Activation of GABAB receptors in central amygdala attenuates activity of PKCδ + neurons and suppresses punishment-resistant alcohol self-administration in rats

Drugs

For systemic injections, racemic baclofen (Sigma-Aldrich) was dissolved in saline and administered intraperitoneally (i.p.) at 0, 1, or 3 mg/kg, in a volume of 1 ml/kg. For intra-CeA injections, baclofen was dissolved in artificial cerebrospinal fluid (aCSF, 3525; TOCRIS) at 1 mM and was administered bilaterally in 0.3 µl, resulting in a dose of 64 ng / CeA. Alcohol solutions were prepared volume/volume (v/v) in tap water from 99% alcohol. Drugs were prepared fresh on the experimental day, and rats were habituated to the route of the administration before the test. For electrophysiology, baclofen was dissolved in milliQ water, and CGP55845 (1248, TOCRIS) was dissolved in 100% dimethyl sulfoxide (DMSO), and they were stocked at −20 °C.

Animals

Male Wistar rats (Charles River), weighing 250–300 g (7–9 weeks) at the beginning of the experiments, were pair-housed in a controlled environment (21 oC, humidity-controlled, reverse 12 h light-dark cycle). Rats were given free access to food pellets and tap water for the duration of the experiment and were weighed at least once a week. All behavioral testing was conducted during the dark phase of the light-dark cycle. Rats were habituated three times to the respective administration route before each experiment. All procedures were conducted in accordance with the European Union Directive 2010/63/Eu, and the protocol was approved by the Ethics Committee for Animal Care and Use at Linköping University.

Alcohol self-administration

Operant training and testing were performed in thirty-two identical operant chambers (Med Associates Inc., St Albans, VT, USA; 30.5 × 29.2 × 24.1 cm) housed in sound-attenuating cubicles. Each operant chamber was equipped with two retractable levers positioned laterally to a liquid cup receptacle. A total of 126 drug-naive rats were trained to self-administer 20% (v/v) alcohol without sucrose/saccharin fading as described previously [25,26,27,28]. Rats were trained initially to lever press on a fixed ratio 1 (FR1) 5 s time-out (TO) schedule to self-administer 20% alcohol during 30 min sessions. Pressing once on the lever associated with alcohol (active) was reinforced by the delivery of a volume of 100 μl of 20% alcohol in water in the adjacent drinking well and initiated a concomitant 5 s time-out period signaled by the illumination of the cue-light above the lever. Responses on the inactive lever and during the time-out period were recorded but had no programmed consequences. Once a stable self-administration baseline was reached, the sessions were conducted under a fixed ratio FR2 until a stable baseline of lever pressing was achieved (defined as a minimum of 15 sessions and no change greater than 15% in the total number of reinforcers earned during the last 3 sessions). Animals that did not acquire the self-administration procedure or earned less than 10 reinforcers in the baseline of the training sessions were excluded from the study [29].

Footshock punished alcohol self-administration

Compulsivity was assessed as responding for alcohol when its delivery was associated with a footshock punishment as previously described [7]. Briefly, conditions were identical to baseline self-administration (i.e 30 min sessions), but each completed FR2 ratio (i.e., 2 responses) was paired with a footshock (0.2 mA, 0.5 s), contingent with the delivery of a volume of 100 µl 20% alcohol in water in the adjacent drinking well. To classify rats as punishment-resistant or sensitive, we calculated a resistance score for each rat, calculated as: (punished alcohol deliveries) / (punished alcohol deliveries + mean alcohol deliveries of last 3 non-punished sessions) [6, 7]. The threshold 0.45 was used to classify shock-sensitive and -resistant rats based on a conservative limit of the shock-resistance distribution peak, identified with Hartigans’ test of unimodality (alpha = 0.001), which corresponds to about 20% decrease from baseline, unpunished alcohol self-administration [7]. Rats with a resistance score > 0.45 were classified as “punishment-resistant” while those with a score < 0.45 were classified as “punishment-sensitive”.

Systemic baclofen injections

Baclofen (0, 1, 3 mg/kg) was administered i.p. 30 min before the beginning of the self-administration session in shock-sensitive and shock-resistant rats (n = 14/group), using a within subject Latin-square design. Punished-alcohol self-administration baseline was re-established between drug tests in 3 consecutive sessions.

Cannula implantation and intracerebral microinjections

Surgeries were performed after 14 days of punished alcohol self-administration. Animals were anesthetized with isoflurane (2–3%, Baxter), and injected with buprenorphine (0.03 mg/kg, s.c.) 30 min before surgery to relieve pain. Ketoprofen (5 mg/kg, s.c.) was injected after surgery and the following day to relieve pain and reduce inflammation. We implanted guide cannulas (26 gauge, Plastics One) 2 mm above the CeA bilaterally using the following coordinates: AP, −2.5 mm; ML, ± 4.5 mm; DV, −6.4 mm. Cannulas were anchored to the skull with jeweler´s screws and dental cement (Paladur, Agnthos, Lidingö, Sweden). Animals were allowed to recover from surgery for one week and were then allowed to re-establish their baseline of punished self-administration.

Rats received baclofen in 0.3 µl/side through 33-gauge injectors (2 mm below the cannula placement) at the rate of 0.15 ul/min, 15 min prior to the punishment session. At the end of the experiments, animals were transcardially perfused, and brains were removed for performing fluorescent immunohistochemistry and verifying the cannula placement.

To verify the injection site and cannula placement, we selected 4 series of sections from each rat, and coronal sections of 40 μm were mounted on slides and stained with Cresyl violet. Placements of the injector were determined through a Leica DMi8 microscope with a 10x objective lens and brightfield images were mapped onto coronal sections of a rat brain stereotaxic atlas [30].

Saccharin self-administration

Saccharin self-administration was performed as previously described [27, 28], under conditions similar to those of alcohol self-administration. Briefly, a total of 36 drug-naive rats were trained to self-administer 0.2% saccharin in 30 minutes sessions under a FR2 5 s time-out schedule of reinforcement. A total of 15 FR2 sessions were performed to reach a stable baseline (no change greater than 15% in the total number of reinforcers earned during the last 3 sessions). The effect of systemic (0, 1, 3 mg/kg) and central (64 ng/0.3 ul) baclofen injections were tested 30 or 15 min before the respective saccharin self-administration session.

Locomotor activity

Baseline locomotor activity was measured in sound-attenuated behavioral chambers equipped with an open field (43 × 43 cm) containing infrared beam detectors (Med Associates, St. Albans, VT) as previously described [27]. Horizontal and vertical movements were examined for 30 min (to match the duration of the alcohol self-administration session) under non-habituated conditions, at ambient light level of 190–210 lux. To assess whether the effects of systemic and central baclofen on alcohol were related to motor impairment, locomotor activity was measured in two separate groups of rats (n = 27; n = 20, respectively) that were pretreated with vehicle or baclofen 30 (systemic) or 15 min (central injections) before the respective locomotor activity session for evaluating the effects of systemic and central baclofen, respectively. To assess whether the interaction of alcohol and baclofen resulted in sedative effects beyond those of baclofen alone, we analyzed the distance traveled (cm) in the operant chambers during the self‐administration sessions, measured using infrared beams built into the operant chambers and spaced 18.1 cm apart. Duplicate events were detected and removed from the total count as previously described [28].

RNAscope in situ hybridization

A group of 6 drug-naive rats were deeply anesthetized with isoflurane and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (PFA). Brains were removed and postfixed in 4% PFA 4 h, then transferred into 30% sucrose solution till sinking. Coronal brain sections (14 µm) were obtained using a Leica cryostat and stored in cryoprotectant (20% glycerol and 30% ethylene glycol in 0.1 M PBS).

Multiplex fluorescent in situ hybridization assay was performed using RNAscope® Multiplex Fluorescent Detection Kit v2 (catalog no. 323110, Advanced Cell Diagnostics) according to the user manual for fixed tissue. Briefly, brain sections were washed in PBS, and mounted on slides. They were post-fixed in 10% neutral-buffered formalin (Thermo Fisher Scientific), dehydrated in 50, 70, 100, and 100% ethanol, and treated with RNAscope Hydrogen Peroxide 10 min at room temperature. Sections were incubated with RNAscope Protease III at 40 °C for 30 min in the HybEZ oven (Advanced Cell Diagnostics), followed by incubation with probes for Prkcd mRNA (catalog no. 441791, GeneBank accession number NM_011103.3, Advanced Cell Diagnostics) and Gabbr1 mRNA (catalog no. 546461-C2, GeneBank accession number NM_031028.3, Advanced Cell Diagnostics) at 40 °C for 2 h. Sections were then incubated with Hybridize AMP1, 2 and 3, after which they were developed for HRP-C1 signal with TSA Plus Cyanine 3 (1:3000 diluted, catalog no. NEL744001KT, PerkinElmer) and HRP-C2 signal with TSA Plus Cyanine 5 (1:3000 diluted, catalog no. NEL745001KT, PerkinElmer). After incubation with DAPI for 30 s at room temperature, slides were cover-slipped with the Antifade Mountant (catalog no. P36961, Invitrogen).

Images of Prkcd and Gabbr1 expression were acquired through a Zeiss LSM 800 upright confocal microscope using a 20x objective lens and cells were identified by DAPI staining. For quantification of Prkcd, and Gabbr1 positive cells (N = 6 rats) we counted the total cells of the fluorescent signal (fluorescent “dots”) from two hemispheres of 2 sections. A single fluorescent dot represents the signal that was amplified from the specific probe of the target mRNA and positive cells expressing Prkcd and/or Gabbr1 were detected above the intensity threshold of the assay, 15163 and 13621 respectively, using ImageJ software [31, 32]. Estimated cell counts were averaged across sections for quantification of Prkcd and Gabbr1 expression levels. Positive cells were counted for the respective probe or their combination using the to determine the overlap in PKCδ and GABAB receptor expression.

Fluorescent immunohistochemistry

Rats were anesthetized using isoflurane 90 min after the start of footshock sessions on day 15, and transcardially perfused with 0.9% saline followed by 4% PFA. Brains were removed and postfixed in 4% PFA for 2 h, then transferred into 30% sucrose solution at 4 °C until sinking. Coronal brain sections (20 µm) of the amygdala (AP bregma level of −1.92 to −2.92 mm) were collected using a Leica cryostat and stored in cryoprotectant at −20 °C until further processing.

For Fos and PKCδ immunofluorescent staining, floating brain sections were washed in PBS 3 × 10 min, then blocked in a solution of 4% bovine serum albumin (BSA) and 0.2% Triton X-100 dissolved in PBS for 1 h at room temperature. For labeling Fos and PKCδ the following antibodies were used: rabbit anti-Fos (1:1000, Abcam, ab190289, RRID: AB_2737414), mouse anti-PKCδ (1:500, BD Bioscience, 610398, RRID: AB_397781). Sections were incubated with primary antibodies overnight at 4 °C. After rinsing in PBS three times, the sections were incubated 2 h at room temperature with following secondary antibodies: donkey anti-rabbit Alexa Fluor 488 (1:200, Thermo Fisher Scientific, A-21206, RRID: AB_2535792), goat anti-mouse Alexa Fluor 568 (1:200, Thermo Fisher Scientific, A-11004, RRID: AB_2534072). Sections were rinsed in PBS three times, mounted on slides, and cover-slipped with Antifade Mountant DAPI (Invitrogen, P36962) or the Antifade Mountant (Invitrogen, P36961).

All Fos and PKCδ immunofluorescence images were acquired through a Zeiss LSM 800 upright confocal microscope using a 20x objective lens. We quantified the total number of Fos and PKCδ positive cells in CeA in a manner blinded to the experimental condition. For each rat, labelled cells were quantified from two hemispheres of 3 sections, and we averaged the counts to give a mean number of each immunoreactive cell type. All images were adjusted to match contrast and brightness in Fiji software; cells were identified by DAPI staining.

Slice preparation and ex vivo electrophysiology

For electrophysiological recordings, behaviorally naïve 8-10 weeks old male Wistar rats (Charles River, Germany) were deeply anesthetized with isoflurane and decapitated. Brains were quickly removed and placed into an ice-cold N-methyl-D-glucamine (NMDG)-based cutting solution [33] containing (in mM): 92 NMDG, 20 HEPES, 25 glucose, 30 NaHCO3, 1.2 NaH2PO4, 2.5 KCl, 5 sodium ascorbate, 3 sodium pyruvate, 2 thiourea, 10 MgSO4, and 0.5 CaCl2 (310 mOsm, pH 7.4 adjusted using HCl). Acute coronal brain slices (250 μm thick) containing the CeA were obtained using a vibratome (Leica VT1200 S, Leica Biosystems Inc., IL, USA). After cutting, slices were transferred to a holding chamber filled with a pre-warmed (∼34 °C) artificial cerebrospinal fluid (aCSF, in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1 MgCl2, 11 glucose, 26 NaHCO3, 2.4 CaCl2 (310 mOsm, pH 7.4). Subsequently, the solution was maintained at room temperature, and after > 1 h of recovery, a single slice was transferred to the recording chamber and continuously perfused with warmed (∼30–32 °C) aCSF solution at a flow rate of ∼2.0 ml/min. All solutions were saturated with 95% O2 and 5% CO2. Neurons were visualized with infrared differential interference contrast (IR-DIC) optics on a Zeiss Examiner A1 microscope (Carl Zeiss AB, Stockholm, Sweden) and recordings were aimed at centrolateral amygdala (CeL) neurons. Electrophysiological recordings were carried out using borosilicate glass patch pipettes (2.5–3.0 MΩ; Harvard Apparatus, MA, USA) containing (in mM): 135 K-gluconate, 20 KCl, 10 HEPES, 0.1 EGTA, 2 MgCl2, 2 Mg-ATP, 0.3 Na-GTP (290 mOsm, pH adjusted to 7.3 using KOH). Recordings were performed using a Multiclamp 700B amplifier (Molecular Devices, CA, USA), digitalized with a Digidata 1440 A (Molecular Devices, CA, USA; 2 kHz low-pass Bessel filter and acquired 20 kHz), and acquired and analyzed with pClamp 10.7 software (Molecular Devices, CA, USA). The estimated junction potential was 11 mV and was not compensated during electrophysiological recordings.

Statistical analysis

All data were analyzed with STATISTICA, Stat Soft 13.0 (RRID:SCR_014213), using Student’s t-test or analysis of variance (ANOVA), with factors and degrees of freedom for the respective analysis indicated in conjunction with its results. Statistically significant difference was set at P < 0.05. Post-hoc analyses were conducted when appropriate using Newman-Keuls test. The data are presented as the mean ± SEM. Prior to ANOVA, data were examined for significant violations for assumptions of homogeneity of variance and normality of distribution using Levene’s and Shapiro-Wilk test, respectively. Where homogeneity of variance or normality were significantly violated, data were square root transformed.

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