Pilot Experiment Proposal

Qi et al. Supplementary information

Supplementary information: Materials and Methods

Plasmid construction, and production and purification of AAV2/5 vectors

The 564bp open reading frame of the rat CRF gene (GenBank accession NM_031019) was generated using gene synthesis and cloned into the HindIII/XhoI sites on the pAAV-cDNA6-

V5His vector. The pAAV-CMV-CRF cis-plasmid was co-transfected with AAV helper plasmids into the HEK 293 cells to produce AAV2/5-CRF stock. The viruses were purified through cesium chloride (CsCl)-gradient centrifugation and the viral titer (AAV2/5-CRF,

7.0x1013 genome copies/ml) was determined using RT-PCR (Vector BioLabs, Philadelphia,

PA, USA). For the control vector, “humanized” (adapted for expression in mammalian cells) green fluorescence protein (GFP) cDNA was cloned into the pTR-UF11 vector (Powell Gene

Therapy Center of University of Florida, Gainesville, FL, USA).1,2 The plasmids containing

GFP cDNA were co-transfected with helper plasmids into HEK 293 cells. After an incubation period, the cells were harvested and the virus was purified. The viral titer (AAV2/5-GFP, 1.2 x 1013 genome copies/ml) was determined using a dot-blot assay.3

Preparation of primary neuronal cultures

Primary neuronal cultures were prepared from the brains of one-day-old Sprague Dawley rats

(Charles River, Wilmington, MA, USA) as described before.4 Briefly, one-day-old rats were deeply anesthetized with isoflurane and then the brains were removed, minced into small pieces, and subjected to trypsin (0.25%) treatment to dissociate the cells. The dissociated

1 Qi et al. Supplementary information cells were washed with Dulbecco's modified Eagle’s medium (DMEM) containing 10% horse serum, and plated in poly-L-lysine precoated 12-well plates at a density of 1 × l06 cells/well.

Starting on day 3, the cultures were treated with 10 µM cytosine arabinoside for 2 days. Then the medium was replaced with DMEM containing 10% horse serum and the cultures were maintained at 37°C with 5% CO2 in a humidified incubator for an additional 2 days before being used for the AAV validation experiments. These neuronal cultures have been well characterized and contain more than 90% neurons.5,6

In vitro validation of AAV2/5-GFP and AAV2/5-CRF vectors

To evaluate the transduction efficiency of the AAV2/5-CRF and AAV2/5-GFP vectors in vitro studies were conducted using primary neuronal cultures. Seven-day-old neuronal cultures were incubated with AAV2/5-CRF (2 doses; 2 µl containing 1.4 × 1011 genome copies; 5 µl containing 3.5 × 1011 genome copies) or AAV2/5-GFP (2 doses; 2 µl containing 2.4 × 1010 genome copies; 5 µl containing 6 × 1010 genome copies). Five days following the infections the cells were collected and subjected to RNA isolation and CRF mRNA levels were determined using RT-PCR. The expression of GFP in the neuronal cultures was viewed with an Olympus BX41 fluorescence microscope (Olympus America, Melville, NY, USA).

Electrode and cannula implantations

Rats were anesthetized with an isoflurane and oxygen vapor mixture (1–3% isoflurane) and placed in a Kopf stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA) with the

2 Qi et al. Supplementary information incisor bar set 3.3 mm below the interaural line (flat skull). Stainless steel cannulas (11 mm in length, 23-gauge) were implanted bilaterally 2.5 mm above the CeA (anterior-posterior

(AP) -2.3, medial-lateral (ML) ±4.0 mm, dorsal-ventral (DV) -4.7 from dura), and the bipolar electrodes (11mm in length, model MS303/2 Plastics One, Roanoke, VA, USA) were implanted in the medial forebrain bundle (AP -0.5 mm, ML ±1.7 mm, DV -8.3 mm from dura) (incisor bar 5 mm above interaural line).

ICSS procedure

Rats were trained on a modified discrete-trial ICSS procedure. First the subjects were trained to turn the wheel on a fixed ratio 1 (FR1) schedule of reinforcement. Each quarter turn resulted in the delivery of a 0.5 second train of 0.1 millisecond cathodal square-wave pulses at a frequency of 100 Hz. After the successful acquisition of responding, defined as 100 reinforcements within 10 minutes, the rats were trained on a discrete-trial current-threshold procedure. Each trial began with the delivery of a non-contingent electrical stimulus, followed by a 7.5 second response window within which the animal could respond to receive a second contingent stimulus identical to the initial non-contingent stimulus. A response during this 7.5 second response window was labeled as a positive response and the lack of a response was labeled as a negative response. During a 2-second period immediately after a positive response, additional responses had no scheduled consequences. The inter-trial interval (ITI), which followed either a positive response or the end of the response window, had an average duration of 10 seconds (7.5 - 12.5 seconds). Responses that occurred during

3 Qi et al. Supplementary information the ITI resulted in an additional 12.5 second delay of the onset of the next trial. During training on the discrete-trial procedure, the duration of the ITI period was gradually increased until animals performed consistently at standard test parameters. The rats were subsequently tested on the current-threshold procedure in which stimulation intensities varied according to the classical psychophysical method of limits. A test session consisted of four alternating series of descending and ascending current intensities starting with a descending series.

Blocks of three trials were presented to the subject at a given stimulation intensity, and the intensity was altered systematically between blocks by 5 µA steps. The initial stimulus intensity was set 40 µA above the baseline current-threshold for each animal. Each test session typically lasted 30 minutes and provided two variables: ICSS thresholds and response latencies. The ICSS threshold for a descending series was defined as the midpoint between stimulation intensities that supported responding (i.e., positive responses on at least two of the three trials), and current intensities that failed to support responding (i.e., positive responses on fewer than two of the three trials for two consecutive blocks of trials). The threshold for an ascending series was defined as the midpoint between stimulation intensities that did not support responding and current intensities that supported responding for two consecutive blocks of trials. Four threshold estimates were recorded and the mean of these values was taken as the ICSS threshold for a specific subject. The response latency was defined as the time interval between the beginning of the non-contingent stimulus and a positive response. Thus, the response latency is the time interval between the first electrical stimulus and the rat’s response on the wheel to obtain the second identical stimulus. Drugs that have sedative effects or induce motor impairments might increase the response latency

4 Qi et al. Supplementary information and stimulants might decrease the response latency.7,8 In the present experiments, the response latency for each test session was defined as the mean response latency on all trials during which a positive response occurred.

Intracerebral microinjection

In experiment 1, the rats were anesthetized with an isoflurane and oxygen vapor mixture, placed in a Kopf stereotaxic frame (flat skull), and were unilaterally injected with AAV2/5-

GFP (6.0x109 genome copies in 0.5 µl) or saline (0.5 µl) into the CeA. In experiment 2, rats were anesthetized and bilaterally injected with AAV2/5-CRF (3.5 x 1010 genome copies) or

AAV2/5-GFP (6.0 x 109 genome copies) with a stainless steel injector that extended 2.5 mm beyond the previously implanted guide cannula. The injection volume was 0.5 µl per side and this was infused over a 2-minute period. The infusion speed was controlled by a Harvard

Apparatus syringe pump (model 975) that was equipped with 10 µl syringes (Hamilton, Rena,

NE, USA). The syringes were connected to the injectors with Tygon microbore PVC tubing

(0.25 mm inner diameter × 0.76 mm outer diameter). The injectors were left in place for 5 minutes to allow diffusion from the injector tip. After the infusions, protective wire stylets were re-inserted into the cannulas.

Osmotic minipump implantation

Rats were anesthetized with an isoflurane and oxygen vapor mixture, and were implanted subcutaneously with osmotic minipumps (model 2ML4, 28 day pumps; Durect Corporation,

5 Qi et al. Supplementary information

Cupertino, CA, USA) filled with either saline or nicotine hydrogen tartrate dissolved in saline

(Sigma-Aldrich, St Louis, MO, USA). The nicotine concentration was adjusted to compensate for differences in body weight and to deliver a dose of 9 mg/kg/day of nicotine salt (3.16 mg/kg/day nicotine base).

Immunohistochemistry

Animals were deeply anesthetized with pentobarbital (150 mg/kg, ip, Sigma-Aldrich, St

Louis, MO, USA) and transcardially perfused with 50 ml of isotonic saline, followed by 250 ml of ice-cold 4% paraformaldehyde in 0.1 M PBS (pH 7.4). Brains were removed and postfixed overnight in the same solution at 4 ºC. The brains were then transferred to 30% sucrose in 0.1 M PBS for cryoprotection. Brains were cut into 40 μm-thick coronal sections using a Leica CM3050 S cryostat (Leica Microsystems GmbH, Wetzlar, Germany) and every fourth section was mounted and processed for immunohistochemistry.

Sections were sequentially processed as follows: (1) incubated in 5% normal donkey serum in TBS (1×, pH 7.4) for 1 h; (2) rinsed in TBS for 30 min; (3) incubated overnight at

4ºC in a mixture of three primary antibodies, chicken anti-GFP (diluted 1:500, Aves Labs,

Tigard, Oregon, USA), mouse monoclonal anti-NeuN to detect neurons (diluted 1:500,

Millipore, Temecula, CA, USA) and rabbit polyclonal anti-CRF (diluted 1:200, EMD

Millipore, Billerica, MA, USA); (4) rinsed in TBS for 30 min; (5) incubated for 2 h in a mixture of three highly cross-adsorbed secondary antibodies, FITC-conjugated donkey anti- chicken IgY, Cy3-conjugated donkey anti-mouse IgG and Alexa Fluor®647 donkey anti-

6 Qi et al. Supplementary information rabbit IgG (diluted 1:500) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA);

(6) incubated in TBS for 30 min. The sections were mounted and coverslipped.

Images were taken with a Zeiss LSM 710 fully spectral laser scanning confocal microscope with 405, 488, 510, 543 and 633 nanometer laser lines and Zeiss ZEN 2009 microscope software (Thornwood, NY, USA). All images that were used for analysis and comparisons were acquired and processed using identical settings with the lasers set below

10% intensity. The mean fluorescence intensity (MFI) of GFP was determined by the average of the MFI of GFP from eight sections per animal. To verify colocalization of GFP and

NeuN, 150-200 GFP positive cells on at least 3 sections per animal were scanned through the z-plane and the percentage of GFP positive cells that unambiguously colocalized with NeuN was calculated. Because only a small portion of neurons expressed CRF, we counted the number of GFP/NeuN positive cells that also colocalized with CRF under a 20-fold magnification in a minimum of three sections per animal. The images shown in the figures

(Supplement Figures S1 and S2) were acquired at measured resolutions of 512 × 512 pixels and were compiled in Adobe Photoshop software on a PC, with image adjustments restricted to brightness and contrast.

Isolation of RNA and RT-PCR

Viral vectors were added to the primary neuronal cultures and five days later the cells were washed with cold PBS and harvested. In order to obtain brain tissue, rats were decapitated and the brains were removed and placed on ice. Brain sections (0.5 mm) were cut using a

7 Qi et al. Supplementary information stainless steel brain matrix (model 69092-C, Electron Microscopy Sciences, Hartfield, PA,

USA). The CeA was punched out according to the rat brain atlas of Paxinos and Watson.9

Brain tissue was snap frozen in liquid nitrogen and stored at -80ºC until further processing.

The harvested cells and brain tissue were homogenized and total RNA was isolated using

RNeasy plus mini kit according to the manufacturer’s instructions (Qiagen, Valencia, CA,

USA). Two hundred nanogram of RNA was reverse transcribed in a 20 μl reaction system with an iScriptTM cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Corticotropin releasing

factor, CRF1 receptor, and CRF2 receptor mRNAs were analyzed by quantitative RT-PCR using target-specific primers and Taqman probes 186816131_1, Rn00578611_m1, and

Rn00575617_m1, respectively (Applied Biosystems, Foster City, CA, USA). For the RT-PCR an ABI Prism 7000 sequence detection system was used (Applied Biosystems) and all cDNA samples were assayed in duplicate. Data were normalized to rat GAPDH mRNA

(Rn01775763_g1, Applied Biosystems).

Western blotting

The Western blots were conducted as described before.10,11 Rats were decapitated and the brains were removed and placed on ice. Brain sections were cut using a brain matrix and the

CeA was punched out (see above). The tissues were homogenized in 20 volumes of RIPA buffer (pH 7.4, containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1M

PBS) added with 1% protease inhibitor cocktail before use. Lysates were mixed with 5× loading buffer (pH 6.8, 1M Tris-HCl, 50% glycerol, 10% SDS, 1% bromophenol blue).

Proteins were separated by SDS-PAGE on 12% Mini-PROTEAN® TGX™ Precast Gel (Bio-

Rad, Hercules, CA, USA), and transferred to a NC membrane. The blots were incubated with rabbit anti-CRF primary antibody (Abcam, Cambridge, MA, USA) and goat anti-rabbit secondary antibody (Sigma–Aldrich, St. Louis, MO, USA), and then exposed to film with

ECL reagents (Pierce Biotechnology, Rockford, IL, USA). The blots were stripped of antibodies and were reincubated with GAPDH antibodies. Protein bands representing CRF were quantified using the NIH ImageJ software (Bethesda, Maryland, USA; http://rsb.info.nih.gov/ij). The CRF level was normalized to GAPDH as described before.12

Statistics

The ICSS parameters (thresholds and response latencies) were expressed as a percentage of the values obtained on the day before the AAV2/5 vector injections, before the mecamylamine injections, or before minipump removal. The effect of the AAV2/5 vectors on

ICSS parameters over a 28-day period was analyzed using a two-way repeated-measures

ANOVA with time as within-subjects factor and AAV2/5 vector (AAV2/5-GFP or AAV2/5-

CRF) as the between-subjects factor. The effect of the overexpression of CRF on mecamylamine-precipitated nicotine withdrawal was analyzed with a three-way repeated- measures ANOVA with the mecamylamine dose as the within-subjects factor and the AAV2/5 vector and pump content (saline or nicotine) as the between-subjects factors. The effect of the overexpression of CRF on spontaneous nicotine withdrawal was analyzed with a three-way repeated-measures ANOVA with time as the within-subjects factor and the AAV2/5 vector

9 Qi et al. Supplementary information and pump content as the between-subjects factors. Newman-Keuls post hoc tests were conducted when the ANOVA analyses revealed statistically significant effects. The western blot and PCR data were analyzed with Student’s t-tests or one-way ANOVAs. For all analyses, P<0.05 was the accepted level of significance. Statistical analyses were performed using IBM SPSS for Windows software.

Supplementary information: L egends to supplementary figures

Figure S1. Time-course of AAV2/5-mediated GFP expression in the CeA. The confocal images depict GFP expression in the CeA, 2 (a), 4 (b), and 8 (c) weeks after the administration of the AAV2/5-GFP vector. GFP levels did not differ between the three time points (d). GFP protein was visualized with a primary antibody to GFP and a secondary fluorescent antibody. Scale bar is 100 µm (applies to a-c). Data are expressed as means ±

Figure S2. AAV2/5-mediated GFP expression in CRF neurons in the CeA. The confocal images show immunofluorescence in the CeA (a). Double stainings for GFP (green) and

NeuN (red) demonstrated that AAV2/5 efficiently transduces neurons in the CeA (97-98%, a, b). Triple stainings for GFP (green), NeuN (red), and CRF (cyan) showed that AAV2/5 transduces almost all CRF neurons (92-95%; c, d). The percentage of GFP-positive neurons and GFP-positive CRF neurons did not change over time (d). Scale bar is 50 µm (applies to a, c). Abbreviations: 2W, 2 weeks; 4W, 4 weeks; 8W, 8 weeks. Data are expressed as means ±

Figure S3. Transduction efficiency of AAV2/5 vector in cultured primary neurons. Native

GFP fluorescence was clearly visible in cultured primary neurons infected with AAV2/5-GFP

(a). Administration of AAV2/5-CRF to the primary neurons led to a 4 (2 µl) to 7 (5 µl) fold increase in CRF mRNA levels (b). One (GFP-5 µl) or two dishes (other groups) with primary neuronal cultures were used for each dose. Scale bar is 100 µm (a). Data are expressed as means ± SEM.

Figure S4. Overexpression of CRF in the CeA does not affect baseline ICSS thresholds and response latencies. The ICSS thresholds (a) and response latencies (b) were assessed daily for

28 days after the administration of AAV2/5-GFP or AAV2/5-CRF. Number of animals per group: AAV2/5-GFP, n = 23; AAV2/5-CRF, n = 23. Data are expressed as means ± SEM.

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