Expression of recombinant proteins

The coding region of murine full-length (1–223) σ1R (AF004927), and the C-terminal region of the glutamate NMDAR NR1 subunit (NM_008169) (residues 827–938), were amplified by RT-PCR using total RNA isolated from mouse brains as the template. Specific primers containing an upstream Sgf I restriction site and a downstream Pme I restriction site were used, as described previously [27]. The PCR products were cloned downstream of the GST coding sequence and the TEV protease site. The sequenced proteins were identical to the GenBank™ sequences. The vector was introduced into E. coli BL21 (KRX #L3002, Promega, Madrid, Spain), and clones were selected on solid medium containing ampicillin. After 3 h of induction at room temperature (1 mM IPTG and 0.1% Rhamnose), the cells were collected by centrifugation, and the pellets were maintained at − 80 °C. The GST fusion proteins were purified under native conditions on GStrap FF columns (GE#17–5130-01, Healthcare, Barcelona, Spain); when necessary, the fusion proteins retained were cleaved on the column with ProTEV protease (Promega, #V605A) and further purification was achieved by high-resolution ion exchange (Enrich Q, BioRad #780–0001) or electroelution of the corresponding gel band (GE 200, Hoefer Scientific Instruments, San Francisco, CA, USA). The sequences were confirmed through automated capillary sequencing.

Animals and drugs

Male albino CD-1 mice and homozygous (σ1R−/−) male sigma receptor knockout mice, backcrossed (N10 generation) onto a CD1 albino genetic background (ENVIGO, Milano, Italy) were used in the study. The mice were maintained at 22 °C on a diurnal 12 h light/dark cycle. Procedures involving mice adhered strictly to the guidelines of the European Community for the Care and Use of Laboratory Animals (Council Directive 86/609/EEC) and Spanish law (RD53/2013) regulating animal research. Each group consisted of eight to ten animals, which were used only once. The compounds used were as follows: morphine sulfate (mu-opiod receptor agonist, Merck, Darmstadt, Germany); NMDA (#0114); CBD (#1570); BD1063 (σ1R antagonist #0883); PRE084 (σ1R agonist #0589); (±)-PPCC oxalate (σ1R agonist #3870), 4-IBP (σ1R ligand #0748); WAY100635 maleate (5HT1A receptor antagonist #4380) were obtained from Tocris Bioscience (Bristol, UK). Progesterone (σ1R antagonist P7556) and pregnenolone sulfate (σ1R agonist P162) were obtained from Sigma-Aldrich (Spain). Test drugs were dissolved in saline except CBD and PPCC, which were prepared in a 1:1:18 (v/v/v) mixture of ethanol:Kolliphor EL (#C5135, Sigma-Aldrich): physiological saline, and injected intracerebroventricularly (icv) 30 min before NMDA administration. To facilitate selective and straightforward access to their targets, the compounds were injected (4 μL) into the lateral ventricles of mice as previously described [79]. Animals were lightly anesthetized and injections were performed with a 10 μL Hamilton syringe at a depth of 3 mm at a point of 2 mm lateral and 2 mm caudal from the bregma. The 4 μL were infused at a rate of 1 μL every 5 s. After this the needle was maintained for an additional 10 s. Mice were randomly assigned to each treatment of the selected compounds (power of 80% to detect statistically significant differences). The use of drugs, experimental design and sample size determination were approved by the Ethical Committee for Research of the CSIC (SAF2015–65420 & CAM PROEX 225/14).

Experimental protocols

In vitro interactions between recombinant proteins: Pull-down of recombinant proteins, effect of drugs on σ1R-NR1 interactions

Having demonstrated that the σ1R does not bind to GST (Z02039; GenScript Co., Piscataway, NJ, USA) [26], we assessed the association of GST-free σ1Rs with GST-tagged NMDAR NR1 C-terminal sequence, which was immobilized through covalent attachment to NHS-activated Sepharose 4 fast flow (FF) (GE#17–0906-01; General Electric Healthcare, Spain) according to the manufacturer’s instructions. The recombinant σ1R (100 nM) was incubated either with NHS-blocked Sepharose 4FF (negative control) or with the immobilized NR1 protein fragment in 200 μL of a buffer containing 50 mM Tris-HCl (pH 7.5) and 0.2% CHAPS in the presence of 2.5 mM of CaCl2. In pilot assays, we determined that after 20 min of incubation the NR1-σ1R association was maximal and that, this period of time was also sufficient for the drugs to promote stable changes in their association. Thus, the samples were mixed by rotation for 20 min at RT, and σ1Rs bound to NR1-Sepharose 4FF were recovered by centrifugation and three cycles of washing. The agarose-attached NR1-σ1R complexes were incubated in the presence of increasing concentrations of the drugs under study for 20 min with rotation at room temperature in 300 μL of 50 mM Tris-HCl (pH 7.5), 2.5 mM CaCl2, and 0.2% CHAPS. In this assay, σ1R ligands dissolved in aqueous solutions display calcium- and concentration-dependent activity in altering σ1R-NR1 associations. If an organic solvent, such as DMSO, is required to incorporate the drug under study, i.e., CBD, DMSO must be kept below 1% in the buffer of the assay. Higher concentrations of DMSO stabilize σ1R-NR1 associations and diminish the disruptive effects of σ1R antagonists. Thus, CBD was initially dissolved in 100% DMSO, and through serial dilutions, the concentrations used in the study were obtained with a final DMSO concentration of approximately 1%. Agarose pellets containing the bound proteins were obtained by centrifugation, washed thrice in the presence of 2.5 mM CaCl2, and solubilized in 2× Laemmli buffer, and the content of σ1Rs was addressed by Western blotting.

The detached σ1Rs from the aforementioned procedure were resolved with SDS/polyacrylamide gel electrophoresis (PAGE) in 4–12% Bis-Tris gels (NuPAGE NP0341, Invitrogen, Thermo Fisher Scientific, Spain) with MES SDS running buffer (NuPAGE NP0002, Invitrogen) and then transferred onto 0.2 μm polyvinylidene difluoride (PVDF) membranes (162–0176; Bio-Rad, Madrid, Spain). The anti-σ1R (#42–3300, Invitrogen) diluted in Tris-buffered saline pH 7.7 (TBS) + 0.05% Tween 20 (TTBS) was incubated overnight at 6 °C. The primary antibody was detected using secondary antibodies conjugated to horseradish peroxidase. The western blot images showing antibody binding, were visualized by chemiluminescence (#170–5061; Bio-Rad) and recorded using a ChemiImager IS-5500 (Alpha Innotech, San Leandro, CA).

Evaluation of antinociception

The response of the animals to nociceptive stimuli was determined by the warm water (52 °C) tail-flick test as previously described [27]. The tail-flick analgesic test applies a thermal noxious stimulus to promote flicking of the mouse’s tail, and opioids given by icv route increase the time elapsed between application of the stimulus and the flick. This response comprises a spinal reflex that is under facilitator drive by the brain stem nociceptive modulating network. Baseline latencies ranged from 1.5 to 2.2 s. A cut-off time of 10 s was used to minimize the risk of tissue damage. Drugs were icv injected and antinociception was assessed at different time intervals thereafter. Saline was likewise administered as a control. Antinociception was expressed as a percentage of the maximum possible effect (MPE = 100 × [test latency-baseline latency]/[cut-off time (10 s)-baseline latency]).

NMDA-induced seizures

Seizures were induced by injection of NMDA (0.3 and 1 nmol/mouse icv, in a volume of 4 μL sterile saline) as described by others [44]. The dose of 1 nmol NMDA was selected as the minimal dose that reliably induced the appearance of tonic seizures in at least 80% of treated mice. Immediately after injection animals were placed in a transparent box (20x20x30 cm) and were observed for a period of 3 min. The seizure activity consisted of a mild myoclonic phase (immobility, mouth and facial movements, tail extension, circling), rearing (violent movements of the hole body, rearing), wild running (episodes of running with explosive jumps), clonic convulsions (characterized by rigidity of the whole body including limbs flexion/extension), followed by continuous/repetitive seizure activity (tonic seizures) and, in approximately 15–20% of the animals, death. The episode typically began a few seconds after injection and evolved to its maximal intensity in less than 1 min. The results are expressed as the percentage of mice exhibiting the aforementioned signs and the mean latencies of the first body clonus.

Permanent unilateral middle cerebral artery occlusion (pMCAO) and the determination of infarct size

Focal cerebral ischemia was induced via pMCAO, as described previously [36]. Briefly, mice were anesthetized and a vertical skin incision was made between the left eye and ear under a dissection microscope. After drilling a small hole in the cranium at the level of the distal portion of the middle cerebral artery, the artery was occluded by cauterization. Flow obstruction was visually verified. Animals showing subdural haemorrhages or signs of incorrect surgery were immediately excluded from the study (< 5% in each group). The mice were returned to their cages after surgery, kept at room temperature, and allowed food and water ad libitum. Strong lesion reproducibility was observed. We exclude mice from further studies if excessive bleeding occurs during surgery, mice fail to recover from anaesthesia within 15 min, or haemorrhage was found in the brain during post-mortem examination. The investigator performing the pMCAO surgery was blinded to treatment group. To determine the infarct size 48 h after surgery, animals were euthanized and their brains were removed, after which six 1 mm-thick coronal brain slices (Brain Matrix, WPI, UK) were obtained. The sections were stained with 2,3,5-triphenyltetrazolium chloride (1% TTC, Sigma-Aldrich). Infarct volumes were calculated by sampling each side of the coronal sections with a digital camera (Nikon Coolpix 990, Tokyo, Japan). The extent of unstained infarct area (expressed in mm2) was integrated from the total area as an orthogonal projection.

Statistical analysis

The data represent the means ± SEM. The Sigmaplot/SigmaStat v.14 package (SPSS Science Software, Erkrath, Germany) was used to generate the graphs, determine parameters (interaction of drugs with σ1R-NR1 complexes), and perform the corresponding statistical analysis. The level of significance was p < 0.05. Data were analyzed using one-way ANOVA followed by Dunnett multiple comparisons as appropriated.