which cells were S100β<sup> + </sup>and which ones were not. We reported the percentage of S100β<sup> +</sup> and S100β<sup> –</sup> cells. Neuron test: we randomly selected a field of view with cell bodies expressing GCaMP6f in BX in anti-NeuN stained brain slices (n=3/mouse, 3 mice). For each slice, we randomly selected a volume of 283 x 283 x 25 μm, 1μm interspaced (1024 x 1024 pixel, averaged 4 times). In this volume we counted the GCaMP6f<sup>+</sup> cell bodies (~6 cells per slice) and identified which cells were NeuN<sup> + </sup>and which ones were not. We reported the percentage of NeuN<sup> +</sup>and NeuN<sup> –</sup> cells. <b>Quantification of astrocyte and neuron density in BX. </b>We used<b> </b>brain slices from mice injected using intersectional strategies to label astrocytes (see intersectional strategy – astrocytes, n = 3 slices/mouse, 4 mice) or neurons (see intersectional strategy – neurons, n = 3 slices/mouse, 3 mice). We used confocal-tile scanning to image GCaMP6f<sup>+</sup> cells in the BX. We manually counted all the cell bodies in the BX column with the most GCaMP6f<sup>+ </sup>cells using Zen Lite software (ZEISS). We divided the number of cells by the volume of each column to estimate the density of astrocytes and neurons. Furthermore, we divided each column into L1, L2/3, L4, and L5/6 based on <i>(56)</i> (~total width: 1125 μm, L1: 37 μm, L2/3: 234 μm, L4: 259 μm and L5/6: 595 μm, scaled)<b>. </b>We divided the number of labelled cells by the volume of each layer to estimate the density of astrocytes and neurons in each preparation respectively. The characteristic barrel-like projection patterns of TurboRFP<sup>+ </sup>thalamocortical neurons was used as assistance in delineating cortical columns and layers. <b>Two photon imaging in behaving animals </b> One week after chronic cranial window implantation, mice were habituated by periodic handling (~20 min daily for ~1 week) and exposure to head-fixation on a cylindrical treadmill (r = 7.4 cm). A vibrissa stimulus was introduced by inserting a wooden rod (toothpick segment) in the treadmill to intercept (one stimulus per rotation, equaling one stimulus every 46.5 cm) the vibrissae contralateral the injection site while the animal run. Animals were briefly anesthetized with 2% isoflurane before head fixation and left to rest and recover from anesthesia for ~10 min before two-photon (2P) imaging. In vivo 2P microscopy was used to image Ca<sup>2+</sup> signaling ~3 weeks after injection (depending on the expression of GCaMP6f). A CCD camera and infrared light source (920 nm, Thorlabs) were used to monitor behavioral activity (focused on the vibrissa pad of the animal) and vibrissa stimulation by the wooden rod. A rotary encoder (E6A2, Omron) was used to record locomotion. <i>In vivo</i> imaging was performed using a custom-built combined wide field and 2P microscope (MOM, Sutter Instruments) with a 25x/NA 1.05 water immersion objective with 2 mm working distance (Olympus). A Ti:sapphire femtosecond pulsed laser (Vision II, Coherent) was used to excite fluorescence at 950 nm (power measured after the objective &lt; 60 mW). The back aperture of the objective was underfilled to elongate the point spread function (PSF) of excitation to ~4-5 μm in axial direction and 1 μm in the imaging plane <i>(39)</i>. A resonant scanner was used to acquire images at 30.9 Hz. Fluorescence was detected by two GaAsP photomultiplier tubes (Hamamatsu) in the spectral range of 490–560 nm (green) and 570–640 nm (red) separated by a 565-nm dichroic mirror (Chroma). Commercial software (MScan, Sutter Instruments) controlled the microscope, analog channels, and CCD camera. Imaging was done ~300 μm below dura (L 2/3) in BX with 512 x 512 pixels and 94 x 94μm<sup>2</sup> field of view. To image the same astrocyte repeatedly, a 5x/NA 0.25 air objective (Zeiss) was used followed by the 25x objective to map the blood vessel pattern on the brain surface directly above the astrocyte of interest. To find the same z-plane over consecutive days (1 - 3 days after the first recording) we used depth information, autofluorescent puncta patterns, the morphology of the astrocyte, and the blood vessel pattern passing through the astrocyte. <b>Multiple pole experiment. </b>3 more mice were used by a different experimenter to investigate the effect of high frequency sti