5-HT_2CR blockade in the amygdala conveys analgesic efficacy to SSRIs in a rat model of arthritis pain

Thresholds of hindlimb withdrawal reflexes evoked by mechanical compression of the knee joint were measured in sham controls (Figure 5A) and arthritic rats (Figure 5B). Compared to controls (n = 6 rats) arthritic rats (n = 7) had decreased thresholds, reflecting increased spinally organized reflexes and mechanical hypersensitivity (see vehicle groups in Figure 5A and 5B). None of the drug regimen had a significant effect in control rats (SB242084, 10 μM, n = 7; fluvoxamine, 30 mg/kg, n = 7; co-application of SB242084 and fluvoxamine, n = 7 rats) and in arthritic rats (SB242084, n = 6; fluvoxamine, n = 5; co-application of SB242084 and fluvoxamine, n = 7 rats). The data suggest that blockade of 5-HT_2CR in the BLA allowed the serotonergic system to affect supraspinally but not spinally organized behaviors.

Since the CeA serves as a major output nucleus for amygdala function related to pain modulation, we tested if 5-HT_2CR also played a role in this nucleus (Figure 6). We only tested the combination of SB242084 and fluvoxamine since the previous results showed that fluvoxamine alone and SB242084 applied into BLA had no effect. We also performed these tests in arthritic animals only since the combination of intra-BLA SB242084 and systemic fluvoxamine had no effect in normal animals. The results show that coapplication of intra-CeA SB242084 (10 μM) and systemic fluvoxamine (30 mg/kg) had no significant effect on audible and ultrasonic vocalizations to (normally) innocuous and noxious stimuli and on spinal reflexes in arthritic rats (n = 5 rats for each parameter).

Male Sprague Dawley rats (225–250 g) were housed in a temperature controlled room and maintained on a 12 h day/night cycle, with free access to food and water. On the day of the experiment, rats were transferred from the animal facility and allowed to acclimate to the laboratory for at least 1 h. At the end of the experiment, the animal was euthanized by decapitation using a guillotine (Harvard Apparatus Decapitator). All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Texas Medical Branch (UTMB) and conformed to the guidelines of the International Association for the Study of Pain (IASP) and of the National Institutes of Health (NIH).

A localized mono-arthritis was induced in the left knee joint by intra-articular injections of kaolin (4%, 80–100 μl) and carrageenan (2%, 80–100 μl) through the patellar ligament. This treatment paradigm reliably leads to inflammation and swelling of the knee within 1–3 h, reaches a maximum plateau at 5–6 h, and persists for several days [106]. Therefore, the 5–6 h time point was selected for measuring behaviors and testing drug effects. In sham control rats, the syringe was inserted into the knee joint cavity under the same conditions as in arthritic animals except that no compound was injected. Vehicle was not injected in sham animals because intraarticular saline injection causes a temporary swelling of the joint [55], which is one of the cardinal symptoms of an inflammation. To avoid any latent effect of increased intraarticular pressure, vehicle (sterile saline) was not injected in sham animals.

On Day 1, a guide cannula for drug (or artificial CSF, ACSF) application by microdialysis was stereotaxically inserted into the right BLA or CeA. On Day 2, 5–6 h after the induction of arthritis (or needle insertion for shams), behavioral experiments were performed 30 min after the systemic (i.p.) injection of the fluvoxamine or its vehicle (0.9% NaCl solution) in combination with an intra-BLA or intra-CeA application of SB242084 (or ACSF vehicle) for 20 min. SB242084 (selective 5-HT_2CR antagonist) and fluvoxamine (SSRI) were purchased from Tocris Bioscience.

Rats were deeply anaesthetized with pentobarbital sodium (Nembutal®, 50 mg/kg, i.p.) on Day 1. A guide cannula (David Kopf Instruments) was stereotaxically implanted into the right BLA or the right CeA, using the following coordinates: BLA, 2.8 mm caudal to bregma, 4.8 mm lateral to midline, 7.6 mm depth; CeA, 2.3 mm caudal to bregma, 4.0 mm lateral to midline, 7.0 mm depth [107]. Guide cannulas were affixed to the skull with dental acrylic (Plastic One, Roanoke, VA). Antibiotic ointment (Solosite Gel, Smith and Nephew) was applied to the exposed tissue to prevent infection. Local application of Lidocaine HCl (1%, 100 μl of 10 mg/ml) was done to minimize surgical pain and to prevent the animal from scratching of the surgical area upon recovery. On Day 2, a microdialysis probe (CMA/Microdialysis 11, Solna Sweden) that extended 1 mm beyond the tip of the guide cannula, was inserted for stereotaxic drug application into the amygdala. The probe was connected to an infusion pump (Harvard Apparatus, Holliston, MA) using polyethylene-50 tubing. Drugs or ACSF (vehicle) were applied for 20 min at a rate of 5 μl/min to establish equilibrium in the tissue. ACSF was oxygenated, equilibrated to pH 7.4 and contained the following (in mM): 125.0 NaCl, 2.6 KCl, 2.5 NaH₂PO₄, 1.3 CaCl₂, 0.9 MgCl₂, 21.0 NaHCO₃, and 3.5 glucose. SB242084 was dissolved in ACSF on the day of the experiment at a concentration 100-fold that predicted to be needed in the tissue based on data in the literature [22, 100‐102] because of the concentration gradient across the dialysis membrane and diffusion in the tissue [45, 48, 84, 85]. At the end of the experiment, rats were decapitated and injection sites were verified histologically after injection of methylene blue (1 μl) and plotted on standard diagrams adapted from Paxinos and Watson [107] (see Figure 1).

All averaged values are given as the mean ± standard error of the mean (SEM). GraphPad Prism 3.0 software (Graph-Pad, San Diego, CA) was used for all statistical analysis. For multiple comparisons, one-way analysis of variance (ANOVA) was used followed by Dunnett’s multiple comparisons tests. Statistical significance was accepted at the level P < 0.05.