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

The subjects in this study were 7–8-week-old female mice, C57BL/6N, obtained from Charles-River (Italy) and CNR-EMMA Mouse Clinic facility (Monterotondo-Rome, Italy). Animals were randomly assigned to standard cages, with four to five animals per cage, and kept at standard housing conditions with a light/dark cycle of 12 h and free access to food and water. Only minipump-implanted mice were housed in isolated cages endowed with special bedding (TEK-FRESCH, Harlan) in order to avoid skin infections around the surgical scar. Since 1 week before immunization, all animals were kindly handled once a day to reduce the stress induced by operator manipulation during behavioral experiments.

All experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals and the European Communities Council Directive of 24 November, 1986 (86/609/EEC).

Chronic EAE was induced in 7–8-week animals as previously described [10, 34]. Furthermore, EAE was induced in female mice totally lacking CB1Rs (CB1R-KO) [35] and respective wild-type (WT) littermate controls. Mice were injected subcutaneously at the flanks with 200 μg of myelin oligodendrocyte glycoprotein 35-55 (MOG_35-55) emulsion to induce EAE by active immunization. The emulsion was prepared under sterile conditions using MOG_35-55 (>85 % purity, Espikem, Florence, Italy) in 300 μl of complete Freund’s adjuvant (CFA, Difco, Lawrence, KS, USA) containing Mycobacterium tuberculosis (8 mg/ml; strain H37Ra, Difco) and emulsified with phosphate-buffered saline (PBS). All animals were injected with 500 ng pertussis toxin (Sigma, St. Louis, MO, USA) intravenously on the day of immunization and 2 days later. Control animals received the same treatment as EAE mice without the immunogen, MOG peptide, including complete CFA and Pertussis toxin (referred to as “CFA”). Animals were daily scored for clinical symptoms of EAE, according to the following scale: 0 = healthy; 1 = flaccid tail; 2 = ataxia and/or paresis of hindlimbs; 3 = paralysis of hindlimbs and/or paresis of forelimbs; 4 = tetraparalysis; and 5 = moribund or death due to EAE. Intermediate clinical signs were scored by adding 0.5 value [10, 35]. The presymptomatic phase was kept in the range 8–11 days post immunization (dpi), before the onset day, when immunized animals showed the first clinical manifestation (12 dpi), as previously shown [34].

One week before immunization, mice were implanted with a minipump in order to allow continuous intracerebroventricular (icv) infusion of either vehicle or interleukin 1 receptor antagonist (IL1-ra) (150 ng/day; R&D Systems) for 4 weeks. Alzet osmotic minipumps (model 1004; Durect Corporation, Cupertino, CA) connected via catheter tube to intracranial cannula (Alzet Brain Infusion Kits 3) delivered vehicle or IL1-ra into the right lateral ventricle at a continuous rate of 0.11 μl/h. The coordinates used for icv minipump implantation were antero-posterior = −0.4 mm from the bregma; lateral = −1 mm; and depth: 2.5 mm from the skull [10, 36].

EAE mice were given intraperitoneal injection of amphetamine sulfate (5 mg/kg) in a volume of 10 ml/kg or vehicle [37] and after 24 h were sacrificed for both electrophysiological and western blot experiments. Control CFA and EAE mice received intraperitoneal injection of saline solution (NaCl 0.9 %). Each experimental group consisted of four to five animals (three sets of experiments).

Behavioral experiments were performed during the presymptomatic phase of the disease (8–9 dpi). The animals were tested during light period (9:00–12:00 am) in a dedicated room with a constant temperature (26 ± 1 °C). All tests were performed in different days with distinct groups of animals. Each session was preceded by at least 1 h habituation in the behavioral room.

Mice were killed by cervical dislocation, and corticostriatal coronal slices (200 μm) were prepared from fresh tissue blocks of the brain with the use of a vibratome [10]. A single slice was transferred to a recording chamber and submerged in a continuously flowing artificial cerebrospinal fluid (ACSF) (34 °C, 2–3 ml/min) gassed with 95 % O2–5 % CO₂. The composition of the control ACSF was (in mM) 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 11 Glucose, and 25 NaHCO3. The striatum could be readily identified under low power magnification, whereas individual neurons were visualized in situ using a differential interference contrast (Nomarski) optical system. This employed an Olympus BX50WI (Japan) non-inverted microscope with 40× water immersion objective combined with an infra-red filter, a monochrome CCD camera (COHU 4912), and a PC compatible system for analysis of images and contrast enhancement (WinVision 2000, Delta Sistem, Italy). Recording pipettes were advanced towards individual striatal cells in the slice under positive pressure and visual control (WinVision 2000, Delta Sistemi, Italy) and, on contact, tight GΩ seals were made by applying negative pressure. The membrane patch was then ruptured by suction and membrane current and potential monitored using an Axopatch 1D patch clamp amplifier (Molecular Devices, Foster City, CA, USA). Whole-cell access resistances measured in voltage clamp were in the range of 5–20 MΩ. Whole-cell patch clamp recordings were made with borosilicate glass pipettes (1.8 mm o.d.; 2–3 MΩ), in voltage-clamp mode, at the holding potential of −80 mV.

To study GABA-mediated spontaneous inhibitory postsynaptic currents (sIPSCs), the recording pipettes were filled with internal solution of the following composition (mM): 110 CsCl, 30 K⁺-gluconate, 1.1 EGTA, 10 HEPES, 0.1 CaCl₂, 4 Mg-ATP, 0.3 Na-GTP. MK-801, and CNQX were added to the external solution to block, respectively, NMDA and non-NMDA glutamate receptors. Drugs were first dissolved in water or in DMSO (HU-210) and then in the ACSF to the desired final concentration. The concentrations of the various drugs were chosen according to previous in vitro studies on corticostriatal brain slices [33, 41] and were as follows (in μM): 10 CNQX, 25 MK-801, and 1 HU-210 (Tocris Bioscience).

Synaptic events were stored by using P-CLAMP 9 (Axon Instruments) and analyzed offline on a personal computer with Mini Analysis 5.1 (Synaptosoft, Leonia, NJ, USA) software. The detection threshold of sIPSCs was set at twice the baseline noise. The fact that no false events would be identified was confirmed by visual inspection for each experiment. Offline analysis was performed on spontaneous synaptic events recorded during fixed time epochs (1–2 min), sampled every 2–3 min (5–12 samplings) [34].

Only data from putative GABAergic medium spiny projection neurons (MSNs) were included in the present study and identified immediately after rupture of the GΩ seal, by evaluating their firing response to the injecting of depolarizing current (typically tonic, with little or no adaptation).

One to five cells per animal were recorded. For each type of experiment and time-point, at least four mice per group were employed. Electrophysiological results from neurons recorded from the same animal were treated as a separate sample and averaged before calculating statistics. One animal per day was used for the electrophysiological experiment. Throughout the text “n” refers to the number of cells, unless otherwise specified.

At least 4 animals per group were included in all western blot (WB) experiments. Mice were sacrificed through cervical dislocation, and both left and right striata were quickly removed and frozen until use. Tissues were homogenized in RIPA buffer plus protease and phosphatase inhibitors cocktail (SIGMA) as previously described [10, 36].

Primary antibodies were used as following: mouse anti-β-actin (1:20,000, 1 h RT; Sigma-Aldrich), rabbit anti-dopamine (DA)- and cAMP-regulated phosphoprotein 32 kDa (DARPP32; 1:50,000, 1 h RT; Abcam), rabbit anti-p-Th34-DARPP32 (1:1000, overnight 4 °C; Merck), goat anti-p-Ser316-CB1R (1:200, overnight 4 °C; SantaCruz), and goat anti-CB1R (1:200, overnight 4 °C; SantaCruz). Membranes were incubated with the following secondary antibodies: anti-mouse IgG HRP (1:10,000; GE Healthcare, formerly Amersham Biosciences), anti-rabbit IgG HRP (1:2000; GE Healthcare, formerly Amersham Biosciences), anti-goat IgG HRP (1:2000; GE Healthcare, formerly Amersham Biosciences), and diluted in 1 % milk for 1 h at RT. Blots were stripped with Restore Western Blot Stripping Buffer (Thermo Scientific), after detection of phospho-sites. Complete stripping was assessed by incubation with proper secondary antibody immunodetection was performed by ECL reagent (Amersham) and membrane was exposed to film (Amersham). Densitometric analysis of protein levels was performed by NIH ImageJ software (http://​rsb.​info.​nih.​gov/​ij/​). Phospho-CB1R band densitometry was normalized respect to β-actin, since in a different blot of the same samples, we did not detect changes in CB1R unphosphorylated protein, while DARPP32 phospho-protein levels were normalized to DARPP32 unphosphorylated protein to account for changes in the amount of DARPP32 unphosphorylated protein. WB results are presented as data normalized to control CFA values.

Total RNA was extracted according to the standard miRNeasy Micro kit protocol (QIAGEN). The RNA quantity and purity were analyzed with NanoDrop 2000c spectrophotometer (Thermo Scientific). The quality of RNA was assessed by visual inspection of the agarose gel electrophoresis images. Next, 250 ng of total RNA was reverse-transcribed using high-capacity cDNA reverse transcription kit (Applied Biosystem) according to the manufacturer’s instructions and 20 ng of cDNA was amplified with SensiMix SYBR Hi-Rox Kit (Bioline; Meridian Life Science) in triplicate using the Applied Biosystem 7900HT Fast Real Time PCR system. Relative quantification was performed using the ΔΔCT method. β-actin was used as internal controls. The following primer sequences were used.

Brain-derived neurotrophic factor (BDNF) (NM_007540): 5′-ACCATAAGGACGCGGACTTGT-3′ (sense); 5′-AAGAGTAGAGGAGGCTCCAAAGG-3′ (antisense); β-actin (NM_007393): 5′-CCTAGCACCATGAAGATCAAGATCA-3′ (sense); and 5′-AAGCCATGCCAATGTTGTCTCT-3′ (antisense).

Data were presented as mean ± SEM. Throughout the text, “n” refers to the number of animals, with the exception of electrophysiological experiments, where “n” refers to the number of the cells. Two-sample comparisons were carried out using the Student’s T test for parametric measures or Mann-Whitney for non-parametric variables, while multiple comparisons were made using one-way ANOVA followed by Tukey’s HSD or non-parametric Kruskal–Wallis test followed by Dunn’s comparisons. The main effects of the two conditions (genotype and EAE) on the dependent behavioral variables and the interactions genotype × EAE were analyzed by performing two-way ANOVAs. The significance level was established at p < 0.05.

We investigated the anxiety-like phenotype associated with MOG-induced central inflammation in presymptomatic EAE mice. In these mice, we assessed anxious-depressive-like responses by using the LDT and the nest building (NB) paradigm, a motivation-based task [39].

Significant differences between EAE and controls emerged at the LDT (Fig. 1a–a”), since the time spent in the light zone (EAE 16.42 ± 4.6 %; CFA: 42.21 ± 4.72 %; unpaired T test; p < 0.01; Fig. 1a) and the number of rearing episodes during LDT (CFA: 23 ± 3.32, n = 8; EAE: 11.38 ± 2.33, n = 8, unpaired T test: p < 0.05; Fig. 1a’) were reduced in EAE, indicating both anxiety-like behavior and reduced motivation-based activity, respectively, in accordance with our previous findings [8]. Next, we measured goal-directed behavior of EAE and control mice by using the NB test. Nesting ability is a natural, instinctive motivation-driven behavior in rodents [39]. Murine NB skills have been found altered during inflammation challenge [42, 43] and, of note, in EAE mice during the acute phase of the disease independently of motor defects [10]. Preclinical EAE mice performed significantly worse than controls in terms of nesting score (CFA 3.5 ± 0.18; EAE 2.07 ± 0.31; Mann-Whitney non-parametric test p < 0.01) (Fig. 1b, b’), again indicating that EAE is associated with reduction of social behavior, which is a characteristic trait of sickness behavior, affecting EAE mice [44].

The endocannabinoid system (ECS) is deeply involved in mood control [25, 26]. We asked whether CB1Rs could be involved in EAE-behavioral syndrome. One week before immunization, we evaluated the spontaneous responses of WT and CB1R-KO mice at LDT, in order to monitor anxiety-like behavior under basal condition. After 2 weeks, during EAE (7–9 dpi), the mice were tested again for anxiety by means of LDT.

CB1R-KO mice spent less time in the lit compartment than WT mice (n = 8 for each groups; F = 13.54, p < 0.01), showing increased anxiety-related behavior as previously reported [30]. Such behavior was exacerbated by EAE induction. EAE induction had a substantial effect on behavioral performance, accounting for 47.01 % of total variance (F = 40.76, p < 0.0001), with a significant interaction with genotype (F = 4.403, p < 0.05) (Fig. 1c), suggesting the role of CB1Rs in anxiety-like behavior associated to EAE.

We have previously reported that the downregulation of CB1R-mediated control of GABA synapses in the striatum represents a reliable synaptic correlate of anxiety induced by chronic psychoemotional stress [45] or by a single intracerebroventricular injection of IL-1β [33]. Of note, in a previous study, we demonstrated that CB1R sensitivity on GABA synapses is lost in the striatum of EAE mice during the symptomatic phase of the disease [46]. We therefore assessed whether EAE-induced anxiety was also associated with alterations of CB1R function. Thus, control and EAE mice were sacrificed to study synaptic transmission in the striatum (9–11 dpi). Here, we found that application of HU210 (1 μM, 10 min) significantly reduced the frequency of sIPSCs in CFA mice (79.8 ± 3.5 %; n = 10; p < 0.01), while this CB1R agonist failed to alter GABA transmission in the presymptomatic EAE mice (96.3 ± 3.4 %; n = 9; p > 0.05), suggesting that this synaptic alteration is precocious and, more interestingly, is linked to behavioral alterations in EAE (Fig. 1d, d’).

Overall, these data indicate that CB1Rs are involved in EAE-mediated anxiety-like behavior occurring in the presymptomatic phase of the disease.

To investigate the role of IL-1β in EAE-induced anxiety, we chronically inhibited IL-1β signaling in these mice by icv delivery of IL-1ra. We have previously shown that this preventive treatment reduced motor disability and inflammatory reaction in the cerebellum, the hippocampus, and the striatum of EAE mice during the symptomatic phase of the disease [10, 36, 47]. Moreover, increased IL1-beta expression in the striatum of EAE mice with preserved motor functions was associated to depressive-like and motivation-based behaviors observed in these mice [10]. Here, we investigated the anxious-like behavior of EAE mice treated with IL-1ra during the preclinical phase of EAE, when motor defects are not detectable.

In the LDT, IL-1ra partially corrected the behavioral alterations of pre-symptomatic EAE mice. The time spent in the lit zone by EAE-IL-1ra mice was not different with respect to both CFA-vehicle and EAE-vehicle (CFA-vehicle 49.90 ± 4.98, EAE-vehicle 27.18 ± 6.34, EAE-IL-1ra 41.34 ± 3.76; one-way ANOVA post hoc comparisons: CFA-vehicle vs EAE-vehicle p < 0.05; CFA-vehicle vs EAE-IL-1ra p > 0.05; EAE-vehicle vs EAE-IL-1ra p > 0.05), although very similar to CFA-vehicle (Fig. 2a, a”). However, rearing activity during the LDT was fully corrected by icv IL-1ra delivery in EAE mice (EAE-IL-1ra: 21.13 ± 2.88, n = 8, vs EAE-VH: 11.38 ± 2.33, n = 8; p < 0.05; Fig. 2a’).

We next addressed the effects of the IL-1ra icv delivery in EAE mice at the NB test. Consistently with the results in non-minipump-implanted animals, the nesting score of EAE mice receiving icv vehicle (9 dpi, n = 14; 1.78 ± 0.15) was worse than CFA-vehicle animals (n = 14; 3.25 ± 0.17), and IL-1ra icv treatment corrected these behavioral alterations (2.67 ± 0.16; n = 14; non-parametric Kruskal–Wallis test followed by Dunn’s comparisons: CFA-vehicle vs EAE-vehicle p < 0.001; CFA-vehicle vs EAE-IL-1ra p > 0.05; EAE-vehicle vs EAE-IL-1ra p < 0.05) (Fig. 2b, b’).

We performed electrophysiological measurements of striatal CB1R sensitivity in these mice 1 week after the behavioral tests in order to recover the effect of the stress induced by behavioral manipulation on endocannabinoid system [48]. Of note, the loss of CB1R sensitivity on GABA synapses is a synaptic dysfunction that occurs throughout the disease progression ([46]; present paper]) and IL-1ra treatment was previously shown to ameliorate both the depressive-like behavior of EAE mice in the acute phase of the disease and the dopaminergic dysfunction affecting the striatum [10].

We observed that the preventive and central treatment with IL-1ra rescued the physiological response of CB1R to HU210 (% of sIPSC frequency pre-HU210: CFA-vehicle 81.60 ± 5.6, n = 8; EAE-vehicle 108.4 ± 5.8, n = 9; EAE-IL-1ra 82.4 ± 3.9, n = 10; one-way ANOVA post hoc comparisons: CFA-vehicle vs EAE-vehicle p < 0.01; EAE-vehicle vs EAE-IL-1ra p < 0.05) (Fig. 2c, c’).

Altogether, these data are consistent with the idea that EAE anxiety-like behavior is linked to IL-1β-mediated CB1R dysfunction.

Striatal CB1R function is regulated by the DA system. Accordingly, pharmacological enhancement of DA signaling in vivo sensitizes CB1R [13] and DA receptor inhibition blocks them [49]. Recent biochemical and electrophysiological experiments demonstrated impaired DA transmission in the striatum of EAE mice [10], raising the possibility that also CB1R dysfunction is mediated by defective DA signaling in this neuroinflammatory condition. Of note, genetic and pharmacological blockade of DA pathway is known to reduce rearing activity in mice [50‐52]. The reduced rearing activity observed in EAE mice confirmed defective DA signaling in EAE mice [10] and suggested that CB1R dysfunction could indeed be secondary to this alteration in EAE.

We tested therefore the hypothesis that defective DA transmission could be responsible of CB1R downregulation in EAE by evaluating striatal CB1R function in EAE mice treated in vivo with amphetamine, to favor DA release from DA nerve terminals. In striatal brain slices prepared from these animals, HU210 caused the expected inhibition of sIPSC frequency (EAE-amphetamine: 80.06 ± 3.2 %, n = 11, paired T test p < 0.05; EAE-vehicle: 97.8 ± 3.6, n = 10; paired T test p > 0.05), confirming the rescue of CB1R function by DA system stimulation in EAE mice (Fig. 3a).

We have recently found that phosphorylation at threonine 34 (P-Th34) of the DA- and cAMP-regulated phosphoprotein 32 kDa (DARPP32), marker of striatal MSNs, is increased in EAE, possibly due to unbalanced signaling through D1- and D2-like receptors [10]. By WB experiments, we checked the phosphorylation of DARPP32 at Th34 site after in vivo treatment with amphetamine to assess whether amphetamine could interfere with DA signaling in MSNs. Amphetamine treatment in CFA mice did not change neither the extent of DARPP32 phosphorylation at Th34 (p-Th34-DARPP32/DARPP32: CFA-vehicle 1 ± 0.06, n = 6; CFA-amphetamine 1.13 ± 0.04, n = 7; unpaired T test p > 0.05; Fig. 3b, b’), nor the total amount of DARPP32 (DARPP32/β-actin ratio: CFA-vehicle 1 ± 0.07 n = 6; CFA-amphetamine 1.17 ± 0.06, n = 7; unpaired T test p > 0.05; Fig. 3b–b”). Amphetamine reduced, although not completely, the hyper-phosphorylation of Th34-DARPP32 induced by EAE (p-Th34-DARPP32/DARPP32 ratio: CFA-vehicle 1 ± 0.25, EAE-vehicle 2.7 ± 0.23, EAE-amphetamine 1.91 ± 0.13; CFA-vehicle n = 5 vs EAE-vehicle n = 5, one-way ANOVA post hoc comparison: p < 0.001; CFA-vehicle vs EAE-amphetamine n = 4; one-way ANOVA post hoc comparison: p < 0.05; Fig. 3c, c’). However, the amphetamine treatment induced a slight increase in the expression of total DARPP32 in the EAE striatum (DARPP32/β-actin ratio: CFA-vehicle 1 ± 0.07, EAE-vehicle 1.03 ± 0.07, EAE-amphetamine 1.34 ± 0.03; one-way ANOVA: CFA-vehicle vs EAE-amphetamine p < 0.01; EAE-vehicle vs EAE-amphetamine p < 0.05) (Fig. 3c–c”), which accounted for the quantification of p-Th34-DARPP32, suggesting the triggering of compensatory mechanism induced by amphetamine to cope with the EAE-induced overstimulation of DA system.

We investigated the possible mechanisms underpinning the link between the CB1R- and DA-mediated neurotransmission. We first assessed the expression of the BDNF, which was previously shown to be a downstream effector of D2Rs in the modulation of CB1R in the mouse striatum [49]. By qPCR, we found that BDNF transcript was significantly increased in the striatum of EAE-vehicle mice (n = 6, fold change mRNA: 5.95 ± 1.90) compared to CFA-vehicle mice (n = 8, fold change mRNA: 1.15 ± 0.22), and that amphetamine treatment was not able to re-establish normal levels of BDNF expression (n = 6, fold change mRNA: 5.23 ± 1.69; one-way ANOVA Tukey post hoc comparisons: CFA-vehicle vs EAE-vehicle p < 0.05, CFA-vehicle vs EAE-amphetamine p > 0.05, EAE-vehicle vs EAE-amphetamine p > 0.05; Fig. 4a). This result indicated that BDNF modulation was not involved in the observed recovery of neurotransmission by amphetamine.

We next asked whether amphetamine treatment could modify the phosphorylation status of CB1R. CB1R undergoes phosphorylation at serine 316 (p-Ser316), and such phospohorylation has been linked to the internalization of the receptor-ligand complex [53]. WB experiments showed that CB1R levels did not differ among the three experimental groups EAE-vehicle (n = 5, CB1R/β-actin ratio: 1 ± 0.02), CFA-vehicle (n = 5, CB1R/β-actin ratio: 1.13 ± 0.28), and EAE-amphetamine (n = 4; CB1R/β-actin ratio: 0.84 ± 0.18; one-way ANOVA Tukey post hoc comparison: p > 0.05 for all comparisons; Fig. 4b, b’). Conversely, compared to control (CFA-vehicle n = 5, p-Ser316-CB1R/β-actin ratio: 1 ± 0.19) EAE striatal lysates (n = 5; p-Ser316-CB1R/β-actin ratio: 0.40 ± 0.12) showed a strong downregulation of the p-Ser316-CB1R, which was not recovered by amphetamine treatment (n = 4, p-Ser316-CB1R/β-actin ratio: 0.43 ± 0.10; one-way ANOVA Tukey post hoc comparisons: CFA-vehicle vs EAE-vehicle p < 0.05, CFA-vehicle vs EAE-amphetamine p > 0.05, EAE-vehicle vs EAE-amphetamine p > 0.05, Fig. 4c, c’), excluding this mechanism for amphetamine-mediated recovery of CB1R sensitivity on GABA terminals.