Chemogenetic activation of astrocytes in the hippocampus and cortex changes the transcriptome of microglia and other cell types

Astrocytes are the most common glial cell type in the brain, yet, it is still not clear how their activation affects the transcriptome of other brain cells such as microglia and neurons. Engineered G protein-coupled receptors called Designer Receptors Exclusively Activated by Designer Drugs (DREADDS) make it possible to selectively activate specific cell types, such as neurons and astrocytes. By combining the selective activation of astrocytes with single cell RNA sequencing, we were able to study transcriptional changes that occur in response to the activation of astrocytes at the single cell level. Interestingly, our data shows that long-term activation of astrocytes in healthy mice results in dramatic alteration in the transcriptome of astrocytes and microglia. Genes that were differentially expressed in these Gq-DREADD-activated astrocytes were involved in neurogenesis and low density lipoprotein particle biology, while those in the microglia were involved in the response to lipoproteins, and the migration and chemotaxis of immune cells. Furthermore, network analysis showed that Gq-DREADD-mediated activation in astrocytes resulted in an upregulation of genes involved in the G protein-coupled receptor signaling pathway and calcium ion homeostasis. This confirmed the activation of astrocytes through the expressed DREADDS. Our findings show the importance of considering the transcriptomic alteration in microglia and neurons after the activation of astrocytes in in vivo models. Therefore, our data will serve as a resource for the broader neuroscience community.


Introduction 44
Astrocytes are the most abundant glial cell in the brain and affect most aspects of neural 45 function. They are responsible for neurotransmitter and ion homeostasis at the level of the 46 synapse, provide trophic support to neurons, regulate blood flow, and provide energy substrates acetylcholine (Armbruster, Li et al. 2007, Rogan and Roth 2011). In this study we used the Gq-63 coupled human muscarinic type 3 receptor (hM3Dq) whose activation results in Ca 2+ 64 mobilization and ERK1/2 phosphorylation (Armbruster, Li et al. 2007). The in vivo functionality of 65 DREADD-mediated astrocyte activation has previously been demonstrated in rats, with 66 excitation of hM3D-expressing astrocytes in the nucleus accumbens core resulting in glutamate 67 gliotransmission and modulated drug seeking behavior (Bull, Freitas et al. 2014, Scofield, Boger 68 et al. 2015). However the effect on astrocyte activation on surrounding cells was not 69 investigated. 70 Recent scRNA seq technologies have made it possible to study transcriptional 71 differences in each cell-type in healthy and diseased brains, as well as in brain injuries such as 72 traumatic brain injury (Keren-Shaul, Spinrad et al. 2017, Arneson, Zhang et al. 2018, Batiuk,73 Martirosyan et al. 2020). In this study, we were interested in the transcriptional changes that 74 occur after astrocyte-specific activation using the Gq-DREADD. To selectively activate 75 astrocytes in our study, we used adeno-associated virus transduction of Gq-DREADD into the 76 hippocampus and cortex of wild type mice under the control of the glial fibrillary acidic promoter 77 (GFAP). We administered CNO to our mice over a two month period to simulate chronic 78 activation of astrocytes instead of initiating an acute injury response. We profiled a total of 79 34,736 cells and showed that Gq-DREADD activation in astrocytes does not only change the 80 transcriptional profile of the astrocytes themselves, but also of neighboring microglia. Network 81 analysis using MetaCore TM showed that the genes that were upregulated in the astrocytes after 82 CNO treatment activation were involved in GPCR signaling and calcium homeostasis, which 83 confirmed Gq-DREADD-mediated activation of astrocytes. No major transcriptional changes 84 were observed in the neurons or other cell types present in the brain such as endothelial and 85 ependymal cells. To the best of our knowledge, our study is the first to study the effect of 86 astrocyte-specific Gq-DREADD activation in surrounding brain cell types at the single cell level 87 using scRNA seq. 88 mg/kg) and xylazine (10 mg/kg) and placed on a stereotaxic instrument. The skin was opened 114 and a hole in the skull was made by a hand held drill. The needle was lowered into the 115 hippocampus (A-P -2 mm; M-L ±1.5 mm; D-V -1.7 mm from Bregma) and the virus (AAV-GFAP-116 hM3D(Gq)-mCherry; Serotype 5; 1.0x10 12 genome copies/ml) was infused at a rate of 0.4 μl/min 117

Material and Methods
(1.5 μl total volume). The needle was allowed to remain in place for 5 minutes then slowly raised 118 0.9 mm to the cortex (A-P -2 mm; M-L ±1.5 mm; D-V -0.8 mm from Bregma) and more virus was 119 infused (0.4 μl/min; 1.0 μl total volume). The needle was again left for 5 minutes before being 120 removed. This was repeated on the contralateral hemisphere for bilateral injection of the virus 121 into both hippocampi and cortex ( Figure 1A). 122

Tissue Preparation and Immunohistochemistry 130
For tissue that was to be assessed histologically, mice were sacrificed by ketamine (100mg/kg 131 i.p.) overdose followed by transcardial perfusion with 4% paraformaldehyde (PFA). Tissue was 132 post-fixed in 4% PFA overnight at 4°C, and then left for 24 h in PBS containing 30% sucrose.

133
Tissue was then embedded in Frozen Section Compound (FSC22; Leica) and sections were cut 134 in the coronal plane (30 μm) using a cryostat. 135 Fluorescence immunohistochemistry was performed on free floating coronal sections to 136 identify mCherry-positive cells and GFAP-positive astrocytes in the hippocampus and cortex. 137 Briefly, sections were blocked for 1 h at room temperature in 5% bovine serum albumin (BSA), 138 and 0.1% Triton-X in PBS followed by incubation overnight in rabbit anti-GFAP antibody 139 ( removal of cells with a mitochondrial count >5%, as well as cells with less than 200 and more 177 than 2,000 detected genes. A total of 34,736 cells remained after quality control filtering steps.

178
The data were log-normalized using a scaling factor of 10,000, scaled and regressed against 179 the percentage of mitochondrial content. Principal component analysis (PCA) was performed 180 with the top 3,000 most variable genes and the number of PCAs to use were determined after 181 1,000 permutations using the ElbowPlot. The first 21 PCAs was used to determine the K-182 nearest neighbor (KNN) and cluster the cells into 15 different cell clusters. The clustering 183 resolution used was 0.5. Marker genes for naming the clusters were determined using the 184 FindConservedMarkers function in Seurat. Cells from the glial and neuronal clusters (astrocytes, 185 microglia I-V, mixed glia I, mixed glia II, neurons) were taken for re-clustering. 186

Analysis of differential gene expression 187
Differential gene expression analysis between the control group (saccharin) and the Gq-188 DREADD group (CNO) was done in Seurat using a likelihood test assuming an underlying 189 negative binomial distribution. Genes with a p-value < 0.05 after Bonferroni correction were 190 considered to be significantly differentially expressed between both groups. The differentially 191 expressed genes were analyzed for functional enrichment using g:Profiler (

Single cell RNA sequencing identifies fifteen different cell types 204
To determine how activation of astrocytes affects the transcriptome of astrocytes and other cell 205 types, we performed scRNA seq on cells isolated from the hippocampus and cortex of four 206 month-old C57BL/6J mice. These mice were injected with AAV-GFAP-hM3D(Gq) at 8 weeks of 207 age ( Figure 1A) and treated with CNO or saccharin for 8 weeks. Two samples per group were 208 sequenced with an average of 442M reads per sample, or an average of 37,000 reads per cell.

209
About 56% of all reads were exonic, while 29% were intronic and only 5% were intergenic. A 210 total of 34,736 cells passed the quality control filters, 16,107 cells in the CNO treated group and 211 18,629 cells in the negative control group (saccharin). Unsupervised clustering resulted in the 212 identification of 15 different cell clusters ( Figure 1D). The cell clusters were annotated manually 213 using marker genes that were conserved between both conditions as astrocytes, microglia, 214 neurons, endothelial cells, ependymal cells, and mixed glia. We observed an enrichment of glial 215 cells with 96% of all cells detected annotated as either astrocytes, microglia or mixed glia. 216 When assessing the cell distribution between the CNO treated and the negative control 217 groups over the different cell types, we noticed large differences in the percentage of cells in the 218 mixed glia I (30.66% in the saccharin group vs. 45.32% in the CNO group), mixed glia II 219 (29.62% in the saccharin group vs. 14.87% in the CNO group) and microglia II clusters (11.39% 220 in the saccharin group vs. 6.60% in the CNO group, Figure 1E). Smaller differences were 221 observed in the astrocyte (7.41% in the saccharin group vs. 5.30% in the CNO activated group) 222 and microglia I (12.37% in the saccharin group vs. 16.74% in CNO group) clusters ( Figure 1E & 223 Supplementary Table 1). 224

Re-clustering of the astrocyte cluster showed the presence of transcriptionally different 225 astrocyte types after chronic activation of astrocytes 226
Since Gq-DREADD was only expressed in astrocytes, we analyzed the effect of Gq-DREADD 227 activation by comparing the genes that were differentially expressed between the saccharin 228 group and the CNO group in the astrocyte cluster. We identified 396 genes that were either up-229 or downregulated in the CNO group compared to the saccharin group ( Figure 2A). Gene 230 enrichment and functional annotation analysis showed that transcripts that were upregulated in 231 the CNO group are involved in myelin sheath biology while the downregulated genes were 232 involved in gene expression and translation (Supplementary Data 1). Network analysis was 233 performed using MetaCore TM . We found that 16 of the genes that were upregulated after 234 activation of Gq-DREADD with CNO were in a 50-gene network represented by CMKLR1, 235 SPARCL1, Rich1, Transcobalamin II, OLFML3 (Supplementary Figure 2). The top processes 236 linked to this network were GPCR signaling pathway, ionotropic glutamate receptor signaling 237 pathway, adenylate cyclase inhibiting GPCR signaling pathway, cellular calcium ion 238 homeostasis, and calcium ion homeostasis. Because these cellular processes are downstream 239 pathways regulated by Gq-DREADD, this analysis confirmed the Gq-DREADD-mediated 240 activation of astrocytes. 241 To investigate how activation of astrocytes alters their gene expression in more detail, 242 we re-clustered the astrocyte cluster ( Figure 2B). Sub-clustering of the astrocytes showed cell 243 types that were observed in the saccharin group (clusters 0, 2 and 3) while nearly absent from 244 the CNO group, and vice versa (clusters 1 and 4, Figure 2B). This was confirmed by the cell 245 distribution over the different clusters. Cluster 0 accounted for 53.26% of all cells in the 246 saccharin group while only 3.96% in the CNO group. Cluster 1 on the other hand represented 247 only 0.87% in the saccharin group, while 66.55% in the CNO group ( Figure 3C, Supplementary 248 Table 2). Gene enrichment and functional annotation analysis of the differentially expressed 249 genes in the astrocytic sub-clusters show different enriched categories for each cluster 250 (Supplementary Data 2). The clusters in the saccharin group (cluster 0, 2 and 3) are enriched 251 for genes involved in regulation of neuronal death, neuroinflammatory response and regulation 252 of synaptic organization, while those of the CNO clusters (cluster 1 and 4) are enriched for 253 genes involved in low-density lipoprotein particles, central nervous system development, 254 gliogenesis, and glial cell differentiation (Supplementary Data 2). 255

Re-clustering of the mixed glia cell clusters showed the presence of both microglia-like 256 and astrocyte-like cells in both mixed glia cell clusters 257
We annotated two cell clusters as 'Mixed Glia' since their conserved marker genes showed an 258 equal likelihood of these cells being annotated as astrocytes or microglia. Activation of 259 astrocytes using CNO led to a 27% increase in cell numbers in the mixed glia I cluster and a 260 56% decrease in the mixed glia II cluster ( Figure 1E & Supplementary Table 1). 261 Differentially expressed genes in the mixed glia I cluster showed that activation of 262 astrocytes by the administration of CNO downregulated genes involved in the immune system, 263 system development, cell migration and response to stress ( Figure 3A, Supplementary Data 1). 264 Genes that were upregulated after CNO treatment are involved in ribosomal processes, as well 265 as neurodegenerative diseases such as Parkinson's disease, Huntington's disease and 266 Alzheimer's disease ( Figure 3A, Supplementary Data 1). In the mixed glia II cluster, gene 267 enrichment and functional annotation of differentially expressed genes demonstrated that 268 downregulated genes were involved in the astrocytic transcription factor AP-1 complex while the 269 genes that were upregulated after hM3Dq activation were mainly involved in ribosomal 270 processes ( Figure 3D, Supplementary Data 1).

271
To investigate what cell type is responsible for these effects, we re-clustered both mixed 272 glia clusters. Re-clustering of the mixed glia I cluster resulted in eight different sub-clusters while 273 the re-clustering of the mixed glia II cluster resulted in six sub-clusters ( Figure 3B and 3E). Cells 274 belonging to cluster 2 in the mixed glia I group were more abundant in the CNO group 275 compared to the saccharin group. 0.78% of the cells in the saccharin group were part of cluster 276 2, while 10.06% of cells in the CNO group ( Figure 3C, Supplementary Table 3). Gene ontology 277 analysis of differentially expressed genes shows that the cells in cluster 2 are involved in 278 lipoprotein particle clearance, lipid transport across the blood-brain barrier and regulation of 279 presynaptic membrane transport (Supplementary Data 3). 280 Re-clustering of the mixed glia II cluster showed more dramatic effects of CNO on the 281 different sub-clusters than was observed in the mixed glia I cluster. Cluster 0 (56.90% in the 282 saccharin group vs. 11.66% in the CNO group), cluster 1 (37.12% in the saccharin group vs. 283 0.87% in the CNO group) and cluster 4 (3.13% in the saccharin group vs. 0.62% in the CNO 284 group) disappeared almost entirely after CNO treatment while cluster 2 (2.69% in the saccharin 285 group vs. 66.26% in the CNO group) and cluster 3 (0.05% in the saccharin group vs. 20.45% in 286 the CNO group) appeared after CNO treatment ( Figure 3E & 3F, Supplementary Table 4).

287
Differentially expressed genes in clusters 0, 1 and 4 were involved in the response to axon 288 injury and extracellular stimuli, corticospinal tract atrophy, cell motility, and regulation of the 289 actin cytoskeleton (Supplementary Data 4). The genes that were differentially expressed in 290 clusters 2 and 3 played a role in vitamin B6 levels, aging and the mitochondrial respiratory 291 complex (Supplementary Data 4). 292

Chronic activation of astrocytes changed the transcriptome of microglia 293
Even though Gq-DREADD was only expressed in astrocytes, we observed a 42% decrease in 294 the number of cells in the microglia II cluster after activation of astrocytes with CNO ( Figure 1E  295 & Supplementary Table 1). Gene enrichment and functional analysis of genes that were 296 upregulated after CNO treatment in the microglia II cluster were mainly involved in ribosomal 297 processes while the downregulated genes were involved in MAPK activation (Supplementary 298 Data 1). 299 Re-clustering of the microglia II cluster resulted in the identification of seven sub-300 clusters. Three sub-clusters were mainly observed in the saccharin group (clusters 0, 2 and 3), 301 two were more common in the CNO group (clusters 1 and 4), and two others (clusters 5 and 6) 302 were present in both groups ( Figure Table 6). 318 Differentially expressed genes in these two clusters were involved in the innate immune system, 319 antigen processing and presentation, and neuronal cell death (Supplementary Data 6). 320 Re-clustering of the microglia III cluster resulted in five sub-clusters ( Figure 5E).

321
Although all of them were observed in both groups, cluster 0 was six times more prominent in 322 the saccharin group (56.08%) compared to the CNO group (8.95%), and cluster 2 was nine 323 times more pronounced in the CNO group (36.24%) than in the saccharin group (3.97%, Figure  324 5E & 5F, Supplementary Table 7). The differentially expressed genes in both clusters were 325 involved in the migration of immune cells like neutrophils, granulocytes, and macrophages 326 (Supplementary Data 7). 327 The microglia IV cluster showed five sub-clusters after re-clustering. Clusters 2 and 3 328 were only observed in the saccharin group, and thus, were completely absent from the CNO 329 group ( Figure 5H & 5I, Supplementary Table 8). Differentially expressed genes in clusters 2 and 330 3 were shown to be involved in negative regulation of chronic inflammatory response, ribosomal 331 processes, and spine synapse organization (Supplementary Data 8). Clusters 0 (14.67% in the 332 saccharin group vs. 45.09% in the CNO group) and 1 (0.80% in the saccharin group vs. 48.61% 333 in the CNO group) were more common in the CNO group compared to the saccharin group 334 ( Figure 5H & 5I, Supplementary Table 8). Differentially expressed genes in these two clusters 335 were involved in cell death, the immune system, and ubiquitination (Supplementary Data 8). 336 Finally, re-clustering of the fifth microglial cluster resulted in four different cell clusters 337 ( Figure 5K). Clusters 0 and 2, showed a 1.7 fold increase and a 50% decrease in cell numbers 338 between the saccharin and the CNO groups, respectively ( Figure 5L, Supplementary Table 9). 339 The gene enrichment and functional annotation of the differentially expressed genes for clusters 340 0 and 2 showed involvement in ribosomal processes, as well as in neurodegenerative diseases 341 such as Parkinson's disease and Alzheimer's disease (Supplementary Data 9). 342

Long-term activation of astrocytes have no major effects on the transcriptome of 343 neurons 344
To study whether Gq-DREADD-induced activation of astrocytes results in transcriptional 345 changes in neurons, we re-clustered the neuronal cluster. Three clusters were identified that 346 were observed in both groups ( Figure 6A). Cluster 0 showed a small decrease and cluster 2 a 347 small increase in number of cells after astrocyte activation with Gq-DREADD, while no 348 difference was observed in cluster 1 ( Figure 6A & 6B, Supplementary Table 10). Genes that 349 were differentially expressed in clusters 0 and 2 are both involved in nervous system 350 development, and specifically in neurogenesis, suggesting that both cell types are not 351 biologically different. The genes that were differentially expressed in cluster 1 are mainly 352 involved in ribosomal processes (Supplemental Data 10). 353

354
To the best of our knowledge, this is the first study to show transcriptional changes in glia that 355 occur after long-term activation of astrocytes with the Gq-DREADD in an in vivo model.

356
Previous studies have expressed the Gq-DREADD specifically in astrocytes using the GFAP wild type mice that selectively expressed Gq-DREADD in astrocytes. Network analysis of the 363 genes that were upregulated after Gq-DREADD activation with CNO in astrocytes showed an 364 involvement of these genes in the GPCR signaling pathway and calcium homeostasis. These 365 findings confirmed the transcriptionally activated state of the astrocytes after long-term 366 activation of Gq-DREADD with CNO (Supplementary Figure 2). Furthermore, our results 367 demonstrate the presence of transcriptionally different astrocyte populations after Gq-DREADD 368 activation with CNO compared to the control group. Although the up-or downregulation of 369 differentially expressed genes was relatively low, we could clearly observe transcriptionally 370 different astrocytic sub-clusters. While some clusters were predominantly present in the control 371 group, other were mainly observed after activation with CNO ( Figure 2B). We also observed that 372 our astrocyte population in the CNO group did not show the transcriptional profile of reactive 373 astrogliosis that is typically seen in neurodegenerative diseases (Supplementary Figure 3) 374 (Kraft, Hu et al. 2013). Therefore, we hypothesize that long-term activation of astrocytes 375 changes their transcriptional profile to a new steady state phenotype. Further research is 376 needed to assess what this new steady state phenotype means to the astrocytes and their 377 environment, and their biological relevance. 378 Gene and functional analysis of the astrocyte subclusters showed a functional difference 379 between the astrocytes present in the control group and after Gq-DREADD activation 380 (Supplementary Data 2). The astrocyte clusters that were more abundant in the control group 381 than in the CNO group (clusters 0, 2 & 3, Figure 2B) were involved in the regulation of several 382 processes such as transport, neuronal cell death, and synapse structure and activity 383 (Supplementary Data 2). The cell clusters that were more abundant in the CNO group (clusters 384 1 & 4, Figure 2B) were important for low-density lipoprotein homeostasis and central nervous 385 system development. Strikingly, gene ontology showed that the differentially expressed genes 386 from the CNO subclusters were enriched for glial cell development, astrocyte differentiation and 387 negative regulation of neuronal apoptotic processes (Supplementary Data 2). While the 388 astrocytes enriched in the control group show a homeostatic phenotype, the phenotype for the 389 astrocytes enriched in the CNO group is less clear (Barres 2008 research is needed to elucidate the exact molecular phenotype of the Gq-DREADD activated-393 astrocytes. 394 Interestingly, the transcriptome of microglia was dramatically affected by the long-term 395 activation of astrocytes (Figures 4 and 5). We observed microglial clusters that were absent in 396 the control group and dominating in the CNO group ( Figures 5B & 5H). Furthermore, the 397 transcriptomic changes that occurred in microglial cells after Gq-DREADD activation were 398 different than those in control microglia (Supplementary Data 5-9). After long-term activation of 399 astrocytes, microglia became involved in synaptic pruning, lipoprotein particle processes, and 400 migration and chemotaxis of immune cells. In addition, our data demonstrated that long-term 401 activation of astrocytes had no significant effect on the transcriptome of neurons. However 402 caution is warranted when interpreting this data since less than 1% of our total cell population 403 subsists of neurons (Supplementary Table 1). 404 Our single cell dissociation protocol shows a significant enrichment of glial cells and a 405 relative depletion of neurons. Possible explanations for this glial enrichment are (1) the fact that 406 we dissociated brain tissue of four months-old mice. Previous studies mostly perform scRNA 407 seq in younger mice while single nucleus RNA sequencing is more common in adult mice 408 hypothesize that ice-cold dissociation was not capable of breaking neuronal connections, while 419 the connections formed by glial cells were easier to break. 420 In summary, our data show for the first time the effect of long-term activation of 421 astrocytes with Gq-DREADD. We have shown that prolonged activation of astrocytes change 422 the transcriptional profile of astrocytes and microglia in the brain, while there is little to no effect 423 on neurons. Our findings are also important for the interpretation of future studies using Gq-424 DREADD activation in astrocytes. University. The content is solely the responsibility of the authors and does not necessarily 450 represent the official views of the National Institutes of Health, National Science Foundation or 451 Eli Lilly and Company. Figure 1A uses an image from Servier Medical Art, Servier Co. 452  showing the six clusters identified after re-clustering the mixed glia II cluster (7,905 cells). The 480

Figure legends
UMAP is split into two groups, the saccharin group (5,504 cells) and the CNO group (2,401 481 cells). 482