Interaction between interleukin-1β and type-1 cannabinoid receptor is involved in anxiety-like behavior in experimental autoimmune encephalomyelitis

Abstract

Background: Mood disorders, including anxiety and depression, are frequently diagnosed in multiple sclerosis (MS) patients, even independently of the disabling symptoms associated with the disease. Anatomical, biochemical, and pharmacological evidence indicates that type-1 cannabinoid receptor (CB1R) is implicated in the control of emotional behavior and is modulated during inflammatory neurodegenerative diseases such as MS and experimental autoimmune encephalomyelitis (EAE).

Methods: We investigated whether CB1R could exert a role in anxiety-like behavior in mice with EAE. We performed behavioral, pharmacological, and electrophysiological experiments to explore the link between central inflammation, mood, and CB1R function in EAE.

Results: We observed that EAE-induced anxiety was associated with the downregulation of CB1R-mediated control of striatal GABA synaptic transmission and was exacerbated in mice lacking CB1R (CB1R-KO mice). Central blockade of interleukin-1β (IL-1β) reversed the anxiety-like phenotype of EAE mice, an effect associated with the concomitant rescue of dopamine (DA)-regulated spontaneous behavior, and DA-CB1R neurotransmission, leading to the rescue of striatal CB1R sensitivity.

Conclusions: Overall, results of the present investigation indicate that synaptic dysfunction linked to CB1R is involved in EAE-related anxiety and motivation-based behavior and contribute to clarify the complex neurobiological mechanisms underlying mood disorders associated to MS.

Keywords: Anxiety; Experimental autoimmune encephalomyelitis; Interleukin-1β; Striatum; Type-1 cannabinoid receptor.

Fig. 1

Anxiety-like behavior in EAE is associated to CB1R dysfunction in the striatum. a Exploratory behavior of EAE mice was investigated firstly by LDT confirmed increased anxiety-like behavior in EAE group as showed by time spent in the light zone of the task. a’ Rear episodes in EAE mice were severely reduced in comparison to CFA mice. a” Representative video-recording tracking of CFA and EAE mice performance in the light zone of the LDT apparatus. b, b’ EAE and CFA control mice were evaluated for their ability to construct nests (rated on a 4-point scale) to investigate motivation-based behavior. Bar graph shows a reduction in the quality of nest in EAE mice (b). b’. Representative photographs of nests built by CFA and EAE mice. c. The performance of CB1R-KO mice at the LD test (% of time spent in the bright compartment) revealed an anxiety-related behavior, which is heavily affected by EAE induction. d, d’ Bath application of the CB1R agonist HU210 on striatal slices induced sIPSC frequency reduction in CFA mice (p < 0.01). In EAE striatal slices, the effect of HU210 was abolished (p > 0.05) (d). Representative electrophysiological traces are depicted in d’. Values are means ± SEM. Statistical differences were analyzed by unpaired T-test or Mann-Whitney test (behavior) and by paired T-test (electrophysiology). *p < 0.05, **p < 0.01. Two-way ANOVA analysis for genotype factor: ##p < 0.01, ### p < 0.0001

Fig. 2

IL-1ra preventive treatment improves EAE behavioral syndrome and restores striatal CB1R function. a–a” At the LDT, the IL-1ra treatment improved the behavioral alterations of EAE mice; the time spent in the light zone, EAE-IL-1ra mice showed values similar to CFA-vehicle, although being not significantly different to both CFA and EAE-vehicle (a). a’ Vertical activity increased in EAE-IL-ra mice, indicating an ameliorated explorative response induced by IL1-ra treatment. Examples of LDT video-tracking are depicted in a”. b, b’ Consistently with the results obtained in non-minipump-implanted animals (shown in Fig. 1b, b’), the nesting score of EAE-vehicle mice was worse than CFA-vehicle animals and IL-1ra icv treatment corrected these behavioral alterations (b). Representative photographs in b’. c, c’. The lack of the depressant effect mediated by HU210 in EAE slices was rescued in EAE mice receiving in vivo treatment of IL-1ra by minipump implantation (c). In c’, there are examples of electrophysiological traces showing the lack of the effect of HU210 only in EAE-vehicle mice. Values are means ± SEM. Statistical differences were analyzed by one-way ANOVA for multiple comparisons (followed by Tukey HSD or Dunn's comparisons) or by unpaired T-test for rear numbers in LDT. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

CB1R function is linked to dopamine system in the EAE striatum. a The inhibitory effect of HU210 on sIPSC frequency of striatal neurons was completely restored in EAE mice receiving i.p. injection of amphetamine (p < 0.05). b–b” Amphetamine treatment did not affect DARPP32 phosphorylation in the striatum of control CFA mice, as shown by the WB image in b and the densitometric analysis in b’. The treatment did not change the amount of unphosphorylated protein, as shown in b and b”. c–c” Representative WB of striatal protein extracts from EAE-vehicle and EAE-amphetamine mice: the densitometric analysis of the bands displays an upregulation of Th34-DARPP32 in EAE-vehicle samples and a partial recovery of such phosphorylation in EAE-amphetamine group, as shown by graph in c’. Amphetamine treatment induced a significant upregulation of DARPP32 expression in EAE striatum (c”). WB data are normalized to CFA values. Values are means ± SEM. Statistical differences were analyzed by paired Student’s T test (electrophysiology) and one-way ANOVA followed by Tukey HSD (WB). *p < 0.05 EAE-amphetamine vs EAE-vehicle

Fig. 4

BDNF and CB1R signaling in EAE striatum. a BDNF mRNA in EAE-vehicle striatum is upregulated with respect to control CFA-vehicle (p < 0.05). Such alteration is not corrected by amphetamine treatment. b shows WB of striatal lysates of CFA-vehicle, EAE-vehicle, and EAE-amphetamine. CB1R levels in the striatum are not affected by EAE, as indicated by WB analysis of protein lysates (b’). Amphetamine given 24 h before the sacrifice of the animals did not affect the total amount of CB1R in the EAE striatal lysates. CB1R signal was normalized to β-actin bands. WB data are normalized to CFA values. c, Representative WB of lysates from CFA-vehicle, EAE-vehicle, and amphetamine-vehicle probed with antibody specific for the carboxy-terminal phosphorylation of CB1R (p-Ser316): the histogram in c’ indicates a remarkable reduction of CB1R phosphorylation, normalized to β-actin, which is not modulated by amphetamine treatment. Values are means ± SEM. Statistical differences were analyzed by one-way ANOVA followed by Tukey HSD (WB and qPCR). *p < 0.05 EAE-vehicle vs CFA-vehicle