Unless otherwise indicated, male Sprague Dawley rats (21–29 days old) were used. Rats were housed under a 12-hr light/dark cycle, with lights on at 7:00 a.m., and food and water available ad libitum. All animal procedures in this report were approved by the Gallo Center Institutional Animal Care and Use Committee and were conducted in agreement with the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996.
Recombinant human GDNF was obtained from R&D System (Minneapolis, MN). TTX, NBQX, MK-801, PD 98059 and LY 294002 were purchased from Tocris (Ballwin, MO). Neuro-DiI was purchased from Biotum (Hayward, CA). Picrotoxin, R(+) Baclofen, 6-hydroxydopamine (6-OHDA), desipramine, and all ingredients of the intracellular and external solutions were obtained from Sigma (St. Louis, MO). The polyclonal p-ERK1/2 antibody used for immunohistochemistry was purchased from Cell Signaling Technology (Beverly, MA). The monoclonal anti-[pThr202/Tyr-204]-p44/42 ERK (p-ERK1/2) was purchased from Cell Signaling Technology (Beverly, MA). The monoclonal anti-TH antibody used for immunohistochemistry was purchased from Sigma, and the polyclonal anti-TH antibody used for post-recording staining was obtained from Chemicon International Inc. (Temecula, CA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The secondary antibodies Alexa Fluor 488-labeled donkey anti-rabbit and Alexa Fluor 594-labeled donkey anti-mouse were purchased from Invitrogen (Chicago, IL). Cy5 anti-rabbit secondary antibody, FITC goat anti-rabbit IgG, and Fluorescein (DTAF) Streptavidin were obtained from Jackson ImmunoResearch Lab Inc. (West Grove, PA). The TRIzol reagent was purchased from Invitrogen (Carlsbad, CA). The oligo(dT) primers and the Reverse Transcription System used for reverse transcription of mRNA into cDNA were obtained from Promega (Madison, WI). Phosphatase inhibitor cocktails I and II were obtained from Sigma (St. Louis, MO). The polyclonal antibodies, anti-Ret, antiphospho-Ret (anti-pRet, Tyr1062) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Protease inhibitor cocktail was purchased from Roche (Indianapolis, IN). The BCA Protein Assay kit was purchased from Pierce Biotechnology Inc. (Philadelphia, PA). The enhanced chemiluminescence (ECL) Plus detection kit was purchased from Amersham (Amersham, UK).
Horizontal sections (150 μm) containing the VTA were prepared with a vibratome in an ice-cold cutting solution containing (in mM): 40 NaCl, 143.5 sucrose, 4 KCl, 1.25 NaH2PO4, 26 NaHCO3, 0.5 CaCl2, 7 MgCl2, 10 glucose, 1 sodium ascorbate and 3 sodium pyruvate, saturated with 95% O2 and 5% CO2. Slices were then incubated in the same solution at 32°C for 45 min, and subsequently maintained at room temperature in the external solution containing (in mM): 125 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgCl2, 1.25 NaH2PO4, 25 NaHCO3 and 10 glucose, saturated with 95% O2 and 5% CO2. For recordings in slices from virus- and DiI-injected animals, rats were intracardially perfused with the ice-cold cutting solution prior to sectioning. Slices were incubated at 32°C in the cutting solution for 25 min and then in a solution containing the cutting and the external solution (1:1 in volume) for an additional 25 min at 32°C. Slices were maintained at room temperature in the external solution until use.
Individual slices were placed in a recording chamber (RC26G, Warner Instruments, Hamden, CT) and viewed with an epifluorescence Olympus microscope (BX50WI). The chamber was superfused with the external solution at a speed of 2 ml/min. The temperature of the chamber solution was controlled at 33–34°C by a dual channel controller (TC-344B, Warner Instruments). DiI-labeled neurons were identified by the red fluorescence of the DiI and patched under the guidance of IR-DIC microscopy. The electrode resistance for all experiments was 4–6 MΩ. The pipette solution contained (in mM): 123 potassium gluconate, 10 HEPES, 0.2 EGTA, 8 NaCl, 2 MgATP, 0.3 NaGTP, and 0.1% biocytin, pH 7.2–7.3, with an osmolarity of 270–280 mOsm. For measuring the spontaneous firing of neurons, cell-attached recordings were conducted in voltage-clamp mode. Data were collected using a MultiClamp 700A amplifier controlled by the pClamp 9 software (Molecular Devices, Union City, CA) Spontaneous spikes were filtered at 2 kHz and digitized at 10 kHz. Neurons with wide spikes and low firing frequencies were selected for data collection. At the end of the cell-attached recording, whole-cell access was obtained for biocytin perfusion into the neuron for later TH staining while simultaneously recording Ih in voltage-clamp mode by a hyperpolarizing step from the holding voltage of −60 mV to −90 mV for 1 sec. We also tested the Ih-mediated “Sag” in current-clamp mode (data not shown). The spike frequency was continuously measured online with Clampex (Molecular Devices, Union City, CA) or afterwards using Clampfit or MiniAnalysis (Synaptosoft, Decatur, GA). For electrophysiological experiments in which GDNF, Adv-shGDNF and their respective controls were used in vivo, the firing rates of neurons were compared in a counterbalanced manner so that the influence of uncontrollable experimental conditions on the neuronal firing were minimized. Specifically, on a single experimental day, the firing rates of VTA neurons were compared between a rat treated with GDNF or Adv-shGDNF, to a rat treated with vehicle or Adv-SCR, respectively. In addition, neurons from rats receiving treatment or control were alternately recorded. For each neuron, the firing rate during a 4-min stable recording was measured. Recordings were performed within 6 hrs after slice preparation and recovery (~ 1 hr).
Measurement of miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs)
For recordings of mEPSCs, 100 μM picrotoxin and 1 μM TTX were present in the bath solution. The pipette solution was the same as described above. mEPSCs were recorded for 5 min with neurons clamped at −70 mV. For recordings of mIPSCs, 10 μM NBQX and 1 μM TTX were present in the bath solution. The intracellular solution contains in mM: 125 KCl, 4 NaCl, 10 HEPES, 1 EGTA, 1 MgCl2, 2 Na2ATP, 0.6 Na3 GTP, 2 Na2CrPO4, 10 QX-314. mIPSCs were recorded for 3 min with neurons clamped at −70 mV. mEPSCs and mIPSCs were analyzed with Mini Analysis Program (Synaptosoft, Fort Lee, NJ), with detection criteria set at greater than 7 pA in amplitude, and verified by eye.
Ex vivo application of GDNF
GDNF was dissolved in a bath solution containing 60 μg/ml bovine serum albumin (BSA). After application, the GDNF solution was re-circulated. The re-cycled solution was aspirated by one channel of a dual-channel mini-pump (P720, Instech Lab, Plymouth Meeting, PA) to a 12.5 ml reservoir where it was re-oxygenated with 95% O2
and 5% CO2
. The re-oxygenated solution was driven by the other channel of the mini-pump to the recording chamber. A similar re-circulating system was previously used to deliver another neurotrophic factor, brain-derived neurotrophic factor (BDNF), into slices for electrophysiological recording (Lauterborn et al., 2007
). Post-recording TH immunostaining
was performed according to Margolis et al. (2008)
. Briefly, immediately after electrophysiological recordings, VTA slices containing biocytin-filled neurons were fixed in 4% paraformaldehyde for 2 hrs and stored at 4°C in PBS. Slices were incubated with anti-TH antibody (1:100) at 4°C for 48 hrs, and then agitated overnight at 4°C with Cy5 anti-rabbit secondary antibody (1:100) for TH detection, and FITC-conjugated streptavidin for Biocytin detection. Images were taken with a Zeiss LSM 510 META microscope. The colors of TH and biocytin immunostaining were switched using the Zeiss LSM program so that TH is green, consistent with the color of TH immunostaining in other experiments (, and ). TH staining of the VTA from GDNF () and DiI-injected animals () was done as described in Carnicella et al. (2008)
Intra-NAc infusion of GDNF causes increased spontaneous firing, altered synaptic drives, and ERK activation in VTA neurons
GDNF increases the firing of the NAc-projecting DA neurons in the VTA
18 days after intra-NAc injection of virus, VTA sections were prepared and permeabilized with 50% ethanol in PBS for 20 min, rinsed in PBS and incubated in TUNEL reaction mixture (In Situ
cell detection kit, Roche Applied Science) for 1 hr at 37°C according to the vendor’s protocol, then rinsed and processed for TH immunostaining as described in Carnicella et al. (2008)
In vivo application of GDNF and Adv-shGDNF
GDNF was infused into the VTA as described previously (Carnicella et al., 2008
). Briefly, GDNF (10 μg in 1 μl PBS) or vehicle (PBS) were bilaterally injected into the VTA (in mm: −4.8 AP, ± 0.75 ML, −7.8 DV) of rats. VTA slices were then prepared for electrophysiological recordings 10 min after the injections.
Rats were anesthetized with isoflurane. The tip of the injector (31G, small parts, Miami lakes, FL) was stereotaxically positioned into the NAc. The injector was connected to a Hamilton syringe (10 μl) that was driven by an automatic pump (Harvard Apparatus, Holliston, MA). The injection speed was 0.2 μl/min. Infusions of GDNF (10 μg in 2 μl saline, or 2 μl saline as vehicle) and viruses (Adv-shGDNF or Adv-SCR 0.35 × 1010
TU/ml, 2 μl) into the NAc were made bilaterally, or unilaterally (for detection of ERK1/2 phosphorylation). For dual-site injections, GDNF or vehicle (saline) was infused into the NAc 7–11 days following intra-prefrontal cortex (PFC) injection of DiI (see below). Each side of the NAc received 4 injections, varying the anterior-posterior and dorsal-ventral axes (in mm: +1.7 AP1, +1.1 AP2, ± 1.3 ML, −7.2 DV1, −6.8 DV2) in order to reach a maximal area in the NAc. Twelve hrs after injections, coronal sections of the NAc were prepared for placement verification, coronal sections of the VTA were prepared for immunostaining of phospho-ERK1/2 and TH using a procedure previously described (Carnicella et al., 2008
), and horizontal sections of the VTA were used for electrophysiology recordings. For retrograde labeling of NAc projecting VTA neurons, DiI (0.3 μl, in 7% ethanol) was bilaterally injected into the NAc (in mm: +1.5 AP, ± 1.3 ML, −7.2 DV). Seven to 10 days after injection, coronal sections of the NAc were prepared for thionin (0.2%)-staining to verify injection placements, coronal sections of the VTA were prepared for TH-staining to examine the overlay of DiI- and TH-labeled cells, and horizontal sections of VTA were used for electrophysiology recordings.
DiI (0.3 μl, 7% in ethanol) was bilaterally injected into the medial PFC (in mm: +2.6 AP, ± 0.78 ML, −4.0 DV).
Cloning and preparation of GDNF shRNA recombinant adenovirus
A 20-nucleotide (20nt) GDNF small interfering RNA (siRNA) sequence (siGDNF), ATG TCA CTG ACT TGG GTC TG, was designed using the online siRNA Retriever http://cancan.cshl.edu/RNAi_central/
. This siGDNF sequence targets the coding domain of the GDNF
mRNA and was used for vector-based small-hairpin RNA (shRNA) expression. Two complementary oligonucleotides were synthesized as follows: 5′-GATCCC (20nt, sense) TTGATATCCG (20nt, antisense), TTTTTT CCAAA-3′ and 3′-GG (20nt antisense) AACTATAGGC (20nt, sense) AAAAAA GGTTTTCGA-5′, flanked by Bam H1 and Hind III residues. The paired oligonucleotides were annealed and ligated into the pRNAT-H1.1/Shuttle (GenScript Corporation, Piscataway, NJ), a GFP-containing adenoviral shuttle siRNA vector. Recombinant sequences were confirmed before subcloning into the adenoviral vector Adeno-X (Clontech, Mountain View, CA). Preparation of adenoviruses was carried out according to the Adeno-X Expression System 1 User Manual (Clontech, Mountain View, CA). The scrambled sequence of siGDNF, GT CTG GGT TCA GTC ACT GTA was designed and prepared in parallel. Viruses were amplified in HEK293 cells, followed by purification using Adeno-X Virus Purification Kit (Clontech, Mountain View, CA) and titered based on GFP-visualized infection. Recombinant viruses were used to infect C6 glioma cells or a stable SHSY5Y cell line that GDNF overexpress (He and Ron, 2006
) at a multiplicity of infection (MOI) of 20, and downregulation of GDNF
after infection was measured thereafter (Figure S1A
Brain sample collection and Quantitative Reverse Transcription – Polymerase Chain Reaction (qRT-PCR)
Eighteen days after viral infusion, brain tissue containing the NAc was dissected. Total RNA was isolated, and reverse transcription of mRNA into cDNA was conducted using the oligo(dT) primers. The reverse transcription reaction was conducted at 42°C for 30 min. The resulting cDNA samples were amplified by TaqMan quantitative PCR using commercially available primer/probe kits from Applied Biosystems for GDNF (Gene Expression Assay Rn00569510_m1) and GAPDH (Gene Expression Assay Rn99999916_s1) as the internal control.
Standard Reverse Transcription – Polymerase Chain Reaction (RT-PCR)
Five or 18 days after viral infusion, brain tissue containing the NAc or the VTA was dissected. Total RNA was isolated with TRIzol reagent and used for reverse transcription with the Reverse Transcription System at 42°C for 30 min. PCR was performed with glyceraldehyde-3-phosphate dehydrogenase
) as an internal control as previously described (Carnicella et al., 2009
). Thirty-two cycles were performed to measure the expressions of GDNF
. Primers were used as follows: rat GDNF
, upstream 5′-GAC GTC ATG GAT TTT ATT CAA GCC ACC 3′ and downstream 5′-CTG GCC TAC TTT GTC ACT TGT TAG CCT-3′; rat TH
, upstream 5′-GAA GCT GAT TGC AGA GAT TGC-3′ and downstream 5′-GCT CAG GTG AAT GCA TAG GTG-3′; rat NGF
, upstream 5′-ACA CTC TGG ATC TAG ACT TCC AGG-3′ and downstream 5′-AGG CAA GTC AGC CTC TTC TTG TAG-3′
Western blot analysis
Midbrain slices were prepared as above. After recovery for at least 1 hr, midbrain slices (150 μm) containing the VTA were dissected. To detect phosphorylation level of Ret, slices were treated with GDNF (400 ng/ml) for 45 min at room temperature in aCSF. To detect the level of phospho-ERK1/2, slices were pretreated with tetrodotoxin (TTX, 1 μM) and MK 801 (20 μM) for 30 min to block activity- and NMDAR-dependent changes in the level of phospho-ERK1/2. Slices were then incubated with GDNF (400 ng/ml) for 45 min in the continuous presence of TTX and MK 801. At the end of the treatment period, the slices were quickly sonicated in RIPA buffer (50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 10 mM NaCl, 1% NP-40, 0.1% deoxycholate, and 0.5% SDS) containing protease and phosphatase inhibitors. Following a 30-min incubation of the lysate on ice, 25μg of protein was resolved in NuPAGE 4–12% Bis-Tris gels (Invitrogen). Anti- phospho-Ret and antiphospho-ERK1/2 antibodies (1:2000) were used to detect the phosphorylation levels of Ret and ERK1/2. ECL reaction was used for detection of signal, which was digitally scanned using a STORM detector. Following detection, membranes were incubated in stripping buffer (25 mM glycine-HCl, 1% SDS, pH 3, for 30 min at room temperature) and re-probed with anti-Ret or anti-ERK2 antibodies (1:1000 for Ret, 1:2000 for ERK2). Densitometric analysis was performed using Image J software (NIH).
Intra-NAc infusion of 6-OHDA
Thirty min before 6-OHDA infusions, rats were administered with desipramine (15 mg/kg, i.p.) to prevent the uptake of 6-OHDA into noradrenergic nerve endings. 6- OHDA was dissolved in saline (8 μg/μl) and ascorbate (0.1 μg/μl). 6-OHDA (1.5 μl) was bilaterally (for electrophysiological recordings) or unilaterally (for immunohistochemical studies) infused into the NAc (see above for coordinates). For electrophysiology experiments, DiI was infused into the PFC (see above for coordinates) to label PFC- projecting VTA neurons. Three weeks later, GDNF (10 μg/2 μl) or vehicle was bilaterally infused to the NAc. Twelve hrs after GDNF/vehicle infusion, animals were anesthetized and brains removed for electrophysiological recordings or immunohistochemical studies.
In vivo microdialysis
Adult male Long-Evans rats (Harlan, 350–400 g) were anesthetized with isoflurane (Baxter Health Care Corporation) or equithesin (1% pentobarbital, 2% magnesium sulfate, 4% chloral hydrate, 42% propyleneglycol, 11% ethanol, 3 ml/kg intraperitoneally). Unilateral guide cannulae for microinjection (26G, Plastics One) were placed dorsal to the VTA (in mm: −5.6 AP, ±0.8 ML, −7.4 DV, relative to the bregma) and guide cannulae for the microdialysis probes (CMA/11, CMA microdialysis) were placed dorsal to the NAc (in mm: +1.7 AP, +1.0 ML, −6.4 DV, relative to the bregma). After the surgery, animals were allowed to recover for 2–4 days.
Microinjection and microdialysis procedure
Microdialysis was performed as previously described (Zapata and Shippenberg, 2005
). In the evening before the experiment, probes (2 mm membrane length) were inserted into guide cannulae and connected to the dialysis system. Rats were housed in the microdialysis chamber with food and water, and the probes were perfused overnight (0.3 μ/min) and 1 hr before experiments (1 μl/min) with artificial cerebrospinal fluid (aCSF) (in mM: 145 NaCl, 2.8 KCl, 1.2 MgCl2
, 1.2 CaCl2
, 0.25 ascorbic acid and 5.4 D-glucose, pH 6.5–7.0 adjusted with NaOH). Fresh aCSF was loaded and probes equilibrated over 1 hr at a flow rate of 0.6 μl/min prior to the beginning of the sample collection. GDNF (10 μg/μl) or PBS and/or U0126 (0.5 μg/μl) or its vehicle (5% DMSO and 6% Tween 80 in PBS) was infused over 2 min into the VTA via injection cannulae (33G, Plastics One) extending 1.0 mm beyond the guide cannula tip. To confirm the functional connectivity between the VTA infusion site and the NAc dialysate collection site, 2.5 hrs later, the GABAB
receptor agonist baclofen (75 ng/μl) was infused into the VTA to inactivate DA neurons. Connectivity is verified by the resulting decrease in NAc DA concentrations. Rats showing no decrease in NAc DA levels after the infusion of baclofen were excluded from the analyses. Dialysis fractions were collected every 15 min, frozen at −80°C, and analyzed for DA content within 48 hrs using HPLC electrochemical detection.
DA level determination
DA levels in the dialysate samples of the first microdialysis experiment (effect of intra-VTA infusion of GDNF on NAc DA overflow, ) were determined as described in (Zapata and Shippenberg, 2005
). The chromatographic system used to determine the DA levels in the dialysate samples of the second microdialysis experiment (including the intra-VTA infusion of the MEK inhibitor U0126, ) consisted of an ESA Model 584 pump and ESA microtiter Model 540 (ESA, Analytical, Chelmsford, MA), an ESA Coulochem III amperometric detector with a Model 5011A dual-detector analytical cell (guard cell = 275 mV, E1 = −150 mV, E2 = 220 mV), and a microbore column (50 mm × 1.5 mm × 3 μm; Shizeido Co., Tokyo, Japan). The mobile phase (150 mM NaH2
, 4.76 mM citric acid, 50 μM EDTA, 3 mM SDS, 8% methanol, 10% acetonitrile; pH 5.6 adjusted with NaOH) was run at a flow rate of 0.2 ml/min. DA was quantified by comparing DA peaks from dialysate samples with external standards. Under these conditions, the retention time for DA was 3 min and the limit of detection was below 0.3 nM (at a signal to noise ratio of 3:1).
Application of GDNF into the VTA causes a rapid and sustained elevation of DA overflow in the NAc that is block by inhibition of the MAPK pathway
Locations of cannulae and microdialysis probe placements were verified in 30 μm-thick coronal slices after cryostat sectioning and subjects with misplaced injectors or probes were excluded from the analyses.
All values are expressed as mean ± SEM unless otherwise stated. Statistical significance of electrophysiological and biochemical data was analyzed by paired or unpaired Student’s t-test unless otherwise stated. The microdialysis data were analyzed by a two-way ANOVA (mixed within-subject design). Significant interaction was further investigated by using the method of contrasts or the Student-Newman-Keuls test.