Decreased Neuronal Excitability in Medial Prefrontal Cortex during Morphine Withdrawal is associated with enhanced SK channel activity and upregulation of small GTPase Rac1

Rationale: Neuroadaptations in the medial prefrontal cortex (mPFC) and Nucleus Accumbens (NAc) play a role in the disruption of control-reward circuits in opioid addiction. Small Conductance Calcium-Activated Potassium (SK) channels in the mPFC have been implicated in neuronal excitability changes during morphine withdrawal. However, the mechanism that modulates SK channels during withdrawal is still unknown. Methods: Rats were exposed for one week to daily morphine injections (10 mg·kg-1 s.c.) followed by conditional place preference (CPP) assessment. One week after withdrawal, electrophysiological, morphological and molecular biological methods were applied to investigate the effects of morphine on SK channels in mPFC, including infralimbic (IL), prelimbic (PrL) cortices and NAc (core and shell). We verified the hypothesis that Rac1, a member of Rho family of small GTPases, implicated in SK channel regulation, modulate SK channel neuroadaptations during opiate withdrawal. Results: One week after morphine withdrawal, the neuronal excitability of layer 5 pyramidal neurons in IL was decreased, but not in PrL. Whereas, the excitability was increased in NAc-shell, but not in NAc-core. In mPFC, the expression of the SK3 subunit was enhanced after one-week of withdrawal compared to controls. In the IL, Rac1 signaling was increased during withdrawal, and the Rac1 inhibitor NSC23766 disrupted SK current, which increased neuronal firing. Suppression of Rac1 inhibited morphine-induced CPP and expression of SK channels in IL. Conclusions: These findings highlight the potential value of SK channels and the upstream molecule Rac1, which may throw light on the therapeutic mechanism of neuromodulation treatment for opioid dependence.

Whole-cell recordings were achieved for mPFC pyramidal neurons and NAc MSNs with the guidance of differential interference contrast microscopy (BX51WI; Olympus, Japan) and a CCD camera (Olympus, Japan). Borosilicate glass micropipettes (3)(4)(5) were prepared by a P-97 horizontal micropipette puller (Axon Instr., USA). The intracellular solution used in whole-cell voltage and current clamp recordings contained (in mM): 130 KOH, 2.8 NaCl, 17 HCl, 20 HEPES, 105 methane sulfonic acid, 0.3 EGTA, 2.5 MgATP, 0.25 GTP ( pH 7.2-7.4, 275-285 mOsm). EGTA was incorporated in the pipette solution to maintain calcium-dependent potassium currents during recordings [2]. To measure firing, we applied current pulses by a patch amplifier in current clamp mode, and applied a sequence of 7-8 current pulses (300 ms duration, 20 pA apart) for every 30 seconds. The minimum current amplitude was attuned for each neuron, in order to make the first pulse just under the spike firing threshold. The resting membrane potential was set to -90 mV before analysis of firing. For SK current measurement, neurons were held at -70 mV, then depolarized for 400 ms to steps ranging from -40 to -10 mV (with 10 mV between steps) prior to being brought back to -70 mV. The SK tail current was observed upon returning to -70 mV. Depolarizing pulses were combined with a 33.3 pA hyperpolarizing pulse to test the input resistance. We utilized the anterior commissure, the lateral ventricles and the dorsal striatum as landmarks for locating the position of the NAc shell, NAc core, prelimbic cortex and infralimbic cortex for the patch clamping. The shape of NAc shell is ring-like in coronal section. The distance between NAc shell to AC is about 4 mm-13 mm. The NAc shell or layer 5 pyramidal cells located in the infralimbic subregion of the mPFC were visually recognized by using an upright infrared differential interference contrast microscope (BX51WI; Olympus, Japan).

Brain stereotaxic injection of TMR into NAc for retrograde tract-tracing
All surgical procedures for 11 rats were deeply anesthetized with pentobarbital sodium (i.p., 40 mg·kg −1 , Cat. # P11011, Merck, Germany). The anesthetized rats were placed onto a stereotaxic frame (NARISHIGE, Tokyo, Japan). According to the stereotaxic coordinates in the stereotaxic atlas of Paxinos and Watson (2007), 0.05 μl of 10% tetramethylrhodamine-dextran (TMR, D3308, 3,000 MW, Molecular Probe, Eugene, OR) dissolved in trisodium citrate solution (pH 3.0) was made stereotaxically into the bilateral NAc core or NAc shell of the rats (NAc core: 1.8 mm anterior to the Bregma, ± 1.4 mm to the midline, and 7.2 mm deep from the brain surface; NAc shell: 1.8 mm posterior to the Bregma, ± 0.8 mm to the midline, and 8.0 mm deep from the brain surface). A glass micropipette (internal tip diameter 15-25 μm) attached to a 1 μl Hamilton microsyringe was used. Each injection was made by pressure over a period of 10 min and the micropipette was left in the place for additional 20 min after the injection.
PP2A activity assay PP2A activity was measured as previously reported using the PP2A Colorimetric Assay kit (GenMed Scientifics, Woodland, CA, USA) [9]. This assay is based on the release of free phosphate from the dephosphorylation of RKpTIRR by endogenous PP2A, which is detected via chromogenic reaction with molybdenum blue produced by ferrous sulfate reduction. The free phosphate concentration was measured at 660 nm on a spectrophotometer (Bio-Rad). The phosphate concentration (μM/L) was converted to PP2A activity/mg protein as described by the manufacturer.

LC-MS/MS iTRAQ analysis
The methods of sample preparation for LC-MS/MS iTRAQ analysis were reported in previous study [10,11]. For the iTRAQ analysis, in each group, fresh mPFC tissues were rapidly dissected from the brains and sampled. To reduce individual variation, 12 SC rats were pooled into 3 samples as saline1, saline2, and saline3, and 12 MW rats were pooled into 3 samples as M1, M2 and M3. The pooled samples were digested according to the FASP procedure and labeled using the 8-plex iTRAQ reagent according to the manufacturer's instructions (Applied Biosystems). The final proteins that were deemed to be differentially expressed were filtered as a p value <0.05 and 1.1-fold changes (>1.10 or <0.91) relative to the SC group. Functional classification and Gene Ontology (GO) enrichment analyses of the DEPs were carried out using DAVID (https://david.ncifcrf.gov/). Proteins were classified by GO category (http://www.geneontology.org), including "biological process," "cell component," and "molecular function." The KEGG (http://www.genome.jp/kegg/) database was employed to identify significantly enriched pathways. Rattus norvegicus was selected as the species and the background. The significance was determined with slight modifications as recommended by the authors of DAVID according to the Benjamini-corrected P value <0.05. Functional protein association networks were explored in STRING v.10.5 (http://string-db.org/).  Figure S2C). Notably, these proteins were found to be enriched in GO terms which associated with potassium channel activity and regulation of Cytoskeletal component (Supplementary Figure S2D).

Assay for Rac1 activity
Active Rac1 pull-downs were performed following the active Rac1 Pull-Down and Detection Kit (catalog #16118, Thermo Scientific™) protocol [12]. Briefly, lysates of the rat mPFC tissue was centrifuged (16,000 × g at 4°C for 15 min), and then the transferred supernatants were added with GTPγS or GDP to incubate at 30°C for 15 min. The mixtures were incubated with glutathione resin beads and glutathione S-transferase-fused Rac1-binding domain of p21-activated kinase (Pak) at 4°C for 1 h. The beads and proteins bound to the fusion protein were washed at 4°C, eluted in SDS sample buffer, and analyzed for bound Rac1 by Western blotting.

Stereotaxic injections of lentivirus into the IL cortex
Animals were deeply anesthetized with an i.p. injection (40 mg·kg −1 ) of pentobarbital sodium (Aoxin Chemical Factory, Yangzhou, China). Lentiviruses (9 × 10 8 to 1 × 10 9 TU/ml) were stereotaxically injected into the IL (2.5 L/site) over 5 min using a glass micropipette (internal tip diameter 15-25 μm) attached to a 5 μl Hamilton microsyringe. The injector was retained in place for another 10 min then withdrawn at 1 mm/min. We applied the injections bilaterally at the following coordinates (as calculated from bregma and the dura mater): 3.2 mm posterior to the Bregma, ± 0.6 mm to the midline, and 5.0 mm deep from the brain surface.

Adeno-associated virus (AAV) construction and infection
In vitro experiments, cortical neurons were transfected with AAV-mediated gene delivery as described in [13]. The transductions were performed at 7 DIV and maintained for 12 DIV. The target shRNA regions were chosen as follows: Rac1-124, GCCAATGTTATGGTAGATGGA; Rac1-219, GCAAACAGACGTGTTCTTAAT; Rac1-340, GGGACGAAGCTTGATCTTAGG; negative control, TTCTCCGAACGTGTCACGT. For the rescue plasmid, the shRNA targeting binding sites of rac1 plasmid were synonymously mutated to prevent the above three shRNAs from interfering with the expression of Rac1. The synonymous mutation sites of rac1 plasmid are as follows: site-124, GCCAATGTAATGGTCGACGGT; site-219, GCAAACAGATGTATTTTTGAT; site-340, GGGACGAAGCTAGACCTGAGA. The AAV-Rac1 shRNA, AAV-Rac1 rescue and AAV-Ctrl-shRNA of AAV2/9 serotype were packaged by Genepharma Technology Co., Ltd (Shanghai, China). shRNA plasmid pSicoR was purchased from Addgene, and oligos coding for the various shRNAs were annealed and cloned into HpaI-XhoI-digested pSicoR vectors. Viral titers over 1×10 12 genomic particles/mL were used. The cells were transduced with AAVs at a multiplicity of infection (MOI) of 10 4 viral genome copies per cell (VGC/cell) for 3 h at 37°C. The media were subsequently completed with B27-supplemented Neurobasal medium.

The assessment of locomotor sensitization
The locomotor activity of each rat was measured 45 min using locomotor activity cage (Noldus Information Technology Co., Ltd, Netherlands) as described previously [14]. Morphine withdrawal responses (such as wet dog shakes) and locomotor activity were simultaneously observed for the same duration. Other withdrawal symptoms (such as the number of fecal pellets, ptosis and diarrhea) were not included here.
Statistical analysis pClamp 10.2 (Axon Instr., USA) and Origin 9.0 (Origin Lab, Northampton, MA, USA) were applied for analyzing results of ex-vivo electrophysiological recordings. Considering varied number of neurons recorded for each rat, we averaged the baseline spike firing and voltage clamp parameters (baseline input/output slope, action potential and input resistance parameters, tail currents, etc.) for all neurons achieved from a given animal, and acquired a specific value of each of these parameters for each individual rat. The data were expressed as means ± SEM for all the tests. All statistics were presented using an unpaired t-tests, otherwise noted. All tests were two-sided and the statistical significance was set at 0.05.