Subjects and apparatus
Female Long–Evans (total n
= 86) rats (National Institute on Drug Abuse breeding program or Charles River Laboratories; 15–16 weeks old at the time of surgery) were maintained on a reverse 12 h light/dark cycle (lights off at 7:30 or 8:00 A.M.). All rats were individually housed after surgical procedures. For experiments 1– 4, rats had ad libitum
access to Purina rat chow and water. For behavioral experiments (experiment 5–7), rats were weighed daily and food restricted to 9 g/d Purina rat chow (~55– 65% of their daily food intake) during the training phase and 14 –16 g/d (to maintain stable body weight) during the extinction and reinstatement test phases; the rat chow was given after the daily behavioral sessions. All procedures followed the guidelines outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals
(eighth edition; http://grants.nih.gov/grants/olaw/Guide-for-the-Care-and-Use-of-Laboratory-Animals.pdf
). A total of 18 rats were excluded because of fiber optic placement outside the dorsal mPFC (n
= 4), poor laser performance during critical test (n
= 1), poor bilateral expression of viral constructs (n
= 5), significant viral expression outside the dorsal mPFC (n
= 3), lost head caps or illness (n
= 4), and failure to meet an extinction criteria (n
Behavioral experiments were conducted in standard self-administration chambers (Med Associates). Each chamber had two levers 9 cm above the floor, but only one lever (the “active,” retractable lever) activated the pellet dispenser, which delivered 45 mg food pellets containing 12.7% fat, 66.7% carbohydrate, and 20.6% protein (catalog #1811155; Test Diet). This pellet type was chosen based on pellet preference tests in food-restricted female rats, using six pellet types (obtained from TestDiet and Bioserv) with different compositions of fat (0 –35%) and carbohydrate (45–91% sugar pellets) and different flavors (no flavor, banana, chocolate, grape).
Construct and AAV preparation
The AAV packaging plasmids pAAV–CaMKIIα
– eYFP and pAAV–CaMKIIα
– eNpHR3.0 – eYFP (Tye et al., 2011
) were provided by K.D. AAV vector stocks were prepared by the National Institute on Drug Abuse/Intramural Research Program Optogenetic and Transgenic Technology Core facility, using the modified triple-transfection method (Xiao et al., 1998
; Howard et al., 2008
). Briefly, 20 15-cm dishes containing HEK293 cells at 85–95% confluency were transfected by CaCl2
method with pHelper (Stratagene), pAAV–CaMKIIa– eYFP and pAAV–CaMKIIa–eNpHR3.0 – eYFP, and pXR1 aka pXX12 (Rabinowitz et al., 2002
), a plasmid containing rep/cap (replication/capsid) genes for serotype 1. Approximately 48 h after transfection, cells were harvested, lysed by freeze–thaw, and purified by centrifugation on CsCl gradient followed by fractionation. Final samples were dialyzed in PBS, aliquoted, and stored at −80°C until use. AAV vector titers were 1–2 × 10 11
vg/ml as determined by quantitative PCR, using eYFP as the target sequence (Howard et al., 2008
Rats in experiments 1, 2, and 4 – 6 were anesthetized with 100 mg/kg ketamine plus 10 mg/kg xylazine and were placed in the stereotaxic frame (David Kopf Instruments). Rats in experiment 3 were anesthetized with intraperitoneal injections of a mixture of sodium pentobarbital and chloral hydrate (60 and 25 mg/kg) before surgery.
Microinjection needles (Hamilton syringe, 10 μ
l with 30 gauge needle) were secured in stereotaxic pumps (UMP4 injector; World Precision Instruments) and inserted bilaterally in experiments 2 and 4 – 6 or unilaterally in experiment 3 (in vivo
electrophysiology), into the dorsal mPFC [coordinates from bregma: +2.5 mm anteroposterior (AP), ±1.2 mm mediolateral (ML) (at 10° angle), and −3.4 mm dorso-ventral (DV)]; these coordinates are based on our previous study in male rats (Bossert et al., 2011
; Nair et al., 2011
) and pilot surgeries in female rats. A total volume of 0.7 μ
l of either AAV1–CaMKIIα
– eYFP or AAV1–CaMKIIα
– eNpHR3.0 – eYFP was injected at a rate of either 0.25 μ
l/min (for in vivo
electrophysiology) or 0.5 μ
l/min (for all other experiments). The microinjector needle was left in place for 3 min, raised 0.1 mm, and left in place 2 min before being removed from the brain. Rats were then removed from the stereotaxic frame and either sutured (if receiving viral injection only) or placed in a different stereotaxic frame for either chronic fiber optic implantation or optrode implantation (below).
Chronic intracranial fiber optic implantation
In experiments 1 and 4 – 6, precalibrated fiber optics (200 μm core fiber optic; Thorlabs) with 2.5 mm stainless steel ferrule (Fiber Instruments) were inserted bilaterally 0.5 mm above the site of the viral injections in the dorsal mPFC [coordinates from bregma: +2.5 mm AP, ±1.2 mm ML (at 10° angle), and −2.9 mm DV] and were secured to the skull using jewelers screws and dental cement (Geristore; DenMat).
Chronic intracranial optrode implantation
In experiment 3, a drivable cannula housing a precalibrated fiber optic assembly (100 μm core; Doric Lenses) set 200 –500 μm above a bundle of eight microelectrode wires (25 μm diameter iron–nickel– chromium wires; A-M Systems) was inserted into the dorsal mPFC [coordinates from bregma: +2.5 mm AP, ±1.2 mm ML (at 10° angle), and −2.4 DV]. The chronic optrode was secured to the skull using jeweler’s screws and dental cement. Before implantation, the microwires were freshly cut with surgical scissors to extend ~0.2– 0.5 mm beyond the fiber optic and ~1 mm beyond the cannula and electroplated with platinum (H2PtCl6; Aldrich) to an impedance of ~300 kΩ. After each recording session, the optrode was advanced in 40 – 80 μm increments to acquire new single-unit activity.
Electrophysiology (ex vivo)
Rats were anesthetized with 40 mg/kg pentobarbital (intraperitoneally) and transcardially perfused with ~30 ml of nearly frozen (~0°C) modified artificial CSF (aCSF) at a rate of ~20 ml/min. The modified aCSF for perfusion contained the following (in mM): 225 sucrose, 119 NaCl, 2.5 KCl, 1.0 NaH2PO4, 4.9 MgCl2, 0.1 CaCl2, 26.2 NaHCO3, and 1.25 glucose. After perfusion, the brain was quickly removed and placed into ice-cold aCSF for 1–2 min. Coronal sections of the mPFC (250 μm) were prepared with VT-1200 vibratome (Leica). Slices were placed in a holding chamber (containing aCSF with 1 mM ascorbic acid added 15 min before brain dissection) and allowed to recover for at least 30 min before being placed in the recording chamber and superfused with a bicarbonate-buffered solution saturated with 95% O2 and 5% CO2 and containing the following (in mM): 119 NaCl, 2.5 KCl, 1.0 NaH2PO4, 1.3 MgCl2, 2.4 CaCl2, 26.2 NaHCO3, and 11 glucose (at 32–34°C).
Recording (ex vivo)
Picrotoxin (50 μM) and CNQX (10 μM) were present throughout the experiment to block inhibitory and excitatory synaptic transmission, respectively. Cells were visualized using infrared differential interference contrast video microscopy. Whole-cell current-clamp recordings were made using a MultiClamp 700B amplifier (Molecular Devices). Electrodes (2.8 – 4.0 MΩ) contained the following: 140 mM potassium methane sulfonate, 10 mM HEPES, 0.05 mM EGTA, 5 mM K-Cl, 2 mM MgCl2, 2 mM Na2GTP, 0.4 mM NaGTP, 1% biocytin, pH 7.2–7.3, 280 mOsm. Series resistance (10 – 40 MΩ) was continually monitored online with a −20 pA, 300 ms current injection given after every current injection step; if the series resistance changed by >20%, data were not included in the analysis. Membrane potentials were not corrected for junction potentials (estimated to be 10 mV). Resting membrane potentials were determined immediately after breaking into the neuron. Membrane potential was then set to −80 mV by injecting direct current through patch amplifier. To determine the input resistance, hyperpolarizing current injections were injected into the cell. Input resistance was taken at the linear part of the trace. For evaluation of light-evoked currents, neurons were voltage clamped at −70 mV and a 200 ms light pulse (>20 mW) of either 532 or 593.5 nm delivered via a 200 μm optical fiber coupled to either a 532 or 593.5 nm solid-state laser (OEM Laser Systems). To evaluate the effect of eNpHR3.0-mediated currents on neuronal firing in the mPFC, 200 ms pulses of either 532 or 593.5 nm light (>20 mW) were applied during direct current injections in a subset of neurons. Data were acquired at 20 kHz and filtered at 2 or 10 kHz using Clampex10.2 software (Molecular Devices).
Electrophysiology (in vivo): optrode recording in awake rats
Optical stimulation and single-unit recording. Three to 8 weeks after surgery, rats were brought to the recording box and connected to a recording cable (Plexon) coupled with a fiber optic patch cable (Doric Lenses). Neural activity was recorded using 16 channel Omniplex System (Plexon), interfaced with a Master-9 Pulse Stimulator (A.M.P.I.), which also controlled a DPSS 593.5 nm laser system (OEM Laser Systems) used for intracranial light delivery. Fiber optic output was precalibrated to 4 – 8 mW from the fiber tip before implantation, and the laser was recalibrated to this power output using an ex vivo power-matched fiber optic before stimulation/recording sessions. Wires were screened for activity daily; if no activity was detected, the rat was removed, and the electrode assembly was advanced 40 or 80 μm. Otherwise, active wires were selected to be recorded, a stimulation session was conducted (detailed below in experiment 3), and the electrode was advanced at the end of the session.
Signals from the electrode wires were amplified 20× by an operational amplifier head stage (HST/8o50-G20; Plexon), located on the recording cable that connected directly to the electrode array. Immediately outside the chamber, the signals were passed through a differential preamplifier, in which the wide-band signal was filtered at 0.5– 8000 Hz. The wideband signal was then sent to the Omniplex chassis, in which it was amplified at 250 –1000× and digitized at 40 kHz. After digitization, the wide-band signal was further filtered at 300 – 8000 Hz and thresholded into a spike waveform segment. Waveforms (>2.5:1 signal-to-noise) were extracted from active channels and recorded to disk by an associated workstation with event timestamps from the stimulator driving laser light delivery. Waveforms were not inverted before data analysis.
In vivo neural data analyses
Units were sorted using Offline Sorter software (Plexon), using a template matching algorithm. Sorted files were then processed in Neuroexplorer to extract unit timestamps and relevant stimulation event markers. These data were subsequently analyzed in MATLAB. Paired t tests were used to measure within-cell differences in firing rate (p < 0.05) during epochs of interest. Bonferroni’s correction was used to account for multiple comparisons in the analyses of population data.
The rats were deeply anesthetized with isoflurane (~80 s) and perfused transcardially with 100 ml of 0.1 M PBS, followed by 400 ml of 4% para-formaldehyde in 0.1 M sodium phosphate, pH 7.4. Brains were removed and postfixed in 4% paraformaldehyde for 2 h before transfer to 30% sucrose in 0.1 M sodium phosphate, pH 7.4, for 48 h at 4°C. Brains were subsequently frozen in powdered dry ice and stored at −80°C until sectioning. Coronal sections (30 or 40 μm) containing dorsal and ventral mPFC (approximately +2.2 to 3.5 mm from bregma) were cut using a cryostat (Leica Microsystems), collected in cryoprotectant (20% glycerol and 2% DMSO in 0.1 M sodium phosphate, pH 7.4), and stored at −80°C until additional processing.
Iba-1 and GFAP immunohistochemistry
Rats were perfused as indicated above (see Histology). Paraformaldehyde-fixed brains were sectioned into 30 μm sections on a Leica cryostat. Sections were collected into phosphate buffer (PB) [16.83 g of NaH2PO4 (140 mM) plus 3.85 g of NaOH, H2O to 1 L], blocked with 4% BSA plus 0.3% Triton X-100 (in PB), and immunostained overnight at 4°C with monoclonal anti-GFAP (clone GA5, 1:300 dilution; Millipore) or polyclonal anti-Iba-1 (019-19741, 1:500 dilution; Wako Pure Chemicals). Alexa Fluor secondary antibodies (Invitrogen) against the appropriate species were used, and nuclei were stained with DAPI (1 μg/ml in PB). Sections were mounted to glass slides with Mowiol 4 – 88 (EMD Chemicals), and images were captured on a Nikon AZ100M (CoolSnap HQ 2 camera, 1× images) or Nikon Eclipse 80i (QImaging Retiga EXi camera, 10× images). For each rat, a single section containing fiber tracts (experiment 1) was selected for analysis. Regions of interest, encompassing the dorsal mPFC, were selected using Nikon Elements software, and the number of immunoreactive cells per square millimeter was quantified and analyzed using Prism (GraphPad Software).
Rats were perfused and tissue prepared as indicated above (see Histology). Immunohistochemical procedures were based on our previous studies (Koya et al., 2009
; Nair et al., 2011
; Bossert et al., 2012
). Immunolabeling for Fos (the protein product of the immediate early gene c-fos
) was used to identify and quantify Fos expression in dorsal mPFC neurons. Sections were thawed and washed three times for 10 min in 0.1 M PBS, incubated for 1 h in 3% normal goat serum (NGS) in PBS with 0.25% Triton X-100 (PBS-Tx), and incubated overnight at 4°C with anti-Fos primary antibody (sc-52, lot I2209; Santa Cruz Biotechnology) diluted 1:4000 in 1% NGS in PBS-Tx. Sections were then washed in PBS and incubated for 2 h with biotinylated anti-rabbit IgG secondary antibody (BA-1000; Vector Laboratories) diluted 1:600 in 1% NGS in PBS-Tx. Sections were washed in PBS and incubated in avidin– biotin–peroxidase complex (ABC Elite kit, PK-6100; Vector Laboratories) in PBS containing 0.5% Triton X-100 for 1 h and washed in PBS. Sections were developed in 3,3′-diaminobenzidine for ~1– 4 min, washed in PBS, mounted onto chromium–potassium sulfate (chrom–alum)/gelatin-coated slides, and air dried overnight. The slides were dehydrated through a graded series of alcohol (30, 60, 90, 95, 100, 100% ethanol), cleared with Citra-solv (Thermo Fisher Scientific), and coverslipped with Permount (Sigma). Bright-field images of the dorsal mPFC were digitally captured using a CCD Camera (Coolsnap; Photometrics, Roper Scientific) attached to a Carl Zeiss Axioskop 2 microscope with a 5× (quantification) or 10× (figure) objective. Labeled Fos-immunoreactive nuclei in two sections (containing fiber tracts) of each left and right hemisphere from each rat were automatically counted using IPLab software (version 3.9.4 r5 for Macintosh; Scanalytics), and the number of immunoreactive cells per square millimeter was quantified. Image capture and quantification of Fos-immunoreactive nuclei were conducted by D.J.C. and A.B.K. in a blind manner.
Verification of virus expression and intracranial fiber optic or optrode placement
Fluorescent images of dorsal mPFC were captured with QImaging Exi Aqua camera attached to a Carl Zeiss Axioskop 2 microscope using a 5× or 10× objective. Rats that had insufficient bilateral expression of eYFP ventral to the fiber tip in dorsal mPFC or rats that had fiber optics that were outside the area of dorsal mPFC expression were excluded. Rats that had bilateral eYFP expression that extended into ventral mPFC were also excluded.
NeuN and GFAP immunohistochemistry
Rats were perfused and tissue prepared as indicated above (see Histology). To confirm that expression of eNpHR3.0 – eYFP was primarily in neurons of the dorsal mPFC, we used NeuN, a marker for neuronal-specific nuclear protein (Mullen et al., 1992
). We determined the proportion of eNpHR3.0 – eYFP-expressing cells that were neurons ~2 weeks after viral injection (ideal time point for visualizing eNpHR3.0 –eYFP expression in cell bodies). Six sections containing dorsal mPFC from three rats were thawed and washed (three times for 10 min each) in Tris-buffered saline (TBS) (0.025 M Tris-HCl, 0.5 M NaCl, pH 7.5) and incubated for 20 min in TBS with 0.2% Triton X-100 (TBS-Tx). Sections were washed in TBS and incubated for 48 h with mouse anti-NeuN primary antibody (1:2000 dilution of MAB377; Millipore) in TBS-Tx. Sections were again washed in TBS and incubated for 1 h with secondary Alexa Fluor 568-labeled goat anti-mouse antibody (1:200 dilution in TBS-Tx; Invitrogen), and nuclei were stained with DAPI (1 μ
g/ml in TBS). Finally, sections were washed in TBS, mounted on chrom–alum/gelatin-coated slides, air dried, and coverslipped with Mowiol fluorescent mounting medium.
To further confirm that expression of eNpHR3.0 – eYFP expression was primarily in mPFC neurons, we used GFAP to determine the proportion of eNpHR3.0 – eYFP-expressing cells that were astrocytes; the assay was performed ~2 weeks after viral injection. Six brain sections containing dorsal mPFC from three rats were thawed and washed with PB [16.83 g of NaH2PO4 (140 mM) plus 3.85 g of NaOH, H2O to 1 L] two times, then blocked with 4% BSA plus 0.3% Triton X-100 (in PB), and immunostained overnight at 4°C with monoclonal anti-GFAP (clone GA5, 1:300 dilution; Millipore). Alexa Fluor secondary antibodies (Invitrogen) against the appropriate species were used, and nuclei were stained with DAPI (1 μg/ml in PB).
Fluorescent images of dorsal mPFC were captured with QImaging Exi Aqua camera attached to a Carl Zeiss Axioskop 2 microscope using a 40× objective. One to two dorsal mPFC 40× images were taken per hemisphere across two sections per rat, and the proportion of eNpHR3.0 –eYFP-positive cells that were NeuN-labeled or GFAP-labeled was quantified using iVision MacOS 10.62 (version 4.0.15) by D.J.C.
Fos + CaMKII and Fos + GAD immunohistochemistry
Two hours after a test for yohimbine-induced reinstatement, the rats were anesthetized and perfused, and their brains were removed and processed as described above (see Histology). We assessed the phenotype of Fos-expressing (Fos +
) neurons by double labeling for Fos and calcium/calmodulin-dependent protein kinase II (CaMKII), a marker of cortical glutamatergic pyramidal projection neurons (Liu and Jones, 1996
) and glutamic acid decarboxylase 67 (GAD67), a marker of GABAergic neurons (Kaufman et al., 1986
Paraformaldehyde-fixed brains were sectioned into 30 μm sections on a Leica cryostat. Sections were incubated for 1 h in a blocking solution (5% NGS and 2.5% bovine serum albumin in PBS with 0.2% Triton X-100) and then incubated for 48 h with the anti-Fos primary antibody (rabbit, 1:400 dilution, sc-52; Santa Cruz Biotechnology) and either anti-CaMKII primary antibody (mouse, 1:1000 dilution, MA1– 048; Pierce Biotechnology) or anti-GAD67 primary antibody (mouse, 1:1000 dilution, MAB5406; Millipore) in blocking solution. After washing, sections were incubated for 2 h in blocking solution with secondary antibodies Alexa Fluor 488-labeled donkey anti-rabbit (1:200 dilution, A-21206; Invitrogen) and Alexa Fluor 568-labeled goat anti-mouse antibody (1: 200 dilution, A-11004; Invitrogen). Sections were then washed, mounted on chrom–alum/gelatin-coated slides, air dried, and coverslipped with Vectashield or mowiol fluorescent mounting medium. Fluorescent images of dorsal mPFC were captured with QImaging Exi Aqua camera attached to a Carl Zeiss Axioskop 2 microscope using a 40× objective. One to two dorsal mPFC 40× images were taken per hemisphere across two sections per rat. The proportion of Fos + cells that were CaMKII labeled or GAD67 labeled was quantified using iVision MacOS 10.62 (version 4.0.15) by A.B.K.
Experiment 1: effect of mPFC light delivery on astrocyte and microglia markers
In experiment 1, we assessed whether continuous light stimulation caused cell inflammation or injury as assessed by astrocyte or microglia activation (summary in ) (Kreutzberg, 1996
; Ridet et al., 1997
). For this purpose, we measured GFAP (a marker of astrocyte activation) and Iba-1 (a marker of microglia activation) 24 h after dorsal mPFC laser light delivery to naive rats (no viral injection). The naive rats (n
= 8) received bilateral fiber optic implants (precalibrated to 20 mW light output from fiber tip) in the dorsal mPFC as described above. Two weeks after surgery, the rats were habituated to upcoming experimental manipulations in two to three sessions in which the rats were connected to a dummy patch cable system and were exposed to the behavioral chamber for 30 –90 min. Three weeks after surgery, the rats were connected unilaterally (right fiber optic) to the laser via a fiber optic rotary joint/patch cable system (Doric Lenses) and intracranial laser light (593.5 nm, recalibrated to 20 mW with power-matched fiber before testing) was delivered unilaterally using one of the following parameters: 30 min of continuous light (n
= 3), 30 min of intermittent light delivery over 1 h (5 s, n
= 3 or 5 min, n
= 2 cycles). The contralateral hemisphere served as the within-subject control for mechanical damage after fiber optic implant. After 24 h, rats were killed, and inflammatory signaling and cell injury were examined by Iba-1 (microglia) and GFAP (astrocyte) immunoreactivity.
Experiment 2: effect of mPFC light delivery on ex vivo neural activity
We examined the effect of 532 or 593.5 nm light delivery on ex vivo dorsal mPFC neuronal activity from rats (n = 5) injected unilaterally with AAV1–CaMKIIα– eYFP virus and contralaterally with the AAV1–CaMKIIα– eNpHR3.0 – eYFP virus. Six to 7 weeks after viral injection, rats were killed and prepared for electrophysiological recordings, which were conducted as described above. To examine whether the basic intrinsic properties of the cells were altered by the membrane expression of the eNpHR3.0 protein, we measured resting membrane potential and input resistance (eNpHR3.0, n = 9; eYFP, n = 4 cells). We also determined the ability of light to evoke outward hyperpolarizing currents and for those currents to block electrically evoked dorsal mPFC neuronal firing.
Experiment 3: effect of mPFC light delivery on in vivo single-unit neural activity in awake rats
We examined the effect of 593.5 light delivery on in vivo dorsal mPFC neuronal activity in rats injected unilaterally with the eNpHR3.0 construct. The rats (n = 3) received unilateral injections of AAV1–CaMKIIα– eNpHR3.0 – eYFP and ipsilateral implantation of the chronic optrode recording device above the site of viral injection. Three to 8 weeks after surgery, rats were brought to the recording box and were connected to a recording cable (Plexon) coupled with a fiber optic patch cable (Doric Lenses). The recording/patch cable was connected to a hybrid electrical/optical rotary joint (Doric Lenses) or a two-part commutator system (electrical: Christ Instruments; optical: Doric Lenses) that allowed freedom of movement in the awake rat. Fiber optic output was precalibrated to 4 – 8 mW from the fiber tip before implantation, and the laser was recalibrated to this power output using a power-matched fiber optic before stimulation/recording sessions. Wires with detectable neural activity were selected to be recorded, and a stimulation session was conducted. To determine whether neurons were light sensitive, the laser was activated in 5 s (40 trials; 10 s intertrial interval), 2 min (five trials, 2 min intertrial interval), and/or 30 min (two trials, 30 min intertrial interval) cycles, and spontaneous activity of neurons was recorded during the light stimulation protocols. The optrode was advanced at the end of the session. Single-unit activity was analyzed as described above (see section In vivo neural data analyses).
Experiment 4: effect of mPFC light delivery on yohimbine-induced Fos induction
We examined the effect of bilateral 593.5 nm intracranial light delivery on water (vehicle)-induced and yohimbine-induced mPFC neuronal activation (as assessed by Fos immunohistochemistry) in rats that received unilateral microinjection of the AAV1–CaMKIIα
– eYFP construct into dorsal mPFC and unilateral microinjection of the AAV1–CaMKIIα
–eNpHR3.0 – eYFP construct into the contralateral side, as well as bilateral fiber optic implants above the site of viral injection. Four to 5 weeks after surgery, the rats were habituated to upcoming experimental manipulations in two to three sessions in which they received sham water (vehicle) injections, were connected to a dummy patch cable system, and were exposed to the behavioral chamber for 30 –90 min. Five to 6 weeks after surgery, in a single session, rats received either vehicle (n
= 8) or yohimbine injections (2 mg/kg, i.p.) (n
= 9) and were immediately connected bilaterally to the laser via a fiber optic rotary joint/patch cable system (Doric Lenses), and intracranial laser light (90 min continuous, 593.5 nm, recalibrated to 4 – 8 mW with power-matched fiber before testing) was delivered bilaterally. The laser was turned off, and the rats were disconnected from the laser patch cable 90 min after the injection. Two hours after the injection, rats were anesthetized and perfused, their brains were removed, and tissue was subsequently processed for Fos immunohistochemistry as described above. We delivered intracranial light for a longer duration (90 min) than that used in our behavioral experiments, because yohimbine-induced mPFC neuronal activation lasts for at least 6 h (Cifani et al., 2012
). The rats were anesthetized and perfused 2 h after yohimbine injections, because Fos induction is often maximal at this time point after neuronal activation (Morgan and Curran, 1991
Experiment 5: effect of mPFC light delivery on ongoing food self-administration
We examined the effect of bilateral 593.5 nm intracranial light delivery on ongoing food self-administration in rats that underwent surgery to inject either the AAV1–CaMKIIα
– eYFP virus (n
= 17) or the AAV1–CaMKIIα
– eNpHR3.0 – eYFP virus (n
= 15) bilaterally into the dorsal mPFC and to chronically implant fiber optics above the injection sites. Fiber optic output was precalibrated to 4 – 8 mW from the fiber tip before implantation, and the laser was recalibrated to this power output using a power-matched fiber optic before behavioral testing sessions. The training conditions were similar to those used in our previous studies (Nair et al., 2011
; Pickens et al., 2012
). Briefly, we gave the rats 3 h “autoshaping” sessions for 2 or 3 d, during which pellets were delivered noncontingently every 5 min into a receptacle located near the active lever. Pellet delivery was accompanied by a compound 5 s tone–light cue, both of which were located above the active lever. Subsequently, we trained the rats to self-administer the pellets on a fixed-ratio-1 (FR-1), 20-s timeout reinforcement schedule. At the start of each 3 h session, the red house light was turned on, and the active lever was extended. Reinforced active lever presses resulted in the delivery of one pellet, accompanied by the compound 5 s tone–light cue. Rats underwent 10 –11 training sessions under these conditions. A subset of the rats in the eYFP (n
= 10) and eNpHR3.0 (n
= 10) groups were tested with intracranial light delivery (593.5 nm, 4 – 8 mW, first 30 min continuous) during a single training session (between sessions 7 and 10), and responding during the first 30 min of this session was compared with responding during the first 30 min of the following session in which intracranial light was not delivered.
Experiment 6: effect of mPFC light delivery on reinstatement of food seeking
We used the same rats as above [two groups of rats: eYFP virus (n
= 14) and eNpHR3.0 virus (n
= 15)] to examine the effect of bilateral 593.5 nm intracranial light delivery on yohimbine- and pellet-priming-induced reinstatement of food seeking. After 10 –11 3-h food self-administration training sessions, all rats underwent extinction sessions that were identical to the training sessions, except that presses on the active lever were not reinforced with a food pellet. The rats were given 11–29 extinction sessions until they reached an extinction criterion (mean active lever responding across the last three extinction sessions of <20% of extinction day 1 responding). Rats that did not reliably extinguish lever responding during the regular 3 h sessions were given four to eight sessions comprising three 1 h mini-sessions, separated by 5 min with the house light off and lever retracted. We then assessed the effect of intracranial light delivery on yohimbine-induced- and pellet-priming-induced reinstatement during 30 min (pellet) or 1 h (yohimbine) sessions. The rats received either water (vehicle) or yohimbine injections (2 mg/kg, i.p.) 30 – 40 min before the start of the test sessions or received four pellets at the beginning of the test session in a counterbalanced order. Each test condition was performed with intracranial light delivery [593.5 nm, 4 – 8 mW, 30-min continuous (pellet) or 1 h continuous (yohimbine)] or without light delivery [30 min (pellet) or 1 h (yohimbine)] in a counterbalanced order. Each rat underwent at least one yohimbine-free and pellet-free extinction session between each test session. Data from the first 30 min of the 1 h yohimbine sessions were used in the statistical analysis to allow for statistical comparisons with the control condition of 30 min light stimulation after vehicle injections and the 30 min pellet-priming manipulation. As described in Results for the 30 min data point, the 60 min light stimulation significantly inhibited yohimbine-induced reinstatement in the eN-pHR3.0 virus condition (40.5 ± 6.7 vs 78.3 ± 18.7 active lever presses per 60 min for the light stimulation and no light, respectively, t(14)
= 2.4, p
= 0.029) but not the eYFP virus condition (43.4 ± 8.0 vs 55.5 ± 9.1 lever presses per 60 min for the light stimulation and no light, respectively, p
> 0.05). [Note that we assessed different durations of light stimulations for pellet priming and yohimbine, because the effect of the former on reinstatement is rarely observed beyond 30 min, whereas yohimbine effects on reinstatement can last for at least 3 h because of the long half-life of the drug (Nair et al., 2009
Experiment 7: phenotypic characterization of dorsal mPFC neurons activated by yohimbine-induced reinstatement
We examined the phenotype of dorsal mPFC neurons that were activated by yohimbine-induced reinstatement using a separate group of rats (n = 8). The rats underwent 10 training sessions, followed by 14 extinction sessions, and a single test for yohimbine-induced reinstatement, as described in experiments 5 and 6. Two hours after the yohimbine injection, the rats were anesthetized and perfused, their brains were removed, and tissue was subsequently processed for Fos/CaMKII and Fos/GAD immunohistochemistry as described above.
The behavioral and molecular data were analyzed by ANOVAs and t tests, and significant main effects and interaction effects (p < 0.05) were followed by Fisher’s protected least significant difference or Bonferroni’s post hoc tests. The dependent measures and the factors used in the statistical analyses are described in Results. Because some of our multifactorial ANOVAs yielded multiple main and interaction effects, we only report significant interaction or main effects that are critical for data interpretation.