All experimental procedures were approved by the Albany Medical College and the University of Maryland School of Medicine Institutional Animal Care and Use Committees, and were conducted according to the USPHS Guide for Care and Use of Laboratory Animals.
Neonatal ventral hippocampal lesion
Pregnant Sprague–Dawley rats were obtained at 18 days of gestation from Taconic Farms (Germantown, NY). At postnatal day (PD) 6, male pups (15–19 g) were randomly separated into two groups to receive vehicle injection (sham) or ibotenic acid injection (lesion). Pups (PD 6–7) were anesthetized with hypothermia for 10–15 min and secured on a styrofoam platform mounted on a stereotaxic frame (David Kopf, Tujunga, CA). A cannula was lowered into the ventral hippocampus (AP: −3.0 mm; L: +3.5 mm; H: −5.0 mm) and 0.3 µl of ibotenic acid (10 µg/µl in mM: 148 NaCl, 3 KCl, 0.2 NaH2PO4, 1.5 Na2HPO4, 1.4 CaCl2, 0.8 MgCl2; pH 7.4) was delivered using a minipump at a rate of 0.15 µl/min. The cannula was left in place for three additional minutes before being removed. This procedure was repeated in the contralateral hemisphere. Sham animals received the same volume of vehicle on each side. After surgery, pups were warmed up and returned to their cages, where they remained undisturbed until weaning except for husbandry. The extent of damage induced by ibotenic acid (i.e., areas with cell loss and cell disorganization) was estimated in all animals by Nissl staining. All rats were maintained on a 12 h light/dark cycle with food and tap water available ad libitum until the time of the experiment.
In situ hybridization for GAD67 and PV
Rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) 15 minutes before being decapitated. Brains were immediately frozen after removal from the skull and stored at −80 °C. Serial coronal sections (12 µm) were cut from +2.7 to + 2.2 bregma (Paxinos and Watson, 1998) and three sections evenly spaced at approximately 160 µm intervals were selected from each animal and used for in situ hybridization studies for each mRNA of interest.
For synthesis of riboprobes, we used a 311 bp fragment for GAD67 mRNA, and a 339 bp fragment for PV mRNA, corresponding to bases 151–461 of the mouse GAD67 mRNA (Y12257) and bases 256–594 of the mouse PV mRNA (X59382). Amplified fragments were subcloned into the plasmid pSTBlue-1 (Novagen, Madison, WI) and antisense and sense probes were transcribed in vitro in the presence of 35S-CTP (Amersham Biosciences, Piscataway, NJ), using T7 or SP6 RNA polymerase.
Hybridization was performed as described previously (Hashimoto et al., 2005
). Following fixation with 4% paraformaldehyde in phosphate-buffered saline, the sections were acetylated, dehydrated through a graded ethanol series, and de-fatted in chloroform for 10 min. The sections were then hybridized with 35
S-labeled riboprobes in hybridization buffer at 56°C for 16 h. The sections were washed in solution containing 0.3 M NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0) and 50% formamide at 63°C, treated with RNase A (20 µg/ml) at 37°C, and washed in 0.1X SSC (150 mM NaCl, 15 mM sodium citrate) at 67°C. Sections were then dehydrated through a graded ethanol series, air-dried, and exposed to BioMax MR film (Kodak, Rochester, NY).
Quantification was performed without knowledge of subject condition by random coding of the sections. Trans-illuminated autoradiographic film images were captured by a video camera under precisely controlled conditions, digitized, and analyzed using a Microcomputer Imaging Device (MCID) system (Imaging Research Inc, London, Ontario). Images of the sections were also captured and superimposed onto the autoradiographic images to draw contours of the full thickness of the prefrontal cortex including the cingulate, prelimbic and infralimbic cortices. Expression levels of each mRNA were determined as optical densities within the contours and expressed as nCi/g of tissue by reference to radioactive standards (Carbon-14 standards, ARC Inc., St. Louis, MO) exposed on the same film.
Because bilateral lesions were made and the mRNA levels were quantified in the PFC of each hemisphere independently, we used a two-factor design analysis of variance (ANOVA) with manipulation (lesion versus sham) as a between-subject factor and laterality (left versus right) as repeated measures for each mRNA.
Brain slice preparation
Rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) 15 minutes before being decapitated. Brains were quickly removed from the skull into ice-cold artificial cerebral spinal fluid (aCSF) oxygenated with 95% O2−5% CO2 and containing (in mM): 125 NaCl, 25 NaHCO3, 10 glucose, 3.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 3 MgCl2 (pH 7.45, 295–300 mOsm). Coronal slices (350 µm thick) containing prelimbic and infralimbic regions of the medial PFC were obtained with a Vibratome (St. Louis, MO) in ice-cold aCSF, incubated in warm (~35°C) aCSF solution constantly oxygenated with 95% O2−5% CO2 for at least 60 minutes before recording. The recording aCSF solution was delivered to the recording chamber with a minipump at the rate of 2 ml/min, and CaCl2 and MgCl2 were adjusted to 2 mM and 1 mM, respectively. Patch electrodes (6–9 MΩ) were obtained from 1.5 mm borosilicate glass capillaries (World Precision Instruments, Sarasota, FL) with a Flaming-Brown horizontal puller (P97, Sutter Instrument Co., Novato, CA), and filled with a solution containing 0.125% Neurobiotin and (in mM): 115 K-gluconate, 10 HEPES, 2 MgCl2, 20 KCl, 2 MgATP, 2 Na2-ATP, 0.3 GTP (pH 7.25–730, 280–285 mOsm). All chemicals and drugs (quinpirole, eticlopride, CNQX, and picrotoxin) were purchased from Sigma (St. Louis, MO), and they were mixed into oxygenated recording aCSF solution in known concentrations.
Whole cell patch-clamp recordings
All experiments were conducted at 33–35° C. Medial PFC pyramidal cells and interneurons from layers V–VI were identified under visual guidance using infrared-differential interference contrast (IR-DIC) video microscopy with a 40x water-immersion objective (Olympus BX51-WI). The image was detected with an IR-sensitive CCD camera (DAGE-MTI; Michigan City, IN) and displayed on a monitor. Whole-cell current-clamp recordings were performed with a computer-controlled amplifier (MultiClamp 700A; Axon Instruments/Molecular Devices, Sunnyvale, CA), digitized (Digidata 1322; Axon Instruments), and acquired with Axoscope 8.1 (Axon Instruments) at a sampling rate of 10 KHz. The liquid junction potential was not corrected and electrode potentials were adjusted to zero before obtaining the whole-cell configuration.
Interneuron excitability was assessed by counting the number of action potentials evoked by a 500 ms duration constant-amplitude depolarizing current pulse before and after drug treatment. Typically, current intensity was adjusted to elicit between 5 and 15 action potentials during baseline. In each neuron, input resistance and membrane potential were also monitored throughout the entire recording session.
Synaptic responses were tested in pyramidal neurons with electrical stimulation of layers I–II with a bipolar electrode made from a pair of twisted Teflon-coated nichrome wires (tips separated by approximately 200 µm) and placed ~1 mm lateral to the vertical axis of the recorded neuron. Stimulation pulses (0.4 to 0.8 mA; 0.3 ms) were delivered every 20 s and the intensity was adjusted to half the current required to evoke an action potential. If synaptic responses exhibited more than 15% variation in amplitude during the initial 10 minutes of recording or the current intensity required was larger than 0.8 mA, the neuron was discarded. Input resistance (measured with hyperpolarizing square pulses), membrane potential, and evoked synaptic responses were analyzed before, during and after drug application.
Standard histochemical techniques were used to verify morphology and location of the neurons recorded with Neurobiotin. After completion of the recording session, the entire slices were fixed in 4% paraformaldehyde for 2 hours. After a series of rinses in 0.1 M PBS, slices were incubated in 2% Triton-X 100 in PBS for 2 hours to enhance penetration, followed by 10–12 hours in Vectastain Elite ABC reagent (Vector Laboratories, Burlingame, CA) at 4°C. Following another series of rinses, slices were reacted with 3,3’ diaminobenzidine and urea-hydrogen peroxide (Sigma FAST DAB set). Slices were then rinsed, mounted on gelatin-coated slides, air-dried for 24 hours, cleared in xylene, coverslipped in Permount and examined on an Olympus CH30 microscope.
All measures are expressed as mean ± SD. Drug effects were compared using Student’s t-test or repeated measures ANOVA, and the differences between experimental conditions were considered statistically significant when P<0.05. In some cases, a two-way ANOVA was used to compare the interactions between experimental groups and the time course of synaptic changes obtained throughout the recording.