Memory Impairment Related to NLRP3 In ammasome Activation and TRPC5 Decrease in Hippocampal Excitatory Synapses in Microglia Knockout IL-10 Mice


 Members of the transient receptor potential canonical (TRPC) protein family are widely distributed in the hippocampus of mammals and exert respective and cooperative influences on the functions of neurons. The relationship between specific TRPC subtypes and neuroinflammation is receiving increasing attention. Here, using Cx3cr1CreER IL-10−/−transgenic mice and their littermates, we demonstrated that Cx3cr1CreER IL-10−/− mice displayed spatial memory deficits in object location recognition (OLR) and Morris water maze (MWM) tasks. The decreased levels of TRPC4 and TRPC5 in the hippocampal regions were verified via reverse transcription polymerase chain reaction, western blotting, and immunofluorescence tests. The expression of postsynaptic density protein 95 (PSD95) and synaptophysin in the hippocampus decreased with an imbalance in the local inflammatory environment in the hippocampus. The number of cells positive for ionized calcium binding adaptor molecule 1 (Iba1), a glial fibrillary acidic protein (GFAP), increased with the high expression of interleukin 6 (IL-6) in Cx3cr1CreER IL-10−/− mice. The nod-like receptor protein 3 (NLRP3) inflammasome was also involved in this process, and the cytokines IL-1β and IL-18 activated by NLRP3 were also elevated by western blotting. The colocalization of TRPC5 and calmodulin-dependent protein kinase IIα (CaMKIIα) significantly decreased TRPC5 expression in excitatory neurons. AAV9-CaMKIIα-TRPC5 was used to upregulate TRPC5 in excitatory neurons in the hippocampus. The results showed that the upregulation of TRPC5 improved the memory performance of Cx3cr1CreER IL-10−/− mice by inhibiting NLRP3 inflammasome-associated inflammatory activity.


Introduction
The TRPC protein family has seven members (TRPC1 to TRPC7) that form homo-and/or heteromorphic tetramers. TRPC is a non-selective cationic membrane channel with Ca 2+ permeability. According to sequence homology, the family can be divided into three subfamilies: TRPC1, C4, and C5; TRPC3, C6, and C7; and TRPC2, which is a pseudogene in humans [1].
The physiological and pathological functions of TRPC, particularly in the central nervous system, are of increasing concern due to the extensive localization of TRPC, especially in the hippocampus. Evidence is converging to show the involvement of TRPC channels in cognitive functions, since multiple TRPC subtypes are highly expressed in the hippocampus. TRPC1 is indispensable for environmental enrichment -induced spatial memory enhancement, which is related to long-term potentiation (LTP) induction and hippocampal neurogenesis [2]. The function of the TRPC1/4/5 subfamily plays a key role in spatial working memory formation [3], since TRPC1/4/5 −/− mice exhibited de ciencies in adapting to a new challenge in a relearning task.
Neuroin ammation is a complex response to brain injury and a major contributor to progressive neuronal damage. IL-10 is a major anti-in ammatory cytokine that maintains the balance of the immune response and is an important molecule in the modulation of neuronal homeostasis and cell survival. At the level of the hippocampus, it has been shown that IL-10 plays a key role in improving the learning and memory ability of animals under physiological and pathological conditions [4]. IL-10 helped to improve spatial memory performance in Sprague-Dawley rats treated with Escherichia coli [5]. Increased IL-10 levels played a role in the process of enriched environment alleviated LPS-induced spatial learning and memory impairment [6]. IL-10 is an important molecule in the modulation of learning and memory dysfunction, as shown by the IL-10 tm1/tm1 mice that exhibited behavioral de cits in the MWM test [7].
Microglial cells are the most investigated innate immune cells in the brain and are the main cytokine producers, including IL-10. Many studies have con rmed that IL-10 secreted by microglial cells plays a key role in the pathological process of neuroin ammation [8,9].
In our study, we focused on the effects of IL-10 induced from microglial cells on animal cognitive and behavioral abilities to explore the role of TRPC in the hippocampus during this process.

Animals.
All experiments were performed on male mice that were 8-10 weeks old and weighed 22-26 g. All animals were on a C57BL/6 background and were maintained in a reversed 12-h light-dark cycle with free access to food and water. Tamoxifen (Sigma-Aldrich, MO, USA) was administered as a solution in corn oil (20 mg/mL) by intraperitoneal injection (80 μL per mouse). All protocols were approved by the Ethics Novel Object Recognition (NOR) task and OLR task Each mouse was gently handled for 3 min every day for three consecutive days before the behavioral test. The test was conducted in a bare square box (48 cm long, 48 cm wide, 36 cm high) made of compressed wood. Brie y, the NOR and OLR tasks consisted of two sessions: the training phase and the test phase. In the NOR task, during the test period, the mice were placed in the empty box and allowed to explore freely for 5 min to adapt to the environment. Two identical objects (plastic boxes, 4-5 cm high) were arranged in a straight line along one side of the wall, 8 cm from the sides. The experimental mice were placed in the box facing the opposite wall and were free to explore and adapt for 5 min. After a 2hour interval, the animals were reintroduced into the experimental box for free exploration during the experimental period. At this point, one of the two objects used during training was replaced by an object of similar size. If any of the mice pointed or touched the new object with its nose within 1 cm, it was considered exploratory. The objects were thoroughly cleaned between trials to avoid olfactory cues. The mice were tracked using a charge-coupled device camera connected to a personal computer (Ethovision 2.0, Noldus, Wagenigeen, Netherlands).
In the OLR task, the experimental procedure was similar to the NOR task in the training stage. The difference was that in the test stage, the two unchanged objects were placed diagonally, as shown in Figure 1.
During the training phase in both the NOR and OLR tasks, if the mouse total exploration time for the two objects was less than 10 s in 5 min, the data was eliminated in a later study. The discrimination index (DI) and object duration were used as the NOR and OLR task evaluation index. The DI is the percentage of time each mouse spent exploring new objects and positions. MWM task and RMWM task.
The mice swam freely in the pool without platform 90s on 1d before the test, so that they could got familiar with the maze environment. In the training stage, each mouse was placed into the water with their heads facing the wall, and randomly selected one of the four starting positions of east, west, south and north. Record the time it takes the animal to nd the underwater platform (escape latency). if the escape latency exceeded 60s, the mouse was guided to the platform and stayed on the platform for 10s.
Training was conducted once a day at each of the four entry points, and the results of the day were statistically analyzed using the mean value of the four escape latencies. The training lasted for four days.
The concealed platform was removed 24 h after the test. After the mice were placed in the water, their swimming trails for 60 s were recorded, and the residence time of the mice in the original platform quadrant and the times of platform crossover were statistically analyzed. The reversal phase started after the test, and the platform was moved to the opposite quadrant of the tank. The platform remained in this northwest quadrant location for all training trials on days 1, 2, and 3, but not for day 4 of the test trial (Figs. 2 and S2). The swimming activity of each mouse was automatically recorded using a video tracking system (Ethovision 2.0, Noldus, Wagenigeen, Netherlands).
Protein sample concentrations were measured using the Pierce BCA Protein Assay Kit (Biosharp, Hefei, China). Equal amounts of proteins (10 μg per lane) were run on 10% or 12% SDS-PAGE and then transferred to PVDF membranes by electroblotting (Bio-Rad, Hercules, CA, USA). The PVDF membranes were blocked with 5% skim milk powder (BD-Difco, USA) diluted with Tween/0.1M PBS (TBST) for one h at room temperature before incubation with the primary antibody.
The membranes were washed with TBST three times for 10 min after the primary antibodies were incubated at 4 °C overnight. The membranes were then incubated with secondary antibodies for 1 h at room temperature, washed three times for 10 min in TPBS, and reacted with chemiluminescent substrate (Biosharp, Hefei, China). The bands were obtained using an ECL luminescence imaging system (Tanon 5200, China). The densities of the target protein bands were measured using Image J and normalized to corresponding β-actin bands. Immunostaining.
Cx3cr1 CreER IL-10 -/-(n=3) and littermate (n=3) male mice were sacri ced and perfused with PBS (pH=7.4), followed by 4% paraformaldehyde in PBS. The samples were immersed in a xed solution overnight and then dehydrated in a gradient solution of 10%, 20%, and 30% sucrose in PBS. Each brain was embedded in OCT and coronal sections of 20μm thickness were prepared using a freezing microtome (Leica CM 1860, Germany). The slices were washed three times with PBS for 5min before staining. The tissue sections were permeabilized in 0.3% Triton X 100 for 30min and then blocked in 10% normal goat serum.
The blocked slices were incubated with the corresponding primary antibodies overnight at 4 °C. The samples were washed in PBS three times for 5 min, and then incubated with uorescent secondary antibodies for 1 h at room temperature. After being washed in PBS three times for 5min, the slices were incubated with DAPI (Beyotime, China, 1:5000). Representative images were obtained using a uorescence microscope (Olympus, BX53, Japan) or confocal microscopy (Olympus, FV1000, Japan).
After the mice were anesthetized (10% chloral hydrate, 3.5 mL/kg), their brains were xed to a stereotactic locator and the virus was injected into the bilateral middle regions of the hippocampus (0.5 μl/ hemisphere) using a 2 μl microsyringe according to the following stereotaxic coordinates referenced in mm from the bregma (AP=−2 mm; ML=±1.4 mm; DV=−1.5 mm). Following injection, the microsyringe was left in place for 5 min to prevent back ow of the solution. After surgery, the mice were single-housed for one week to recover well.
Data analysis.
All data were expressed as mean ±S.E.M. One-way or Two-way ANOVA were used for data analysis based on different experimental designs or data sets followed by multiple comparison test or by unpaired, twotailed t test. Values of P<0.05 were considered statistically signi cant.

Results
Microglial cell knockout (KO) IL-10 impaired the learning and memory ability of mice.
Before the experiment, all mice were detected by PCR and classi ed (Fig. S1a). Colocalization of microglial markers Iba1 and IL-10 was also examined after tamoxifen injection (Fig. S1b). To test the learning and memory abilities of mice, we tested NOR and OLR in our study. Figure 1a shows the experimental procedure. There was no difference between the two groups during NOR detection in both the training and test stages, not only in the discrimination index, but also in object duration time history detection (P>0.05, n=8, t test, Fig 1.b-d).
There was no difference between the two groups in NOR, but there was a difference in OLR, indicating that the two groups had differential sensitivity in spatial location recognition [10]. We further detected the learning and memory abilities of the two groups using MWM. The results showed that both the escape latency to the platform (F (1, 14) = 10.96, P<0.01, Two-way ANOVA, Fig.2 Fig.2.f). In the reversal MWM test, the KO mice showed a decreased ability in the learning stage, but not in the memory stage (Fig. S2).
Since spatial learning relies heavily on hippocampal activity, in addition to TRPC2, other TRPC subtypes were detected via RT-PCR since TRPC2 is not expressed in the hippocampus [11]. RT-PCR results showed that the mRNA levels of TRPC1,3,4 and 5 decreased in the hippocampus (Fig.3.a). Using western blot to detect the protein expression levels of these TRPC subtypes, also revealed that TRPC4 (t=3.130, P=0.020<0.05, n=4, t test) and TRPC5 (t=5.910, P=0.001<0.01, n=4, t test) were decreased in the hippocampus (Fig.3 b-f). Immuno uorescence results in the CA3 region further veri ed the results ( Fig.3.g).
Hippocampal synaptic proteins are one of the structural bases of spatial learning and memory. We tested the expression of glutamate receptors NR2A and NR2B, synaptic protein postsynaptic density protein 95 (PSD95), and synaptophysin in the hippocampus.  Figure 4f, which was consistent with that of western blotting. The uorescence intensity was reduced in both PSD95 and synaptophysin in the KO group.
Synaptophysin is an integral membrane protein of small synaptic vesicles and has been identi ed as a useful marker for synaptic density [12]. PSD95 is a protein localized to the postsynaptic density of synapses [13] and plays a key role in synapse stabilization and plasticity [14], suggesting that microgliaderived IL-10 may play a role in synaptic protein synthesis through underlying mechanisms, thus affecting the spatial learning and memory ability of Cx3cr1 CreER IL-10 -/mice.
To determine whether changes in IL-10 levels in microglia affect in ammation in vivo, the levels of glial marker Iba1 (for microglia) and GFAP (for astrocytes) were tested. Our results showed increased protein expression of GFAP (t=4.583, P=0.005<0.01, n=4, t test,) and Iba1(t=2.698, P=0.036<0.05, n=4, t test, Fig.5 a,b and c), which was consistent with enhanced immuno uorescence levels of Iba1 and GFAP-positive cells in the hippocampal CA3 region (Fig 5.i). The increased expressions of GFAP and Iba1 suggested that the behavioral changes in KO mice were involved in in ammation activation.
The balance between the central inhibitory and facilitatory systems may serve as a principal mechanism of memory activation and regulation [15]. TRPC is expressed in both the inhibitory and excitatory neurons of the CNS [16]. We explored how downregulation of TRPC in Cx3cr1 CreER IL-10 -/mice affects memory by affecting network excitation and inhibitory balance. Our results showed that gene KO did not affect the content of GAD67 (t=1.430, P=0.20>0.05, n=4, t test, Fig.6. a,b) but could enhance the content of Pv (t=13.53, P<0.0001, n=4, t test, Fig.6. a,c), which is a speci c subtype of gamma aminobutyric acid (GABA) interneurons that may subserve distinct behavioral functions and behavior-dependent network activities [17]. Inhibition of the GABAergic system has memory-facilitating effects, whereas stimulation produces memory impairment. These results suggest that the effect of TRPC4 or 5 downregulation on learning and memory in Cx3cr1 CreER IL-10 -/mice mainly acted on excitatory neurons. Immuno uorescence showed increased TRPC5 colocalization in the hippocampus with CaMKIIα (an excitatory neuron marker) (Fig.6. d).
CaMKIIα was used as the promoter of the AAV9 virus, speci cally upregulating TRPC5 in excitatory neurons ( Fig.7. b, c). After injection of the virus for four weeks, we performed MWM tests in all groups of mice. The results of MWM showed differences in escape latency to the platform (F (3, 16)=8.137, P<0.01) and average swimming distance F (3, 16)=13.00, P<0.001) during the learning phase. The KO mice treated with AAV-9 TRPC5 injection showed an improved behavioral performance compared to KO-GFP group (P<0.05, Fig.7d, e). In the training stage, the KO mice treated with TRPC5 exhibited alleviated memory damage in both platform crossover times (F (3, 16)=7.053, P<0.01, Fig.7. g) and in time spent in the target quadrant (F (3, 16)=12.81, P<0.001, Fig.7 h).

Discussion
IL-10 is a key cytokine that represses excessive in ammatory responses and is linked to antiin ammatory and protective functions in the CNS [18]. In the CNS, IL-10 is mainly produced by astrocytes and microglia. Our results showed that Cx3cr1 CreER IL-10 −/− mice showed impaired spatial cognition, suggesting that microglia-targeted production of IL-10 plays an important role in hippocampal function.
This result is similar to the results of other relevant studies, in which IL-10 tm1/tm1 male mice with a low expression of IL-10 exhibited defective learning and memory behaviors in the MWM test [7]. A recent study showed that IL-10 produced from microglial cells in non-learned helpless mice is necessary to maintain learning and memory [19].Intranasal administration of IL-10 increased dendritic spine density by 2.0-and 4.3-fold in the dentate gyrus of non-learned helpless and learned helpless mice [19].
We detected mRNA levels of TRPC subtypes in the hippocampus other than TRPC2, a pseudogene in humans. TRPC1, 3, 4, and 5 mRNA levels decreased. Further results con rmed that TRPC4 and TRPC5 protein levels were downregulated. This result could be linked to another related study, in which the TRPC1/4/5 channels were relevant to synaptic transmission for working memory formation and in relearning tasks in the hippocampus [3]. Controversially, TRPC1/4/5 KO did not affect mouse reference memory in that study. Based on the synaptic plasticity de cit, it could be di cult to explain how TRPC1/4/5 KO can speci cally affect working memory without affecting the reference memory [3].
Notably, during the training phase in the MWM task, TRPC1/4/5 −/− mice showed a similar decrease in learning ability. This difference may also be related to the interaction of TRPC subtypes, since the expression of TRPC1 protein in our study did not change in mice.
The cognitive impairment caused by Cx3cr1 CreER IL-10 −/− mice is closely related to a decrease in synaptic transmission. Synaptic transmission involves the release of neurotransmitters from presynaptic neurons, which then bind to speci c postsynaptic receptors. Synaptic proteins PSD95 and synaptophysin were tested in our study, and an obvious decrease in presynaptic synaptophysin and postsynaptic density protein PSD95 suggested that the memory de cits of Cx3cr1 CreER IL-10 −/− mice depend on structural changes in synaptic associated proteins, which is a dual mechanism involving presynaptic and postsynaptic processes. Notably, the glutamate receptors NR2A and NR2B were not involved in this process.
IL-10 is a key cytokine that has been shown to inhibit excessive in ammation associated with antiin ammatory and protective functions in the CNS. IL-10 exerts anti-in ammatory effects by inhibiting monocyte/macrophage-derived cytokines, including TNF-α, IL-1β, IL-6, and IL-8. It has been reported that immune dysfunction is commonly associated with the progression of many CNS diseases, such as neuropsychiatric disorders and neurodegenerative disorders. [18,23]. Moreover, the role of cytokines is of particular interest because they are involved in cognitive impairment in hippocampal-dependent memory [24,25].
The increase in the number of GFAP-and Iba1 positive cells indicates an imbalance in local neuroin ammation in the hippocampus. Our results con rmed the upregulation of the in ammasome NLRP3 signaling pathway, accompanied by the upregulation of IL-6. These results suggest that KO IL-10 from microglia may affect the behavioral performance of animals by altering the balance of local proin ammatory and anti-in ammatory networks. The NLRP in ammasome has been identi ed as a multiprotein complex that plays a pathogenic role in nervous system diseases. Among these types of in ammasomes, NLRP3 has been implicated in several chronic in ammatory responses and is associated with many CNS diseases [27]. Neuroin ammation can trigger cognitive impairment, and the role of in ammasome NLRP3 processes involved in recognition impairment in a variety of nervous system diseases, has been supported by experimental evidence, especially in recent years. Several studies have shown that NLRP3 can be a target for improving memory impairment in diabetes [28][29][30][31], sepsis-associated encephalopathy [32], hypoxemia [33], epilepsy [34,35], AD [36,37], intracerebral hemorrhage [38], cerebral ischemia [39], and aged [40]. Baicalin increases the performance of APP/PS1 transgenic mice in MWM by suppressing NLRP3 in ammasomes to alleviate microglia-mediated neuroin ammation [41]. Activation of the NLRP3 in ammasome plays a role in the Gastrodin-induced amelioration of cognitive impairment in diabetic rats [28], and NLRP3 in ammation, a molecular marker involved in in ammatory response, is known to play a key role in the development of cognitive impairment.
NLRP3 activation can trigger IL-1β and IL-18 production and secretion [42], and enhanced IL-1β production in astrocytes is associated with the pathogenesis of major depressive disorder. Our results showed an increase in NLRP3, IL-1β, and IL-18 in microglial cells knocked out in IL-10 mice compared to their littermates, suggesting that the NLRP3 pathway could play an important role in memory impairment in Cx3cr1 CreER IL-10 −/− mice.
TRPC channels are widely expressed in the brain and are related to a variety of neuronal functions [43]but in general, TRPC4 and TRPC5 are the predominant subtypes in the rodent brain [44] .In our study, we observed that both TRPC4 and 5 decreased in hippocampi of KO mice, which indicated that both channels could contribute to the spatial memory impairment of Cx3cr1 CreER IL-10 −/− mice, although it is di cult to judge which of these channels contributes to cognitive impairment. TRPC5 is highly expressed in the hippocampus [45,46]. TRPC channels have been implicated in presynaptic and postsynaptic neuronal processes. To date, the physiological function of TRPC channels in the brain is unknown. In cultured neurons, TRPC5 insertion and TRPC5-mediated Ca 2+ in ux are important determinants of hippocampal neurite growth rate and growth cone morphology [47,48].
In our study, we used CaMKIIα as a promoter to speci cally express TRPC5 in pyramidal neurons. The results showed that high expression of TRPC5 could improve spatial cognitive impairment in IL-10 KO mice. Furthermore, the results suggested that the effect of TRPC5 on behavioral improvement in IL-10 KO mice might be related to its inhibition of neuroin ammation. Increasing the expression of TRPC5 in excitatory neurons can reduce the high expression of GFAP and Iba1 in Cx3cr1 CreER IL-10 −/− mice. Several other studies have also focused on the role of TRPC5 in in ammation. Growing evidence has linked the activation of TRPC5 complexes to in ammation. TRPC5 −/− mice showed enhanced synovitis and local in ammation, and the TRPC4/5 antagonist ML204 increased the levels of TNF-α and IL-10 in synovial uid. In TRPC5 KO and wild-type mice treated with TRPC4/5 antagonists, IL-10 secretion was found to be elevated to regulate a highly in ammatory response. The absence or antagonism of TRPC5 increases the local secretion of many key pro-in ammatory cytokines, such as TNF-α and IL-1β [49]. TRPC5 −/− mice pretreated with thioredoxin also showed that cytokines (TNF-α and IL-6) in the peritoneum were exacerbated in the systemic in ammatory response [50]. There have also been con icting studies on the relationship between TRPC5 and in ammation. Expression of TRPC5 in nasal polyps was positively correlated with the number of eosinophils, IL-6 expression and in ammation [51], suggestting that these pathways may respond differently to different in ammatory responses. There are few studies on the relationship between TRPC and NLRP3.TRPC1 downregulation affects IL-1β release through the Caspase-11 pathway, a process associated with NLRP3 activation [52].
TRPC5 may also directly affect the learning and memory ability of animals by improving the e ciency of synaptic transmission. Some studies have shown that TRPC5 regulates synaptic plasticity by changing the presynaptic Ca 2+ homeostasis of hippocampal neurons [53] and both TRPC4 and 5 channels contribute to persistent ring in CA1 pyramidal cells [54]. In another study, TRPC5 channels, profoundly regulate synaptic plasticity and elevate the rate of spontaneous release, indicating a key role of TRPC5 in short-term plasticity. In addition, the speci c activation of TRPC4/5 induced a signi cant increase in the mEPSC frequency in hippocampal neurons of wild-type mice [53]. TRPC5 is also an important determinant at neurite outgrowth rates, growth cone morphology [48] and plateau potentials of excitatory neurons in the hippocampus [55].

Conclusions
Our results using Cx3cr1 CreER IL-10 −/− mice indicated the involvement of TRPC4 and 5 channels in recognition impairment. Speci c high expression of the excitatory neuron TRPC5 can improve the behavioral performance of KO mice. However, a limitation in our present study does not preclude the role of TRPC4 in the process. Future research should focus on resolving the contradictory observations mentioned above and determining the molecular mechanisms between TRPC and the in ammasome

Consent to participate
Informed consent was obtained from all individual participants included in the study.

Consent for Publication
All individual participants have consented to the submission of the manuscript to the journal. Table   Due to technical limitations, table 1 is only available as a download in the Supplemental Files section. Figure 1 Cx3cr1CreER IL-10-/-mice showed a decrease recognition impairment in OLR task. a: Schematic of experimental design and schedule. The animal experimental protocol indicated the time course of various interventions utilized during the experiment. b: Representative movement traces from the two groups on the test stage of the NOR task. There were no signi cant differences between the two mice in the different groups. There was no signi cant difference between the two groups in the discrimination index (c) and the new object duration (d). e: Representative movement traces from the two groups on the test stage of the OLR task. There was a signi cant decrease in the location of new objects in the Cx3cr1CreER IL-10-/group. There was a signi cant decrease in the two groups in discrimination index (f) and new object duration (g) in the test stage in the Cx3cr1CreER IL-10-/-group. Each dot represents a mouse. Bars represent mean±SEM. n=8 in each group. Signi cant differences were established by t test, *P<0.05.

Figure 2
Cx3cr1CreER IL-10-/-mice showed a decrease recognition impairment in MWM task. a: Representative movement traces from two groups during the training stage of the MWM task. Cx3cr1CreER IL-10-/-mice had more dispersed paths in the training stage, suggesting impairments in learning ability. There was a signi cant increase in escape latency (b) and average distance (c) in Cx3cr1CreER IL-10-/-mice in the training stage, while Cx3cr1CreER IL-10-/-mice swam faster in the MWM task (d). e: Representative movement traces from the two groups on the test stage of the MWM task. Cx3cr1CreER IL-10-/-mice had more dispersed paths in the test stage, suggesting memory impairments. There was a signi cant decrease in both the platform crossover times (f) and time spent in the target quadrant (g) in the Cx3cr1CreER IL-10-/-group in the test stage, while the swimming speed was similar between the two groups during the test stage (h). Each dot represents a mouse. Bars represent mean±SEM. n=8 in each group. Signi cant differences were established by two-way ANOVA (b-d) and t test in other bar graphs, *P<0.05.

Figure 3
The decrease of both TRPC4 and TRPC5 in the hippocampi of Cx3cr1CreER IL-10-/-mice. a: RT-PCR results of TRPC isoforms in the hippocampus (n=3 in each group, t-test ***P<0.001). b: western blotting bands of TRPC1, TRPC3, TRPC4, and TRPC5 in littermates and in Cx3cr1CreER IL-10-/-mice. Bar graphs represent densitometric plots of protein expression in littermate and Cx3cr1CreER IL-10-/-mice in TRPC1 (c), TRPC3 (d), TRPC4 (e), and TRPC5 (f). Each dot represents a mouse. Bars represent mean±SEM. n=4 in each group, Signi cant differences were established by t test. *P<0.05, **P<0.01. g: Expression of TRPC1, TRPC3, TRPC4, and TRPC5 in the CA3 region of mouse hippocampal slices. Immuno uorescence images were captured with a 20× objective, green, immunoreactivity of TRPC1, TRPC3, TRPC4, and TRPC5; blue, nuclei stained with DAPI. Merged images of each TRPC isoform and DAPI staining Immuno uorescence images were captured with a 20× objective, green, immunoreactivity of TRPC1, TRPC3, TRPC4, and TRPC5; blue, nuclei stained with DAPI. The merged images of PSD95, synaptophysin, and DAPI staining Neuroin ammatory activity enhanced in the hippocampus in Cx3cr1CreER IL-10-/-mice a: western blotting bands of in ammation-related molecules in littermates and Cx3cr1CreER IL-10-/-mice. Bar graphs represent densitometric plots of protein expression in littermate and Cx3cr1CreER IL-10-/-mice in GFAP (b), Iba1 (c), NLRP3 (d), IL-1β (e), IL-18 (f), IL-10 (g), and IL-6 (h). Each dot represents a mouse. Bars represent mean±SEM. n=4 in each group. Signi cant differences were established by t test, *P<0.05, **P<0.01. i: Expression of GFAP and Iba1 in CA3 region in mice hippocampal slices. Immuno uorescence images, captured with a 10× objective, Green, immunoreactivity of GFAP and Iba1; blue, nuclei staining with DAPI. The merged images of GFAP, Iba1 and DAPI staining. The effects of knocking out IL-10 from Microglia in interneuron and the increase of TRPC5 in excitatory neuron a: western blotting bands of GAD67 and parvalbumin in littermates and in Cx3cr1CreER IL-10-/mice. Bar graphs represent densitometric plots of protein expression in littermate and Cx3cr1CreER IL-10-/-mice in GAD67 (b) and parvalbumin (c). Each dot represents a mouse. Bars represent mean ± SEM. n=4 in each group, Signi cant differences were established by t test, ***P<0.001. d: colocalization of TRPC5 and CaMKIIα in CA3 region in hippocampal slices. Immuno uorescence images, captured with a 20× objective, Green, immunoreactivity of TRPC5; red, immunoreactivity of CaMKIIα, a marker of excitatory neurons, blue, nuclei staining with DAPI. The merged images of TRPC5, CaMKIIα and DAPI staining.