Potential neuronal IL-1R1 expression throughout the brain

First, we determined the general expression patterns of potential neuronal IL-1R1 in the brain of adult male mice. The Il1r1 reporter mouse (Il1r1GR/GR) co-expresses Il1r1 mRNA and tdTomato [34] and was previously used to identify Il1r1 expressing cell types in the brain [33]. In regard to potential neuronal IL-1R1 (nIL-1R1) expression, nIL-1R1 was found to be highly expressed in the dentate gyrus (DG) of the hippocampus. Beyond the hippocampus, we identified and annotated potential Il1r1 expressing nuclei throughout the entire brain of the Il1r1GR/GR mice. An anti-RFP antibody, which is cross-reactive to endogenous tdTomato, was used to amplify the tdTomato signal that is co-expressed with Il1r1 mRNA and was pseudo-colored green for visualization purposes. Sagittal sections of Il1r1GR/GR brains reveal distinct neuronal populations which express nIl1r1 (Fig. 1A). White boxes indicate regions where nIl1r1 was annotated, such as the dorsal raphe nucleus (DRN), dorsal tenia tecta (DTT), external plexiform layer of the olfactory bulb (EP), dentate gyrus (DG), and cerebellum (Fig. 1A, below). Further annotation of nIl1r1-expressing regions throughout the brain was performed using the QUINT pathway [62] and the Allen Brain Atlas (Allen Reference Atlas – Mouse Brain [brain atlas]. Available from atlas.brain-map.org). Linear and non-linear registrations of coronal sections were performed on tilescan images of tdTomato immunofluorescent labeling in Il1r1GR/GR mice, revealing additional nIl1r1 expressing nuclei (Fig. 1B). White dashed boxes indicate regions with prominent Il1r1 expression; for example, the auditory cortex region (AUD), granule layer of the dorsal DG (dDG), ventral DG (vDG), dorsal endopiriform nucleus (EPd), the amygdalohippocampal area (AHi ) and cornu ammonis field 3 strata oriens (CA3so) but not the CA3 strata lacunosum-moleculare, radiatum, lucidum, or pyramidale (CA3slm, ra, sp, luc, py) (Fig. 1B, below). Table 1 lists all regions of the brain in which potential nIL-1R1-expressing nuclei were detected, ordered from anterior to posterior. Annotated coronal brain sections from Il1r1GR/GR mice following non-linear registration were used to generate Table 1.

Fig. 1

Representative images of neuronal IL-1R1 expression in the brain. Representative tilescan images of tdTomato labeling of a sagittal (A) and coronal (B) section from an Il1r1GR/GR mouse. Areas where high levels of neuronal IL-1R1 were found are marked with dashed squares and shown at higher magnification below (A) or to the right of the low-magnification image (B). Scale bar: 500 μm. DRN, dorsal raphe nucleus; DTT, dorsal tenia tecta; EP, endopiriform nucleus; dDG, dorsal dentate gyrus; vDG, ventral dentate gyrus; AUD, auditory cortex; CA3, Cornus ammonus 3; AHi, hippocampal-amygdalar transition area

Due to the tdTomato reporter being under the control of endogenous Il1r1 promoters, some of the neuronal tdTomato expression was weak and could be misconstrued as background if not compared with a negative control. To prevent errors in our reporting, we confirmed that the nIl1r1 expression was not an artifact of our genetic mouse model or labeling methods by restricting IL-1R1 expression to endothelial cells with the Tie2-Cre promoter in our Il1r1r/r mouse line. Analysis of the whole brains of Tie2-Cre-Il1r1r/r mice show endothelial tdTomato expression is present throughout the entire brain. However, reported regions of nIl1r1 expression observed in Il1r1GR/GR were not present in the Tie2-Cre-Il1r1r/r sections such as the Pir, SSp, and DTT (Supplementary Fig. 1A-B).

Table 1 Annotation of all nIl1r1-expressing nuclei in the Il1r1GR/GR mouse brain

As proof of concept that IL-1R1 is typically expressed in specific sub-populations of neurons and can be selectively restored, we injected an adeno-associated virus co-expressing Cre and GFP (AAV2-Cre-GFP) into the major IL-1R1 expressing regions of the brain, the DG and DRN, of the Il1r1r/r mice. Following unilateral injection of AAV2-Cre-GFP, expression of IL-1R1 can be restored on one half of the DG as indicated by tdTomato fluorescence (Supplementary Fig. 1C, left side of image) and was absent in the non-injected contralateral side (right side of image). Furthermore, this targeted Cre expression in the DG or DRN restored IL-1R1 expression as indicated by tdTomato expression in those neurons (Supplemental Fig. 1D and E). However, not all GFP expressing neurons were tdTomato+, indicating that not all neurons which express Cre co-express nIL-1R1. Therefore, these data indicate that only neurons which typically use endogenous Il1r1 promoters in these discrete brain regions have the ability to express IL-1R1 and that viral-mediated nIL-1R1 restoration strategies can be implemented to decipher the function of nIL-1R1 in various brain regions. To gain an understanding of the specificity of the neuronal Il1r1-expressing circuits we analyzed the nIl1r1 distribution data from two perspectives: (1) functional neuroanatomy and (2) neurotransmitter-usage.

nIL-1R1 is primarily expressed on sensory and emotional processing circuits

All nIl1r1-expressing brain regions (n = 46) identified within the Il1r1GR/GR mice were classified as one of the following categories based on functional neuroanatomy: (1) sensory detection, relay, and processing; (2) emotional regulation, (3) spatial and cognitive Processes, (4) neuroendocrine response and (5) miscellaneous. Based on total nIl1r1 nuclei, 50% were related to sensory detection, relay, and processing; 26% were related to emotional regulation; 8.7% were related to spatial and cognitive processes, 5.5% were related to neuroendocrine responses, and 9% were determined miscellaneous (Supplementary Fig. 1F). These data point towards the sensory processing and emotional regulation nuclei as the key brain regions in which nIl1r1 can modify.

nIl1r1 is expressed in various, but limited, neurotransmitter systems

We next aimed to determine whether particular neurotransmitter systems express nIl1r1. To confirm the observed potential nIl1r1 expression are indeed neurons in specific neurotransmitter systems we (1) co-localized Il1r1 (tdTomato) and various neurotransmitter markers in the Il1r1GR/GR mice and (2) restored IL-1R1 expression with specific neurotransmitter-related Cre expression. In the dentate gyrus and basolateral amygdala of the Il1r1GR/GR mice, immunofluorescent labeling of tdTomato and vesicular glutamate transporter 2, Vglut2, is colocalized (Fig. 2A and Supplementary Fig. 2A, respectively). Restoration of IL-1R1 expression to excitatory forebrain neurons, using Camk2a-Cre-Il1r1r/r mice, revealed neuronal tdTomato expression in regions such as the Pir, DG, and AUD in the absence of endothelial Il1r1 (Fig. 2B). These data establish the nIl1r1 is primarily expressed in glutamatergic neurons.

Fig. 2

Neuronal IL-1R1 is expressed in diverse types of neurons classified by different neurotransmitters. (A) Representative images of tdTomato and Vglut2 labeling in Il1r1GR/GR hippocampal sections. Dashed squares mark the area shown at higher magnification on the bottom right. (B) Representative images of tdTomato from different CamK2a-Cre-Il1r1r/r brain sections. Pir, Piriform cortex; DG, dentate gyrus; AUD, primary auditory cortex. (C) Representative images of tdTomato and Tph2 labeling in Il1r1GR/GR dorsal raphe sections. A dashed square marks the area shown at higher magnification on the bottom right. DRN, dorsal raphe nucleus. (D) Representative images of tdTomato and Tph2 labeling in ePet1-Cre-Il1r1r/r dorsal raphe sections. Dashed squares mark the area shown at higher magnification on the bottom right. (E) Representative images of tdTomato from different Gad2-Cre-Il1r1r/r brain sections, white dotted lines denote granule cell layer of the DG. DG, Dentate gyrus, VHC, ventral hippocampus, CTX, cortex. Scale bar: 200 μm

IL-1 is known to signal within serotonergic neurons [2][67], therefore we examined whether IL-1R1 was expressed in serotonergic neurons. Specifically, the dorsal raphe nucleus (DRN) is known as the primary serotonergic nuclei of the brain and can respond to IL-1 stimulation. To confirm IL-1R1 is expressed in serotonergic neurons, Il1r1GR/GR coronal brain sections containing the DRN were immunofluorescently co-labeled for tdTomato and tryptophan hydroxylase 2 (Tph2), the rate limiting enzyme in serotonin production. Most, but not all, neurons of the DRN were both Tph2+ and tdTomato+ (Fig. 2C). Restricting IL-1R1 expression to serotonergic neurons using ePet1-Cre-Il1r1r/r mice, DRN neurons were found to co-express tdTomato and Tph2 via immunofluorescent labeling. Other serotonergic brain regions such as the raphe pallidus nucleus (RPa) and raphe nucleus magnus (RMG) also show colocalization of both tdTomato and Tph2 (Supplementary Fig. 2C); however, these regions had fewer Il1r1+ cells compared to the DRN. These data indicate that subsets of serotonergic neurons express nIl1r1.

Other neurotransmitter systems which were thought to be influenced directly by IL-1 are cholinergic [59], dopaminergic [38], and GABAergic neurons [3, 4, 68]. Colocalization of choline acetyltransferase (ChAT) with tdTomato in the horizontal limb of diagonal band (HDB) was not observed (Supplementary Fig. 2B). Further, in dopaminergic nuclei of the brain, the DRN, ventral tegmental area (VTA), substantia nigra (SN), colocalization of tyrosine hydroxylase (TH) and tdTomato expressing neurons was not found (Supplementary Fig. 2D-G). After restoring IL-1R1 expression to only GABAergic neurons, by crossing a Gad2-Cre with the Il1r1r/r mouse, tdTomato expression throughout the brain was sparse but identifiable. tdTomato+ GABA-ergic neurons were identified in the DG hilus, but not the granule cell layer, ventral hippocampus (VHC), and throughout the cortex (CTX, Fig. 2E). In Gad2-Cre-Il1r1r/r mice, qPCR of Il1r1 mRNA expression in the hippocampus show detectable but significantly lower levels of Il1r1 mRNA compared to WT controls (Supplementary Fig. 2H). This data shows IL-1R1 is not expressed in acetylcholine or dopaminergic neurons; however, IL-1R1 is sparsely expressed in subsets of GABAergic neurons.

Confirmation of neuronal IL-1R1 expression from scRNAseq databases

As an unbiased confirmation of nIl1r1 distribution, we analyzed publicly available single cell RNA sequencing (scRNAseq) of various brain regions in mice [50]. The predominate cellular expression of Il1r1 in most brain regions were endothelial and choroid plexus cells, however, astrocyte and mural cell Il1r1 expression was detected (Supplementary Fig. 3A). Out of all the cells analyzed, neuronal Il1r1 was detected throughout the brain in the hippocampus (2%), followed by the thalamus (0.5%), then frontal cortex (0.3%). These subsets of neurons were then classified by their neurotransmitter-related gene expression. Within the hippocampus, frontal and posterior cortex, and thalamus, Il1r1 expressing cells were predominately glutamatergic (Supplementary Fig. 3B, grey). Other regions show sparse GABAergic expression of Il1r1 such as the entoperduncular region, globus pallidus and substantia nigra (Supplementary Fig. 3B, orange). scRNAseq data from the mouse DRN conducted by Huang et al. was analyzed for Il1r1 expression patterns [26]. Il1r1 expressing cells were primarily vascular cells and astrocytes. Of all the total cells analyzed, only 1.5% of the cells expressed detectable transcripts of Il1r1. Of all the neurons isolated, 7% expressed Il1r1 mRNA and a majority (86%) of the Il1r1+ neurons were serotonergic. The DRN is comprised of a variety of serotonergic neurons subtypes and these subtypes are known to project to different brain regions, which could inform how IL-1 can alter neurocircuitry and behavior. Of the serotonergic neurons, Il1r1 expression is highest in 5-HT subtype II (7%, orange), followed by 5-HT subtype IV (6%, yellow), 5-HT subtype I (2%, blue), and then 5-HT subtype III (1%). Il1r1 expression was not identified in 5-HT subtype V (light blue) (Supplementary Fig. 3C). scRNAseq from human prefrontal cortex (PFC) and hippocampus (HC) confirm Il1r1 expression is indeed expressed in human excitatory neurons of the PFC and dentate gyrus (Supplementary Fig. 3D); however, one of the major differences in humans is that Il1r1 is expressed in inhibitory neurons of the PFC suggesting species-specific Il1r1 expression [24].

Detailed mapping of glutamatergic neuronal IL-1R1 expression throughout the brain

Our immunofluorescence and scRNAseq analysis of multiple brain regions strongly suggests that nIL-1R1 is primarily expressed on glutamatergic neurons. To verify and detail which of the brain nuclei express nIL-1R1 in glutamatergic neurons, Vglut2-Cre-Il1r1r/r mice were used to restore IL-1R1 only in glutamatergic neurons. Immunohistochemical labeling of endogenous tdTomato in sections of Vglut2-Cre-Il1r1r/r mice is required as tdTomato expression in neurons is weak (Supplemental Fig. 7G). QUINT pathway analysis was used to annotate glutamatergic nIL-1R1 expression throughout the brain. Without confounding non-neuronal IL-1R1 expression and using higher contrast IHC techniques, glutamatergic nIL-1R1 was detected in 49 individual brain regions. Glutamatergic nIL-1R1 was detected in many anterior brain regions such as the olfactory regions of the anterior olfactory nucleus (AON) and piriform cortex (PIR) along with other motor regions of the primary motor cortex (MOp) (Fig. 3A). Other IL-1R1 expressing regions that were detected in the Il1r1GR/GR mice were not found to express tdTomato in Vglut2-Cre-Il1r1r/r such as the dorsal raphe nucleus (DRN), lateral septum (LS), fimbria (fi) and central nucleus of the raphe (CS) (Supplementary Fig. 4A-D). These regions are well known to be comprised of serotonergic or GABAergic neurons. nIl1r1 expression in the PIR extends from anterior (+ 2.3 mm from bregma) to posterior (-0.5 mm from bregma) (Supplementary Fig. 4E). More posteriorly, glutamatergic nIl1r1 was found to be expressed in regions of the somatosensory cortex (SSp, SSs, VISC) but not in the adjacent motor cortex (Fig. 3B). More apparent labeling of thalamic Il1r1 expression was detected in the Vglut2-Cre-Il1r1r/r sections. Nuclei within the thalamus were detected to have clear cell bodies in the anterior dorsal nucleus (AD), medial dorsal (MD), and paraventricular nucleus of the thalamus (PVT) (Fig. 3C). In the PVT, glutamatergic neuronal tdTomato expression was found be highly expressed in the anterior (-0.25 mm relative to bregma) and posterior (-1.5 mm relative to bregma) PVT, whereas the medial (-0.5–1.0 mm relative to bregma) PVT shows fewer tdTomato + cells (Supplemental Fig. 4F). This suggests that some nuclei can have differential expression of nIl1r1 in subregions of a nuclei. Other glutamatergic nIl1r1-expressing regions identified in the Il1r1GR/GR mice, such as the dentate gyrus (DG), claustrum (CLA) and basomedial amygdala (BMA) were verified in the Vglut2-Cre-Il1r1r/r (Fig. 3D).

Fig. 3

Representative images of IL-1R1 expression on glutamatergic neurons in different brain sections. (A) Representative images of tdTomato immunolabeling in Vglut2-Cre-Il1r1r/r mice, highlighting the cortex and olfactory areas. MOp, primary motor area; ORBvl, orbital area, ventrolateral part; Alv, agranular insular area, ventral part; PIR, piriform area; AON, anterior olfactory nucleus. The dashed squares indicate areas with high neuronal IL-1R1 expression and are magnified. Scale bar = 200 μm (B) Representative images of tdTomato immunolabeling in the Vglut2-Cre-Il1r1r/r somatosensory areas, visceral areas, and piriform area. SSp, primary somatosensory area; SSs, supplementary somatosensory area; VISC, visceral areas; PIR, piriform area. Scale bar: 200 μm (C) Representative images of DAB labeling in various thalamic nuclei of the Vglut2-Cre-Il1r1r/r mouse. AD, anterodorsal nucleus; MD, mediodorsal nucleus of thalamus; PVT, paraventricular nucleus of thalamus; AM, anteromedial nucleus. Scale bar: 200 μm (D) Representative images of tdTomato immunolabeling in Vglut2-Cre-Il1r1r/r mouse dentate gyrus (DG), central lateral nucleus of the thalamus (CL), and basomedial amygdalar nucleus (BMA). Scale bar = 200 μm

Fig. 4

IL-1R1 protein expression is localized across the entire neuron of the dentate gyrus. (A) Representative tilescan image of immunofluorescent labeling of HA-tag in the hippocampus of glyoxal-fixed Vglut2-Cre-Il1r1r/r mice. (B-C) 20x micrograph of immunofluorescent labeling in the DG of DAPI, tdTomato, and HA-tag in Il1r1r/r (B) or Vglut2-Cre-Il1r1r/r (C) mice. (D) 20x micrograph of immunofluorescent labeling in the CA3 of DAPI, tdTomato, and HA-tag in Vglut2-Cre-Il1r1r/r (D) mice

Similar to the analysis conducted in Il1r1GR/GR mice, functional neuroanatomical analysis was conducted in the Vglut2-Cre-Il1r1r/rmice. Brain regions were categorized as (1) sensory detection, relay, and processing; (2) emotional Regulation, (3) spatial and cognitive processes, (4) neuroendocrine response and (5) miscellaneous. Based on all total nIl1r1+ nuclei in Vglut-Cre-Il1r1r/r mice, 49% were related to sensory detection, relay, and processing; 20% were related to emotional regulation; 12% were related to spatial and cognitive processes, 8% were related to neuroendocrine responses, and 10% were determined as miscellaneous (Supplementary Fig. 1G). A fully annotated list containing all glutamatergic nIl1r1 expression is provided in Table 2.

Protein expression of IL-1R1 in glutamatergic neurons

We next sought to determine the IL-1R1 protein expression patterns in brain regions with high nIl1r1 expression. Immunohistochemical labeling of IL-1R1 has been difficult in previous studies. Using the Vglut-Cre-Il1r1r/r mice where Il1r1 mRNA is tracked via cytosolic tdTomato expression and a 3xHA tag is fused to the intracellular region of the IL-1R1 protein, we have successfully labeled IL-1R1 protein via the HA tag in DG neurons using glyoxal-based fixatives (Fig. 4A). In an IL-1R1 knockout (Cre negative Il1r1r/r littermates) no tdTomato (Il1r1 mRNA) or HA tag (IL-1R1 protein) was detected (Fig. 4B). In the Vglut2-Cre-Il1r1r/r mice concordant tdtomato (red) and HA tag (cyan) was detected in the DG neurons (Fig. 4C). TdTomato filled the entire cellular processes of DG neurons (Fig. 4C, red) whereas HA tag was localized on the cellular membrane (Fig. 4C, cyan). Mossy fiber terminals from the DG and projecting to the CA2/3, express both tdTomato and HA tag, however, no IL-1R1 expression was found in the CA2/3 pyramidal cells (Fig. 4D). These data show, nIL-1R1 protein is concordant with Il1r1 mRNA expression and is expressed throughout the entire dentate gyrus neuron.

IL-1R1 expression does not alter gross neuronal structure or excitatory synaptic density in the dentate gyrus

Since IL-1R1 is expressed by most, if not all, dentate gyrus neurons we examined whether the presence of nIL-1R1 can alter the structural anatomy of DG neurons. To address this, we used Golgi staining (FDNeuroTech) to measure neuron complexity and synaptic spine density in male 12wk old WT (n = 4) and Il1r1r/r (n = 3) mice. Individual Golgi-stained neurons within the dentate gyrus were imaged and then traced using Neurolucida360 (MBF Neuroscience). Branch order analysis of these neurons was conducted to analyze the dendritic complexity. Segments of secondary dendrites from each neuron were analyzed for spine density. There were no differences in branch order (Supplementary Fig. 5A-E) or synaptic spine density (Supplementary Fig. 5F-G) between WT and Il1r1r/r mice in the dentate gyrus. These data suggest that global elimination of IL-1R1 does not lead to altered dendritic or synaptic structure in the dentate gyrus of adult male mice.

IL-1R1 expressing neurons do not utilize canonical NFκB signaling pathways

Although nIL-1R1 is distributed throughout the brain in discrete glutamatergic neurons, it is unknown whether glutamatergic nIL-1R1 would respond to an IL-1β stimulus like that of other IL-1R1 expressing cells (e.g. endothelial). Typically, in non-neuronal cells, IL-1/IL-1R1 signaling induces the canonical NFκB pathway activation, however, in neurons the signaling pathways are disputed. To determine whether IL-1R1 expressing neurons initiate canonical NFκB signaling following IL-1 stimulation, we crossed the NFκB-GFP reporter mouse to our global IL-1R1 reporter (Il1r1GR/GR) to generate a double reporter mouse (Il1r1GR/GRNFκBGFP). NFκB expression was examined throughout the brain at 2, 4, 8, and 24 h following intracerebroventricular IL-1 (20 ng) or PBS administration via immunofluorescent labeling of GFP and tdTomato. The timepoint where NFκB-GFP was first detected, by a given cell type was reported. In the PBS sham surgeries, no NFκB-GFP was detected at any timepoint, whereas tdTomato was, as previously described, detected in DG and DRN neurons, endothelial cells, and ependymal cells (Supplementary Fig. 6B-D). Following i.c.v. IL-1β, NFκB-GFP was detected in the ependymal cells lining the 3rd ventricle at 2 h (Supplementary Fig. 6E), in infiltrating leukocytes near blood vessels at 4 h (Supplementary Fig. 6F), in choroid plexus epithelial cells at 8 h (Supplementary Fig. 6G) and in brain vasculature at 24 h post injection (Fig. 5A-B). No GFP expression was detected in Il1r1 expressing neurons of the DG or DRN across all timepoints; however, GFP was detected in the hippocampal and DRN endothelial cells at 24 h indicating IL-1β reached these nIL-1R1 expressing regions (Fig. 5A-B). This indicates that neuronal IL-1R1 does not utilize the canonical NFκB signaling pathway.

Direct stimulation of nIL-1R1 in Vglut2-Cre-Il1r1r/r mice does not lead to acute downstream gene expression

Transcriptional pathways in nIL-1R1-expressing neurons related to direct IL-1 stimulus have yet to be determined. To determine which signaling pathways nIL-1R1 may use, Vglut2-Cre-Il1r1r/r mice were injected i.c.v. with PBS or IL-1β (20 ng), 3 h later the DG was microdissected, RNA was isolated, and RNA sequencing of the tissue was performed. Gene expression was analyzed using the DESeq2 Bioconductor package in R as previously reported [14, 69]. The threshold of a log2Fold Change > 1 was set as a biologically significant gene. Of the top differentially expressed genes compared with PBS controls, none reached our criteria (Fig. 5C). However, some genes reached statistical significance below a Log2Fold change of 1: 1500015A07Rik, Hddc3, and Ubc were downregulated genes and Pdp1, Hmgn2, Ppig, Trove2, Hrh3, Rpph1, Pter, Mir6236, Sox 11, Slc17a6 were marginally upregulated genes. Following GO pathway analysis, these genes did not associate with any specific cellular, molecular, or biological pathways. At this acute timepoint, we conclude, stimulation of nIL-1R1 with IL-1β does not change transcriptional activity.

Next, we performed an exploratory study to determine which genes and pathways are influenced by the restoration of DG nIL-1R1 for an extended period of time. Bilateral AAV injections were performed in Il1r1r/r mice in which AAV-Cre-GFP was injected into the right DG (to restore IL-1R1 expression in neurons) and AAV-GFP vector control was injected into the left DG. Injected mice were allowed for nIL-1R1 expression to occur for 1 month (n = 3). To verify that AAV-Cre would indeed restore IL-1R1 protein to only one hemisphere of the brain, triple immunofluorescent labeling for GFP, tdTomato, and HA tag was conducted. GFP, indicating successful viral transfection, was detected in DG neurons on both sides of the hippocampus, whereas, tdTomato and HA tag were detected only on the AAV-Cre, but not in the AAV-GFP, side (Supplemental Fig. 7E-F). AAV-injected brains were collected and GeoMx spatial transcriptomics (Nanostring) was performed to isolate GFP+NeuN+ neurons from either the right and left hippocampus (Fig. 5D-E), respectively, in order to identify nIL-1R1-associated genes and pathways following restoration of IL-1R1. Volcano plot of gene expression revealed genes associated with AAV-CRE (i.e. the Il1r1 expressing DG) contained complement (C1ra, C1qa) and immune-related genes (ADAM18, Cd300a) (Fig. 5F). GO pathway analysis of all genes above a -Log10pvalue of 1.3 (p < 0.05) that were increased with the AAV-CRE injected DG showed no enrichment for any specific GO pathway. However, GO pathway analyses (molecular function, cellular components, and biological processes) identified significantly enriched pathways in the AAV-GFP injected DG (i.e. the Il1r1-null DG). These include RNA and mRNA binding pathways (Fig. 5G, red bars), synaptic regulation pathways (Fig. 5H, red bars) and synaptic transmission, formation, and adhesion pathways (Fig. 5I, red). Thus, while IL-1β did not induce acute gene expression in the DG neurons, the observed transcriptional effects from long term nIL-1R1 restoration indicates nIL-1R1, nonetheless, modulates neuronal gene expression over time.

Fig. 5

Neuronal IL-1R1-mediated signaling is independent of NF-κB pathway and chronic restoration of DG neuronal IL-1R1 reveals negative regulation of synaptic-related pathways. (A-B) Representative images of GFP and tdTomato labeling in Il1r1GR/GRNFκBGFP DG (A) and DRN (B) sections. The dashed square marks the area shown at higher magnification on the top right. Dashed lines mark the dentate gyrus granule cell layer. DG, dentate gyrus; DRN, dorsal raphe nucleus. Scale bar = 200 μm. (C) Log2 fold change of top 13 differentially expressed genes of microdissected dentate gyrus following i.c.v. IL-1 (20 ng) compared to PBS. (D-E) Immunofluorescent labeling of NeuN (Red), GFP (Green), and DAPI (Blue) in Il1r1r/r mice injected with AAV-CMV-CRE-GFP into the right dentate (D) and AAV-CMV-GFP injected into the left dentate (E). (F) Volcano plot of differentially expressed genes between AAV-Cre versus AAV-GFP injected dentate gyrus neurons within Il1r1r/r mice (n = 3). Vertical dotted lines indicate a log2 value of 1 and horizontal dotted lines indicate a -Log10 p value of 1.3. Red data points were used in GO pathway enrichment analysis in G-I. (G-I) GO Molecular function (G), Cellular component (H), and Biological Process (I) pathway analysis of differentially expressed genes in the AAV-CMV-GFP injected Il1r1r/r DG neurons

Dentate gyrus neurons utilize a unique splice variant of the IL-1R1 accessory protein (IL-1RAcP)

Due to the lack of acute neuronal transcriptional activity, following direct IL-1β stimulation, alternative pathways for IL-1R1 signaling were hypothesized. Typically, the IL-1R1 signaling cascade requires an accessory protein (AcP), named IL-1RAcP, to initiate intracellular signaling. In the central nervous system, neurons use a CNS-restricted isoform, called IL-1RAcPb, that blocks canonical MyD88 binding and can possibly signal though other transcription factors [27, 53] or act as trans-synaptic adhesion molecules [61, 65]. It is unclear whether IL-1RAcPb is expressed in the same regions which express nIL-1R1 and whether neurons solely express IL-1RAcP variant 1 (AcP, exon 10–11), IL-1RAcPb variant 2 (AcPb, exon 10–12), or a combination of the two isoforms. IL-1RAcP and IL-1RAcPb gene fragments were quantified from bulk RNA sequencing data throughout various brain regions such as the cerebellum, prelimbic cortex, hippocampus, midbrain, and isolated dentate gyrus granule cells. The Sashimi plot reports the frequency of exonal usage and connection and mapped exonal usage is reported in Fig. 6A. The ratio of AcPb: AcP expression by brain region was analyzed. Within the cerebellum and midbrain, AcPb: AcP ratio was near 1, suggesting an even distribution pattern. However, in other regions such as the prelimbic cortex and whole hippocampus, the ratio of AcPb: AcP increased 2–4 times indicating that the primary expressed isoform is the alternative spliced AcPb. Isolated dentate gyrus granule cells show high specificity for only AcPb, as the AcPb: AcP ratio was near 12 (Fig. 6B). Confirmation of this expression pattern was conducted with Basescope in situ hybridization (ACDBio). Probes were generated that selectively bind to either the exon junction between exon 10 and exon 11 which encodes AcP or between exon 10 and 12 which encodes AcPb. Puncta of AcP (Blue-Green) and AcPb (Red) mRNA were detected in brain sections of the hippocampus (Fig. 6C). AcPb dominant regions reside in the neuronal layers of the CA1 (Fig. 6D), CA3 (Fig. 6E), and strongest expression in the DG (Fig. 6F). AcP puncta were detected sparsely throughout the CA3 region but not DG or CA1 cell layers. AcP-dominant expression was identified in the corpus callosum (CC, Fig. 6G) and choroid plexus (CP, Fig. 6H). AcPb KO mice (Amgen) were used to validate specificity of the probes and determine whether the expression pattern of AcP changes when AcPb is eliminated. AcPb KO mice showed complete elimination of AcPb mRNA expression in the DG, CA1 and CA3 neurons (Supplementary Fig. 7A-C) while AcP was still detected in the CC and ChPlx (data not shown). All samples were run in tandem with positive and negative control probes (Supplementary Fig. 7D). Interestingly, elimination of AcPb did not permit AcP expression in the dentate, suggesting restricted AcPb expression in neurons.

Fig. 6

IL-1R1 expressing neurons utilize alternatively spliced accessory protein, IL-1RAcPb. (A) Sashimi plots from RNAseq data analyses of different brain areas and schematics showing the last two exons of Il1rap. (D) Quantification of AcPb/AcP RNA transcripts ratio in different brain areas. Dashed line separates two different RNAseq experiments. Error bars represent the mean ± SEM. (C) Photomicrograph of the WT hippocampus following BaseScope in situ hybridization of exon junction regions for IL-1RAcP (Blue-Green) and IL-1RAcPb (Red). Scale bar = 300 μm Dotted lines indicate magnified regions presented in D-G. (D-F) Photomicrographs of predominantly IL-1RAcPb expression regions, such as the CA1 (D), CA3 (E), and DG (F), which are magnified images to the right of each corresponding image. (G-H) Photomicrographs of predominantly IL-1RAcP expressing regions of the corpus callosum (G) and the choroid plexus (H), which are magnified images to the right of each corresponding image. DG, dentate gyrus; CC, corpus callosum; CP, choroid plexus. Scale bar = 100 μm