Chronic but not acute nicotine treatment ameliorates acute inflammation‐induced working memory impairment by increasing CRTC1 and HCN2 in adult male mice

Abstract Background Systemic inflammation in which lipopolysaccharide (LPS) is released into circulation can cause cognitive dysfunction and we have previously shown that LPS impaired working memory (WM) which refers to the ability to guide incoming behavior by retrieving recently acquired information. However, the mechanism is not very clear, and currently, there is no approved strategy to improve inflammation‐induced WM deficit. Notably, epidemiological studies have demonstrated a lower occurrence rate of inflammatory‐related diseases in smoking patients, suggesting that inflammation‐induced WM impairment may be improved by nicotine treatment. Here, our object is to investigate the effect and potential mechanisms of acute and chronic nicotine treatment on LPS‐produced WM deficiency. Methods Delayed alternation T‐maze task (DAT) was applied for evaluating WM which includes both the short‐term information storage and the ability to correct errors in adult male mice. Immunofluorescence staining and immunoblotting were used for assessing the levels and distribution of CREB‐regulated transcription coactivator 1 (CRTC1) and hyperpolarization‐activated cation channels 2 (HCN2) in the medial prefrontal cortex (mPFC) and hippocampus. Quantitative PCR and ELISA were employed for analyzing the mRNA and protein levels of TNF‐α and IL‐1β. Results Our results revealed that administration of LPS (i.p.) at a dose of 0.5 mg/kg significantly produced WM impairment in the DAT task accompanied by an increase in IL‐1β and TNF‐α expression in the mPFC. Moreover, intra‐mPFC infusion of IL‐1Ra, an IL‐1 antagonist, markedly alleviated LPS‐induced WM deficiency. More important, chronic (2 weeks) but not acute nicotine (0.2 mg/kg, subcutaneous) treatment significantly alleviated LPS‐induced WM deficiency by upregulating CRTC1 and HCN2. Of note, intra‐mPFC infusion of HCN blocker ZD7288 produced significant WM deficiency. Conclusions In summary, in this study, we show that chronic nicotine treatment ameliorates acute inflammation‐induced working memory deficiency by increasing CRTC1 and HCN2 in adult male mice.


| INTRODUC TI ON
Prefrontal cortex (PFC) and hippocampus-dependent working memory (WM) refer to the ability to possess relevant information online in coordinating goal-directed behavior. 1WM deficiency would disrupt the capacity to instruct incoming behavior through retrieval of recently acquired information.It could be disrupted under various pathological conditions such as Alzheimer's disease, 2 schizophrenia, 3 and stress. 4stemic inflammation, which releases lipopolysaccharide (LPS) into circulation, 5 can cause emotional and cognitive dysfunction through the release of a substantial quantity of inflammatory factors and the activation of immune cells. 6We have shown that acute LPS treatment induced significant WM deficiency in the DAT task, accompanied by decreased synaptophysin levels. 7wever, it is not known how LPS induced WM deficiency and there is currently no effective strategy for inflammation-induced WM deficiency.CREB-regulated transcription coactivator 1 (CRTC1), a key regulator of gene transcription driven by the cAMP response element (CRE), 8 plays essential roles in memory consolidation 9 and reconsolidation. 10CRTC1 dysfunction is associated with memory deficiency caused by ischemic stroke, 11 Alzheimer's disease, 12 and LPS. 13 Nevertheless, it remains uncertain whether CRTC1 is implicated in inflammation-induced WM damage.In addition, LPS has been demonstrated to regulate the activity of hyperpolarization-activated cation channels (HCN), 14 a channel that plays important roles in synaptic plasticity 15 and memory. 16For example, HCN is involved in memory deficiency induced by chronic cerebral hypoperfusion 17,18 or chronic morphine exposure. 19In addition, it has been shown that inhibition of cAMP-HCN in PFC enhanced WM. 20 However, it is unclear whether HCN is related to inflammation-produced WM deficiency.
Although the detrimental health consequences of tobacco smoking are widely reported, epidemiological studies have also revealed a lower occurrence rate of several neurodegenerative diseases 21 and inflammatory diseases 22 in smoking patients.In addition, chronic smoking, but not acute nicotine treatment, affected neural correlates of WM. 23 Furthermore, chronic nicotine treatment reduced sepsis-produced oxidative damage of multiple organs 24 and we have demonstrated that acute nicotine administration (0.2 mg/kg) improved the performance of spatial working memory in adult male rats in DAT task. 25Therefore, in this study, we examined the effects of both acute and chronic nicotine treatments on WM deficiency induced by acute LPS treatment using the DAT task and showed that chronic (2 weeks) but not acute nicotine (0.2 mg/kg) treatment improved LPS-produced WM deficiency through upregulating CRTC1 and HCN2 in adult male mice.

| Subjects
Male C57BL/6 mice (8-10 weeks) were obtained from SLAC Laboratory Animal (Shanghai, China).They were raised in specific pathogen-free (SPF)-level environment with constant temperature (23 ± 1°C) and were subjected to a restricted diet for 1 week before the commencement of the experiment to maintain 85% of their body weight.All experimental designs were approved by the Animal Care Committee of Soochow University and were carried out following the NIH Guide for the Care and Use of Laboratory Animals.All designs followed the principle to reduce and minimize the number of animals and their suffering.

| Surgery
The mice were anesthetized by intraperitoneal (i.p.) injection of pentobarbital sodium (5 mL/kg), followed by the bilateral implantation of 23-gauge stainless-steel guide cannula.The mPFC coordinates were obtained from the Paxinos and Watson 1986 atlas (anterior-posterior, +1.8 mm; medial-lateral, ±0.45 mm; and dorsalventral, 2.1 mm from skull surface). 26Sterile stainless-steel screws and dental cement were employed to anchor the guide cannula to the cranium.To prevent infection, a sterile dummy cannula was inserted into the guide cannula to maintain sterility.Subsequently, following surgery, the mice underwent a 5-to 7-day recovery period.

| Drugs and drug administration
Nicotine hydrogen tartrate (Sigma Chemical Company, St. Louis, MO, USA) dissolved in saline (pH = 7.4) was injected subcutaneously at 0.2 mg/kg.Nicotine tartrate was used as the term to represent all nicotine doses throughout the text.

Conclusions:
In summary, in this study, we show that chronic nicotine treatment ameliorates acute inflammation-induced working memory deficiency by increasing CRTC1 and HCN2 in adult male mice.

K E Y W O R D S
CRTC1, HCN2, lipopolysaccharide, mice, nicotine, working memory Lipopolysaccharide (LPS, Sigma, St. Louis, MO, USA) was dissolved in sterile saline and intraperitoneal injected for 4 h before testing with a dose of 0.5 mg/kg which can produce WM deficiency in DAT task in T maze. 7N channel blocker ZD7288 (Sigma, St. Louis, MO, USA) was intra-PFC infused with 1.5 μg/μL, or 15 μg/μL 15 min before testing. 27-1 receptor antagonist IL-1Ra (Techno Gene, 1 μg/1 μL/day, dissolved in saline) was intra-PFC infused through a guide cannula 5 h before testing.28

| Delayed alternation T-maze task
There were three free shuttle arms in the mouse T-maze: an initiation arm (30 × 10 × 30 cm) and two detection arms (35 × 10 × 30 cm each).
As we described previously, mice were trained using the delayed alternation task (DAT). 7The daily food of mice was limited, but water was sufficient.First, the mice had to acclimatize in the T-maze for 2 days, and the trials began on the third day.Each daily session began with one initial trial, followed by nine normal trials.Briefly, mice were trained to visit the two detection arms in an alternating pattern, receiving a reward for each correct choice.In the initial trial, there was food at the end of both arms so that the mice could achieve food by choosing either arm.Mice were placed in the starting zone, the gate was opened, and after the mice reached the end of either arm to obtain reward, mice were replaced into the starting zone again.During normal trials, mice only received the reward when they entered the opposite arm.Otherwise, the mice had to return to the starting zone to restart the train trial.The mice can proceed to the next normal trial only when they can successfully get the reward.The T-maze would be cleaned with alcohol to remove odors between sessions, at a 10-s interval.
T-maze training lasted for about 1 week.The formal trial began when the mice achieved an 80% success rate for 3 consecutive days (the correct choice accounted for a percentage of all choices, and the correct choice was always 9).Four hours after LPS treatment, each mouse was tested 10 times and the numbers of two types of errors were recorded: win-shift failure (incorrect choice following a previous correct choice) and lose-shift failure (incorrect choice following a previous incorrect choice).Specifically, the number of winshift failures reflected the spatial working memory of mice, and the number of lose-shift failures reflected the error correction ability of mice. 7

| Measurement of locomotor activity
The behavior response of mice in the T-maze during the WM test was reported by a camera and a computer connected to it with Anymaze software (Shanghai Xinruan Information Technology Co. Ltd).Based on the time taken by the mouse and the distance traveled, speed was calculated.
After perfusion with PBS/PFA, the brain tissue was removed and preserved for subsequent experimental analysis (see Figure 6A for detailed experimental procedures).

| Immunoblotting for CRTC1 and HCN2
Mice received overdose anesthesia with pentobarbital and were perfused with precooling PBS after the behavioral test.The expression levels of HCN2 and CRTC1 in the mouse hippocampus and mPFC were analyzed.Tissues were homogenized and lysed in a lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl 2 •2H 2 O, 1% triton X-100, and 0.05% Brij-35, pH = 7.6).The protein concentration was measured using a BCA protein assay kit (Beyotime, Haimen, China).Based on our published paper previously, 29 equal amounts (30 μg of total protein) of each homogenate aliquot were boiled at 100°C for 5 min and then the proteins were separated by electrophoresed in 10% or 12.5% SDS-PAGE gels, transferred onto PVDF membranes (0.45 μm, Millipore, Billenca, USA).
Membranes were subsequently blocked within TBS-T (Tris buffer saline and 0.1% Tween-20) containing 5% nonfat milk for 1 h, and incubated with primary antibodies CRTC1 (1:1000, Cell Signaling Technology, USA), HCN2 (1:1000, Alomone USA), or β-actin (1:5000, Boster, China) at 4°C for 12 h.After TBS-T washing, the membranes were incubated with the corresponding secondary antibodies (Boster, China) for 2 h.Finally, the membranes were subjected to incubation with the SuperSignal West Pico HRP substrate reagents (Thermo Fisher, USA) and imaging capture.The protein expression levels of HCN2 and CRTC1 were quantified using Image J software and normalized with β-actin as the loading control.

| Immunofluorescence staining for HCN2 and CRTC1
Immediately after the behavioral test, mice received overdose anesthesia with pentobarbital and were subsequently perfused with PBS and fixed by 4% PFA.The brain tissues were then sectioned into 20μm-thick cryosections for the evaluation of HCN2 and CRTC1, as previously described. 30Briefly, nonspecific component was removed with a blocking buffer containing 0.3% Triton X-100, 1% BSA, and 5% goat serum.The blocked sections were incubated first with primary antibodies against CRTC1 (1:200, Cell Signaling Technology, USA), HCN2 (1:200, Alomone), or NeuN (1:500, Millipore) overnight at 4°C, and then with the secondary antibody (anti-rabbit Alexa Fluor 488 conjugated, diluted at 1:800) for 1 h at room temperature.The staining of mPFC sections was captured using a Zeiss LSM 700 microscope.

| ELISA to detect TNFα and IL-1β
Mice received overdose anesthesia with pentobarbital and were perfused with PBS immediately after the behavioral test.The medial prefrontal cortex (mPFC) was collected for ELISA (Boster, China).Tissues were homogenized, lysed, and quantified.The total protein concentration of the brain homogenates was adjusted to 1 mg/mL, and the concentrations of TNFα and IL-1β in the samples were determined according to the kit instructions.Briefly, the protein supernatant, containing potential TNFα and IL-1β, was incubated at 37°C with the coated antibody 1.5 h and then corresponding antibody for 1 h in 96-well plates, respectively.After washing, avidin-biotin-peroxidase complex (ABC) was added and incubated for 30 min at 37°C.Following another washing step, tetramethylbenzidine (TMB) substrate was added and incubated for 15 min at 37°C.The absorbance of each well was measured at 450 nm, and the concentrations of TNFα and IL-1β were determined using a standard curve.

| Real-time RT-PCR
Total RNA was extracted from brain tissue using the TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions.in mRNA expression were determined by ΔΔCt, as previously described, 13,31 and expressed as 2 −ΔΔC t values.

| Statistical analysis
Formal tests for normality were used to assess data distribution.
Data are shown as the means ± SEM.
Statistical analysis was performed using the SPSS 18.0 software (SPSS, Chicago, IL, USA).Either one-way ANOVA or two-way ANOVA, followed by Bonferroni post hoc tests, was used for data analysis.p < 0.05 was considered statistically significant.

| Effect of acute nicotine on LPS-induced WM impairment in DAT task
LPS (0.5 mg/kg) treatment significantly impaired mice WM in the DAT task, 7 and acute nicotine (0.2 mg/kg) treatment can improve rats' spatial WM in the DAT task. 25Using DAT task, we detected the impact of acute nicotine treatment on LPS-produced WM deficit in DAT task in mice.Figure 1A

| Effect of chronic nicotine on LPS-induced WM deficit in DAT task
Chronic nicotine can selectively improve short-term memory in the G72 schizophrenia mouse model. 32In this study, Here, we examined the effect of chronic treatment with nicotine on LPS-produced WM deficiency.The detailed experimental procedures are shown in Figure 2A, and systemic LPS treatment significantly impaired mice performance in the T-maze task (Figure 2B).A two-way ANOVA revealed a main effect of LPS 52) = 4.448, p < 0.01).This implies that nicotine treatmentproduced improvement in WM may be attributed to enhanced proficiency in employing the lose-shift strategy.There were no significant alterations in mice motor activity following either LPS or nicotine treatment (Figure 2E), as indicated by a twoway ANOVA showing no main effect of LPS (F(1, 52) = 3.777, p > 0.05), nicotine (F(1, 52) = 0.007, p > 0.05), and no interaction between LPS and nicotine (F(1, 52) = 0.229, p > 0.05).Therefore, the performance deficit in the T-maze during the test was not owing to the effect on motor activity (Figure 2E).

| Pretreatment with nicotine reduced LPS-induced decrease in HCN2
HCN channels are crucial in the formation of working memory 20 and upregulating the expression of HCN2 in the PFC of rats inhibited WM deficiency caused by bilateral common carotid artery occlusion. 17Co-staining of NeuN and HCN2 was done to show the distribution of HCN2 in mPFC.As depicted in Figure 4A, HCN2 was found in neurons in mPFC, and LPS treatment notably downregulated the expression of HCN2 in mPFC, and nicotine pretreatment significantly ameliorated such reduction (Figure 4B).Western blot results confirmed the HCN2 expression change in PFC (Figure 4C), and a two-way ANOVA showed a main effect of LPS (F(1, 11) = 6.772, p < 0.05), a main effect of nicotine (F(1, 11) = 8.999, p < 0.05), and a main effect of interaction of LPS × nicotine (F(1, 11) = 5.733, p < 0.05).

| Pretreatment with nicotine significantly reduced LPS-induced mRNA and protein upregulation of IL-1β and TNFα in the medial prefrontal cortex (mPFC)
LPS can impair WM in rats 33 and D-galactose-impaired rat's WM and decreased spontaneous alternation in the Y-maze by increasing TNFα and IL-1β. 34Here, we detected the mRNA (Figure 5A,B) and protein (Figure 5C,D) levels of IL-1β and TNFα.As can be seen in Figure 5A,B, mice treated with LPS significantly increased the mRNA level of IL-1β (Figure 5A) and TNFα (Figure 5B) in the PFC, and 2-week nicotine treatment significantly reduced the upregulation.In addition, ELISA results showed that LPS treatment upregulated the protein of IL-1β (Figure 5C) and TNFα expression (Figure 5D) in PFC, and 2-week nicotine treatment inhibited such increase.The above experimental results indicate that nicotine treatment may improve LPS-induced WM deficiency by decreasing TNFα and IL-1β in mPFC.

| Effect of blockade IL-1 β on LPS-induced WM impairment in DAT task
We next explored whether blocking IL-1β could reduce LPS-induced WM deficiency.The detailed experimental procedures are shown in Figure 5E. Figure 5F shows that mice treated with LPS significantly reduced the accuracy, and intra-mPFC-infused antagonist IL-1Ra markedly increased the accuracy of mice in DAT and a two-way ANOVA revealed a main effect of interaction of LPS × IL-1Ra (F(1, 55) = 7.319, p < 0.01), but no main effect of LPS (F(1, 55) = 1.469, p > 0.05) and no main effect of IL-1Ra (F(1, 55) = 2.895, p > 0.05), suggesting that IL-1Ra treatment significantly ameliorated systemic LPS-impaired correct choice in mice.More interesting, analysis of the error types showed that systemic LPS treatment disrupted loseshift but not win-shift strategy and IL-1Ra treatment did not improve the ability to apply win-shift strategy (Figure 5G) as there is no main effect of LPS (F(1, 55) = 0.113, p > 0.05) and IL-1Ra (F(1, 55) = 1.013, p > 0.05), and no interaction of IL-1Ra × LPS (F(1, 55) = 3.616, p > 0.05).
A two-way ANOVA analysis on lose-shift failure (Figure 5H

| DISCUSS ION
In this study, we demonstrated that 2 weeks but not acute nicotine treatment significantly ameliorated LPS-produced WM deficiency.Specifically, the treatment enhanced the ability to apply the lose-shift strategy, but not the win-shift strategy.We demonstrated that LPS treatment significantly produced WM deficiency accompanied by the upregulation of both IL-1β and TNFα in mPFC.Additionally, intra-mPFC infusion of the IL-1 antagonist IL-1Ra markedly alleviated LPS-induced WM deficiency.More importantly, chronic but not acute nicotine treatment had a significant effect on alleviating LPS-induced WM deficit by upregulating CRTC1 and HCN2 (Figure 7).Of note, intra-mPFC infusion of HCN channel blocker ZD7288 induced significant WM deficiency.
F I G U R E 4 Chronic nicotine treatment significantly inhibited LPSinduced HCN2 reduction in PFC.(A, B) Immunofluorescence results showed that HCN2 (green) and NeuN (red) are co-localized and LPS significantly reduced the expression of HCN2 in the PFC (***p < 0.001 vs. the saline group, n = 3), and chronic nicotine treatment markedly inhibited such effect ( ### p < 0.001 vs. the LPS group; n = 3; scale bar = 200 μm).(C) Western blot results showed that LPS significantly reduced level of the HCN2 in the PFC (*p < 0.05 vs. the saline group, n = 6), and chronic nicotine significantly inhibited such effect ( # p < 0.05 vs. the saline group; n = 6).(D) Western blot results demonstrated that neither LPS nor nicotine significantly affected the expression of HCN2 in hippocampus (p > 0.05; n = 3).
The T-maze is used to detect spatial working memory, in which animals inherently tend to switch arm exploration, that is, spontaneous alternation during successive experiments.While the selection of the target arm is based on remembering the last explored target arm, that is, spatial working memory, therefore, the correct alternate choice of the target arm by the animal is a manifestation of the ability of spatial working memory. 35,36The T-shaped maze fully utilizes the foraging nature of animal exploration, which can minimize the factors that affect the experimental results, and compared with other detection methods, the T-shaped maze is easier to detect.Our previous results showed that acute nicotine (salt, 0.2 mg/kg, s.c.) ameliorated WM in DAT task in male rats, 25 but in the current study, acute nicotine treatment at the same dose did not significantly alleviate LPS-induced WM deficit (Figure 1).Improvement of WM under physiological and pathological conditions is very different and maybe higher dose or combination treatment is required for acute nicotine treatment to improve LPS-induced WM deficit.In addition, Wei et al. have shown that acute nicotine treatment reduced LPS-produced cognitive deficit through upregulating BDNF expression and decreasing neuroinflammation in rat hippocampus. 38The difference may be dependent on the complexity of the behavior task and nicotine dose; in Wei's study, they used water maze task to detect spatial memory, whereas in our study, we used DAT T-maze task to detect WM which includes both the hippocampus-dependent spatial working memory and PFC-dependent ability to correct error.In addition, they used a higher dose of nicotine, 0.5 mg/kg versus the dosage, used in this study (0.2 mg/kg).
We demonstrated that chronic nicotine treatment could significantly improve LPS-produced WM deficiency (Figure 2).
Nicotine treatment has been reported to ameliorate maternal LPS exposure-induced schizophrenia-like cognitive deficits which were detected by prepulse inhibition, latent inhibition, and delayed nonmatching to sample. 39In addition, in the G72 schizophrenia mouse model, chronic nicotine treatment has been shown to selectively improve short-term memory. 32Chronic treatment could mimic the smoking status because it is reported that the nicotinic receptor has a critical role in cigarette smoking-induced improvement in schizophrenia-associated spatial working memory and attentional deficits.

1
Effect of acute nicotine treatment on LPS-induced WM deficit in DAT task.(A) The diagram illustrated the procedure in the experiment.(B) LPS treatment showed significant downregulation of the correct choice rate of mice in the DAT task (**p < 0.01 vs. the saline group), and acute nicotine treatment failed to dramatically improve the reduction of the correct choice (p > 0.05).(C, D) LPS treatment had no notable impact on the win-shift failure (p > 0.05), but significantly increased lose-shift failure (**p < 0.01 vs. the Saline group), and acute nicotine treatment did not significantly improve LPS's effect (p > 0.05; n = 9).(E) LPS and acute nicotine treatment had no significant impact on motor function (p > 0.05, n = 7).F I G U R E 2 Effect of chronic nicotine treatment on LPS-induced WM deficit in DAT task as well as the levels of proinflammatory factors IL-1β and TNFα in the PFC.(A) Diagram of the experimental procedure.(B) LPS treatment markedly reduced the correct choice rate of mice in DAT (**p < 0.01 vs. the saline group) and chronic nicotine treatment significantly alleviated this reduction (##p < 0.01 vs. the LPS group).(C) Neither LPS nor chronic nicotine had significant differences in the win-shift failure in the DAT test (p > 0.05).(D) LPS increased the lose-shift failure of mice in the DAT test (**p < 0.01 vs. the saline group) and chronic nicotine markedly inhibited such effect (#p < 0.05 vs. the LPS group).(E) Neither LPS nor nicotine had a significant effect on the motor function of the mice (p > 0.05; n = 11).

F I G U R E 5
Pretreatment with nicotine significantly reduced LPS-induced IL-1β and TNFα increase in PFC and blockade of IL-1 β alleviated LPS-induced WM deficit in the DAT task.LPS induced a significant effect on increasing the mRNA levels of pro-inflammatory factors IL-1β (A) and TNFα (B) in the PFC (**p < 0.01 vs. the saline group; n = 5), and chronic nicotine treatment significantly inhibited such effect ( # p < 0.05 vs. the LPS group; n = 5).The protein levels of pro-inflammatory factors IL-1β (C) and TNFα (D) in the PFC were significantly increased by LPS treatment (**p < 0.01 vs. the saline group; n = 5), and chronic nicotine treatment markedly alleviated such effect ( # p < 0.05, ## p < 0.01 vs. the LPS group; n = 5).(E) Experimental procedure.(F) LPS significantly reduced the correct choice rate of mice in the DAT task (**p < 0.01 vs. the saline group, n = 14), and IL-1Ra significantly suppressed such reduction ( # p < 0.05 vs. the LPS group; n = 14).(G) Neither LPS nor IL-1Ra significantly affected the win-shift failure (p > 0.05; n = 14).(H) LPS markedly increased the lose-shift failure (*p < 0.05 vs. the saline group, n = 14), and IL-1Ra significantly alleviated such effect ( # p < 0.05 vs. the LPS group; n = 14).(I) Western blot results demonstrate that LPS significantly downregulated the level of CRTC1 expression in the PFC (**p < 0.01 vs. the saline group, n = 3) and IL-1Ra significantly suppressed such effect ( ## p < 0.01 vs. the LPS group, n = 3).(J) Western blot results demonstrated that neither LPS nor IL-1Ra significantly affect the expression of CRTC1 in hippocampus (p > 0.05; n = 3).(K) Western blot results demonstrated that LPS significantly reduced level of the HCN2 in the PFC (*p < 0.05 vs. the saline group, n = 6), and IL-1Ra had a significant effect on inhibiting such effect ( ## p < 0.01 vs. the saline group; n = 6).(L) Western blot results demonstrated that neither LPS nor IL-1Ra significantly affected the expression of HCN2 in hippocampus (p > 0.05; n = 3).

F I G U R E 7
Summary.LPS significantly induced WM deficit in mPFC, accompanied by IL-1β and TNFα upregulation.Nicotine treatment significantly alleviated LPS-induced WM deficit by upregulating CRTC1 and HCN2.