Microglial calcium signaling is attuned to neuronal activity in awake mice.

Key resources table

Reagent type (species) or resource Designation Source or reference Identifiers Additional information Strain, strain background ( M. musculus ; male and female) C57BL/6J Jackson labs #016962 Strain, strain background ( M. musculus ; male and female) CX3CR1 CreER-IRES-eYFP Jackson labs #021160 Strain, strain background ( M. musculus ; male and female) GCaMP6s (cytosolic) Jackson labs #024106 Strain, strain background ( M. musculus ; male and female) Lck-GCaMP6f Jackson labs #029626 Transfected construct ( M. musculus ) pENN.AAV.CaMKII.GCaMP6f.WPRE.SV40 Addgene Cat #: 105537-AAV9; RRID: Addgene_100834 Transfected construct ( M. musculus ) pAAV.CaMKIIa.hM3D(Gq)-mCherry Addgene Cat #: 50476-AAV5; RRID: Addgene_50476 Transfected construct ( M. musculus ) pAAV.CaMKIIa.hM4D(Gi)-mCherry Addgene Cat #: 50477-AAV5; RRID: Addgene_50477 Chemical compound, drug Clozapine N-Oxide Cayman Cat #: 16882 Chemical compound, drug Isoflurane Terrell Cat #: 66794-019-10 Chemical compound, drug Kainic acid Tocris Cat #: 0222 Chemical compound, drug Tamoxifen chow Envigo Cat #: TD.130856 Software, algorithm Excel Microsoft Software, algorithm ImageJ NIH Java 1.8.0_172 Software, algorithm Prism GraphPad Version 8 Other 5.2 mm Headplate Neurotar Model 2 Other Closed-loop temperature controller Physitemp TCAT-2DF Used during isoflurane imaging Other VivoScope Galvo Multiphoton Scientifica Other Mai-Tai DeepSee Spectra-Physics MAI TAI HP DS 2-P laser Other Mobile HomeCage Neurotar Cat #: NTR000289-01 Air table for in vivo imaging Other UMP3 Ultra Micro Pump WPI Cat #: UMP3-4 Used in AAV delivery Mice GCaMP6s (Rosa26-CAG-LSL-GCaMP6s; 024106), Lck-GCaMP6f (Rosa26-CAG-LSL-Lck-GCaMP6f; 029626), and CX3CR1 CreER-eYFP (021160) knock-in mouse lines can be obtained from the Jackson Laboratory. Neuronal calcium activity was studied in C57BL/6J WT mice using AAV injections. Both male and female offspring were used across all studies at an age ranging from 3 to 5 months. Mice were group housed in an AAALAC-approved facility in climate-controlled rooms with a 12 hr light/dark cycle (lights on at 6am) and had free access to food and water. All experimental procedures were approved by the Mayo Clinic’s Institutional Animal Care and Use Committee (IACUC). Administration of tamoxifen in chow In CreER lines, tamoxifen was administered through chow to activate GCaMP expression in microglia. Mice were weaned at P21 and then provided tamoxifen in chow for a two-week period (250 mg tamoxifen per 1 kg of chow; Envigo). Notably, chronic window studies occurred no sooner than 4 weeks after tamoxifen administration ended, which is sufficient to label microglia but not peripheral CX3CR1 cell types due to differences in their turnover rates ( Parkhurst et al., 2013 ).

Stereotaxic delivery of AAV

Under isoflurane anesthesia (4% induction, 1.5–2.5% maintenance), AAVs were injected into the somatosensory cortex (AP: −4.5, ML: +2.0) using a glass pipette and micropump (World Precision Instruments). AAVs were targeted to both layer V neurons (DV: −0.5) and layer II/III neurons (DV: −0.2). A 250 nL volume was dispensed at each level at a 40 nL/min rate followed by a 10-min rest period for diffusion. pENN.AAV9.CaMKII.GCaMP6s.WPRE.SV40, AAV5.CaMKIIa.hM3D(Gq)-mCherry, and AAV5.CaMKIIa.hM4D(Gi)-mCherry were either injected alone (1:1 dilution in PBS) or in conjunction (1:1 ratio of DREADD and GCaMP virus). All viruses were used at a titer of 10 12 GC/mL and acquired from Addgene.

Cranial window surgery

Cranial windows were implanted following standard techniques. Under isoflurane anesthesia (4% induction, 1.5–2.5% maintenance), a circular craniotomy (9 µm 2 ). Second, each process was visually inspected in the T-series for extension, retraction, or stability. Finally, processes needed to display at least a 50% area enlargement to be considered an extension (e.g. 10 µm 2 to 15 µm 2 ) or 50% area decrease to be considered a retraction (e.g. 10 µm 2 to 5 µm 2 ). Stable processes had less than a 50% change in area in either direction. After confirmation, these ROIs were also used to generate ∆F/F calcium traces to determine signal area and associated calcium activity based on each motility characteristic ( Figures 3I , 5G , 6K and L ). A process was considered to be associated with calcium activity if it had a signal area of >5 ∆F/F∙s. To determine temporal correlations between process extension/retraction and calcium events ( Figure 5H ), processes were selected as previously described using a manual ROI. We determined the start and end time of an extension/retraction event by using the threshold tool to create a frame-by-frame assessment of process area. The start of extension was defined as the first frame in a series of frames where process area became sequentially larger, while the end of extension was defined by a plateau in process area. The start of retraction was defined as the first frame in a series in which the process area began to reduce, while the end of retraction was defined by a plateau at a minimum value. We determined the temporal relationship between this extension/retraction start time and calcium events by determining the closest calcium event before and after the start time (if present, see Figure 5—figure supplement 2 for examples). A calcium event needed to have a peak ∆F/F value of 0.5 to be considered, and only the nearest event before or after was considered. The latency between the peak of these calcium events relative to the start of extension/retraction was calculated for all extending/retracting processes in the Figure 5 analysis and then binned by processes with an