Background
Extinction of fear is defined as a reduction in conditioned fear when a conditioned stimulus (CS) is repeatedly presented in the absence of an unconditioned stimulus (US). The inability to extinguish intense fear memories is an important clinical problem in patients with psychiatric disorders involving dysregulation of fear, such as specific phobias, panic disorder, and post-traumatic stress disorder [
1‐
6]. Increasing interest has developed for the role of innate immune cytokines in impaired neuronal function and cognition that arise with trauma, infection, and/or disease. Several clinical studies have shown that levels of the innate immune cytokines, such as interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α, are correlated with impaired fear extinction [
7‐
9]. However, the mechanism underlying the correlation between cytokines and psychiatric disorders remains unclear.
Behavioral tests designed to model this aspect of mental disorders are based on Pavlovian principles of associating an innocuous cue, such as a tone or light (CS), with a painful or aversive stimulus, such as an electrical foot-shock (US). Conditioned fear responses can then be indexed through various outputs, such as conditioned freezing [
10]. Rodent models of Pavlovian conditioning have been widely used to study consolidation, extinction, and reconsolidation of fear memory [
11]. Extinction in fear conditioning studies involves exposing rodents to a fear eliciting cue(s) or context without the aversive US [
12,
13]. The fear extinction behavior is thought to be an active learning process and not simply “forgetting” a conditioned behavior that reverses the original learning [
14].
It has been established that the basolateral amygdala (BLA) is a key brain structure for extinction learning [
10]. To further reveal the pathological roles of cytokines in impaired fear memory, we directly tested whether microinfusion of interferon (IFN)-α into the amygdala affects auditory fear extinction in rats. IFN-α is an innate immune cytokine with antiviral and anti-proliferative activities and is therefore used to treat infectious diseases and cancers [
15,
16]. The involvement of IFN-α in the brain function has been demonstrated by clinical studies showing that IFN-α induces high rates of behavioral disturbance, including depression, in 30–50 % of IFN-α-treated patients [
10,
17‐
24]. Some experimental studies have explored the potential mechanisms of IFN-α-induced depression by systemic or intra-cerebroventricular injections of IFN-α in rodents [
25‐
28]. However, no study has investigated the effects of IFN-α on fear extinction.
In this study, we first determined that directly infusing exogenous IFN-α into the amygdala impaired the extinction of conditioned fear in rats. Because glial cells play active roles in initiating and maintaining the inflammatory process in the brain, we further examined the activation of microglia and astrocytes in the amygdala following the IFN-α infusion. Next, we determined whether administering minocycline, an inhibitor of microglial activation, ameliorated IFN-α-induced impairment of fear extinction.
Methods
Subjects
This study conformed with the policies and procedures detailed in the “Guide for the Care and Use of Laboratory Animals” of the National Institutes of Health. The animal experimental protocols of the “Guide” and the treatment procedures were reviewed and approved by the Animal Care and Use Committee of China Medical University (No. 2014195). Male Wistar rats (weight, 250–300 g) from our own colony were housed in a humidity- (50–55 %) and temperature-controlled (22–24 °C) facility under a 12-h light/dark cycle (lights on at 7:30 a.m.). The animals had free access to food (standard laboratory rat chow) and water. All surgeries were performed under anesthesia, and all efforts were made to minimize animal suffering.
Behavioral apparatus
The rats were fear conditioned in a 25 × 29 × 28-cm chamber (context A) constructed of aluminum and Plexiglas walls (Coulbourn Inst., Allentown, PA, USA). The floor consisted of stainless steel bars that could be electrified to deliver a mild shock, and illumination was provided by a single overhead light. The chamber had a speaker mounted on the outside wall and was placed inside a sound-attenuating box. The fear conditioning chambers were cleaned with 5 % ethanol each time a rat was removed from the chamber. Fear conditioning of the rats was extinguished and tested in context B, wherein the chamber was modified by introducing a black Plexiglas floor washed with peppermint soap. The wall pattern was changed to black and white stripes, and three house lights were installed. The CS was a 5-kHz tone with a 20-s duration and 75 dB intensity. The US was a 1.0-mA foot shock of 0.5-s duration, which co-terminated with the tone during the conditioning phase.
Drugs
Recombinant human IFN-α was obtained from PeproTech Inc. (#300-02AB; Rocky Hill, NJ, USA) and was dissolved in artificial cerebrospinal fluid (ACSF; glucose, 5 mM; CaCl2, 1 mM; NaCl, 125 mM; MgCl2, 1 mM; NaHCO3, 27 mM; KCl, 0.5 mM; Na2SO4, 0.5 mM; NaH2PO4, 0.5 mM; and Na2HPO4, 1.2 mM). Rat serum albumin (1 mg) was added to 1 ml of 2 × 107 IU/ml IFN-α. The rats received bilateral infusions of ACSF (vehicle) or IFN-α at doses of 100, 200, or 400 IU/μl (1 μl/side).
Minocycline hydrochloride (#M9511; Sigma, St. Louis, MO, USA) was dissolved fresh in 0.9 % NaCl and administered intragastrically (i.g.) once daily at a dosage of 90 mg/kg rat body weight for 3 days prior to the IFN-α treatment. The dose was selected on the basis of previous studies showing the beneficial effects of this dosage in animal models of cerebral brain ischemia, multiple sclerosis, and Parkinson’s disease [
29‐
33].
Cannula implantation and microinjections
The rats were anesthetized with sodium pentobarbital (50 mg/kg) intraperitoneally (i.p.) and mounted on a stereotaxic apparatus (SR-5R; Narishige, Tokyo, Japan) for surgery. Two cannulae consisting of a 22-gauge stainless steel tubing were implanted bilaterally into the BLA. The coordinates were AP, −2.3 mm; ML, ±4.5 mm; and DV, −7.0 mm according to the Paxinos and Watson brain atlas [
34]. Three jewelry screws were implanted in the skull as anchors, and the entire assembly was affixed to the skull with dental cement. A 28-gauge dummy cannula was inserted into each cannula to prevent clogging. After the surgical procedure, the rats were monitored daily and given 1 week to recover prior to the experiment. The microinjections were performed at 10:00–12:00 a.m. IFN-α was dissolved in sterile ACSF and injected into the BLA via a 28-gauge infusion cannula connected with polyethylene (PE 20) tubing to a 10-μl Hamilton microsyringe (Hamilton Co., Reno, NV, USA). The infusion cannula protruded 0.5 mm beyond the guide cannula. An infusion volume of 1 μl was delivered using a Harvard PHD2000 syringe pump (Harvard Apparatus, Holliston, MA, USA) over the course of 10 min (at a rate of 0.1 μl/min). The infusion cannula remained in place for at least 1 min after the infusion before being pulled out to prevent backflow of the injectate through the guide cannula.
After all behavioral tests were completed, the rats were anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and transcardially perfused with paraformaldehyde (4 %, pH 7.4). The brains were removed and sectioned, and slides were prepared and examined under a light microscope to verify that the cannulae were placed in the BLA. Only rats with proper placement of the cannulae were included in statistical analyses.
Fear conditioning and extinction and drug application
Acclimation to the experimental conditions is an important measure to reduce unpredictable effects on behavior. Before the behavioral experiments, the rats were acclimated to handling and to the laboratory for 5 days. The rats were habituated to the test chamber for 30 min (contexts A and B for 15 min each in a counterbalanced manner) on day 5. The rats were placed in the context A chamber the next day and received a tone habituation session consisting of five CS presentations (5 kHz tone, 75 dB, 20 s). The rats’ behavior was measured as the baseline response. Next, the rats received seven tone–shock pairs in context A (conditioning session) for auditory fear conditioning.
We only selected rats for the extinction experiment that showed equivalent levels of behavioral response during the conditioning session to exclude the potential effects of the rat’s internal characteristics, surgery, or other health differences. The conditioned rats were divided into four groups. Each group received an infusion of either vehicle or IFN-α into the BLA (100, 200, or 400 IU/side, bilaterally) and was returned to their cage. The rats were placed in context B 8 h later and received an extinction session consisting of 15 tone–alone trials of 20 s each. The extinction session was designed to test the rat’s fear memory to the auditory CS rather than to the environment. Thus, we changed the wall pattern (blank to striped), floor material (steel to Plexiglas), illumination (one to three lights), and smell (ethanol to peppermint soap). The freezing behavior of all rats was scored by the same experimenter who was blind to the experimental conditions to reduce subjective error, and behavior was scored through a video camera. Freezing responses were judged as the absence of all movement except those related to respiration [
35,
36]. The total duration of the freezing response during tone presentation (20 s) was recorded and transformed into a percentage of freezing (seconds spent freezing/20-s CS).
To evaluate whether minocycline could modulate the effects of IFN-α on extinction memory, the rats were assigned randomly to either the vehicle or minocycline group. Before the fear conditioning and extinction experiments, the rats received daily administration of the vehicle or minocycline at a dose of 90 mg/kg i.g. for 3 days. After the rats completed auditory fear conditioning in context A, they received an intra-amygdala infusion of IFN-α (400 IU/side). The rats underwent extinction training in context B 8 h later. Another group of rats received only minocycline without IFN-α to assess the effect of minocycline on fear extinction.
Immunohistochemistry
Immediately following the behavioral experiments, the rats were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and perfused via the ascending aorta with cold 0.9 % NaCl followed by chilled 4 % paraformaldehyde in 0.01 M phosphate-buffered saline (PBS). The brains were post-fixed in the same fixative for 24 h at 4 °C and embedded in paraffin for sectioning at 5 μm. Serial coronal sections were cut through the amygdala (at a level corresponding to 2–3 mm posterior to the bregma) [
34].
Immunohistochemical staining was performed using the avidin–biotin–peroxidase complex detection kit and diaminobenzidine substrate. Microglial activation was measured using an antibody to ionized calcium-binding adaptor molecule 1 (Iba1; 1:100, goat polyclonal; Abcam, Shanghai, China). Astrocytic activation was measured using an antibody to glial fibrillary acidic protein (GFAP). Sections were incubated with their primary antibodies for 16 h at 4 °C. Negative control sections were incubated with PBS instead of primary antibodies. The sections were incubated with the appropriate avidin–biotin complex solutions (Zhongshan Golden Bridge, Beijing, China) at 37 °C for 20 min. All sections were counterstained with Harris’s hematoxylin.
Cell quantification
To minimize any potential confounding effects from immunohistochemistry, the sections were prepared, stained, and imaged at the same time as their relevant control. Furthermore, the cell number was counted in a predefined area of the brain. Nine sections among the serial coronal sections of the amygdala were selected from each brain, which were centered at the site of the cannula tip and separated by 10 sections (50 μm). The areas of the amygdala were captured using an Olympus BX51 automatic microscope (Tokyo, Japan). The total numbers of cells stained with GFAP, Iba1, or neuronal nuclear antigen (NeuN) in a 400 × 400 μm area (cannula tip centered) were marked by an operator who was blinded to the identity of the sections, and an automated cell count was generated using an image analysis system. Only morphologically intact and clearly identifiable cells were counted in the regions. As no obvious difference in cell profiles was detected between the two hemispheres, the right and left hemisphere values were averaged for each rat. The number of cells in each section was averaged to obtain a mean value for each animal (nine sections/rat). The mean values obtained from five rats in each group were used for the statistical analysis.
Measurement of proinflammatory cytokines
IL-1β and TNF-α levels in the amygdala were measured using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions (Neobioscience, Shenzhen, China). The rats were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.), and the brain was removed rapidly and frozen at −20 °C for 20 min. To reduce the possible error caused by brain sampling, we placed the frozen brain on a rat brain section mold (# 68709; RWD Life Science, Shenzhen, China) and carefully dissected the brain tissues corresponding to the amygdala (AP −1.0 to −4.0 mm, ML 4.0–6.0 mm, and DV 7.0–9.0 mm) using a sharp steel blade. Brain tissues from both hemispheres were mixed and homogenized on ice in 0.01 M PBS (pH, 7.4) and centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatants were collected and stored at −80 °C until the measurement of IL-1β and TNF-α by ELISA. All samples were measured in duplicate and adjusted according to the protein content determined using an enhanced BCA Protein Assay kit (Beyotime, Harman, China). The results are expressed as picogram per milligram protein.
Statistical analyses
Statistical analyses were performed using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). Depending on whether data were normally distributed or not (determined using the Kolmogorov–Smirnov test), either parametric or nonparametric test was used for statistical evaluation. Two-way repeated-measures analysis of variance (ANOVA) was performed on the freezing response data among the different groups and trials. Differences in the immunohistochemical data and ELISA results were detected by one-way ANOVA. Each ANOVA reporting significant effects was followed by Tukey’s post hoc test of multiple comparison. A p value of <0.05 was considered statistically significant.
Abbreviations
ACSF, artificial cerebrospinal fluid; ANOVA, analysis of variance; BLA, basolateral amygdala; CS, conditioned stimulus; ELISA, enzyme-linked immunosorbent assay; GFAP, glial fibrillary acidic protein; Iba1, calcium-binding adaptor molecule 1; IFN, interferon; IL-1β, interleukin-1β; IL-6, interleukin-6; NeuN, neuron-specific nuclear protein; PBS, phosphate-buffered saline; TNF-α, tumor necrosis factor-α; US, unconditioned stimulus
Acknowledgements
Not applicable.