C57BL/6 mice were purchased from Harlan (Bicester, UK), and ranged in age from postnatal day (P)6 to P102. Mice were kept on a 12-h light/dark cycle and fed ad libitum. All experiments were performed under the animal care guidelines of the UK Animals (Scientific Procedures) Act 1986.
Thalamocortical Slice Preparation
Thalamocortical slices (350–400 μm) containing the barrel subfield of somatosensory cortex were prepared as previously described (Agmon and Connors 1991
; Mierau et al. 2004
). Briefly, mice were decapitated under isoflurane anesthesia in accordance with UK Animals (Scientific Procedures) Act 1986. The brain was rapidly removed in ice-cold artificial cerebrospinal fluid containing (in mM): NaCl 126; KCl 3; NaH2
26; glucose 10; pH 7.2–7.4; bubbled with carbogen gas (95% O2
). Slices were cut on a vibrating microtome (VT 1000S; Leica, Wetzlar, Germany) and maintained in a submerged-style recording chamber at room temperature (22–27 °C) until used (1–6 h).
Slices containing the barrel subfield were identified under a stereomicroscope by the presence of three to five 200- to 400-μm-wide barrels in layer 4. Whole-cell patch-clamp recordings were made from layer 2/3 pyramidal neurons in one of the barrel columns under visual guidance by infrared differential interference contrast (DIC) microscopy. All recordings were made in a submerged-style recording chamber at 25–29 °C between 1 and 6 h after slice preparation. Current-clamp recordings were made with patch pipettes (5–7 MΩ) pulled from standard-wall borosilicate tubing and filled with a solution containing (in mM): potassium gluconate 110; HEPES (4-[2-hydroxyethyl]piperazine-1-ethanesulfonic acid) 40; NaCl 4; ATP-Mg 4; GTP 0.3, pH 7.2–7.3. In some experiments 5 mg/mL biocytin was included in the pipette solution to enable post hoc identification of the recorded neuron. Voltage-clamp recordings in Supplementary Figure S2
were made with an internal solution containing (in mM): CsCl 140; EGTA (ethylene glycol-bis[β-aminoethyl ether]-N,N,N′,N′-tetraacetic acid) 0.2; HEPES 10; ATP-Mg 2; GTP 0.3; and QX-314 5. All recordings were low-pass filtered at 2 kHz and acquired at 5 kHz using an ITC-16 AD board (Instrutech, Port Washington, NY) and custom-made software procedures programmed in Igor Pro (Wavemetrics, Lake Oswego, OR). Series resistance was monitored by adjusting the bridge balance at regular intervals throughout the experiment. Cells were rejected if series resistance changed by more than 15%.
Timing-Dependent LTD and LTP Induction Protocols
Excitatory postsynaptic potentials (EPSPs) were evoked alternately in 2 input pathways, test and control, each at 0.2 Hz by brief current pulses (50 μs, 5–50 μA) via 2 monopolar stimulation electrodes placed within the base of a barrel in layer 4, vertically aligned to the site of recording. while studying cross-columnar layer 2/3-to-layer 2/3 synapses, one stimulating electrode was placed in layer 2/3, just above the identified barrel structure and another electrode was placed on the opposite side of the recording site and served as control. After a stable EPSP baseline period of 10 min, the test input was paired 100 times with a single postsynaptic spike. The control pathway was not stimulated during the pairing period. To induce t-LTD, the postsynaptic action potential was evoked within 10–15 ms before the onset of the EPSP, whereas the postsynaptic action potential was evoked 10 ms after the onset of the EPSP to induce t-LTP. Both EPSP slopes and peak amplitudes were monitored for at least 20 min after each pairing episode. Presynaptic stimulation frequency remained constant throughout the experiment. Interleaved control t-LTD and t-LTP experiments were performed for each pharmacological blocker tested.
The slope of the EPSP was measured as a linear fit between time points on the rising phase of the EPSP corresponding to 25–30% and 70–75% of the peak amplitude during control conditions. For statistical comparisons, the mean EPSP slope was calculated from 60 consecutive sweeps immediately before the start of pairing (baseline) and compared with 60 sweeps corresponding to 25–30 min after pairing. Data analysis was carried out using Igor Pro software. Data are given as mean ± SEM, unless otherwise stated. Statistical comparisons were made using one-sample or 2-sample 2-tailed Student's t-test as appropriate. One-sample tests are reported in the text, 2-sample tests in figure legends. P values less than 0.05 were considered statistically significant.
D-2-amino-5-phosphonopentanoic acid (D-AP5), (−)-bicuculline methiodide, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) disodium salt, 2-(4-benzylpiperidino)-1-(4-hydroxyphenyl)-1-propanol (ifenprodil) hemitartrate, (αR,βS)-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidinepropanol (Ro 25-6981) maleate, (2S*,3R*)-1-(phenanthren-2-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA), and N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) were purchased from Tocris Bioscience (Bristol, UK). (2R*,3S*)-1-(Phenanthrenyl-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP141) was purchased from Ascent Scientific (Weston-Super-Mare, UK). (R)-[(S)-1-(4-bromophenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077) was a gift from Novartis Pharma AG (Switzerland). All drugs were bath applied.
Cytochrome Oxidase Staining
Cortical slices were flat mounted between glass slides separated by 1- to 1.5-mm spacers and post fixed in 4% paraformaldehyde (PFA) for about 6 h. They were then transferred to 30% sucrose in phosphate buffer (PB) and left overnight. Slices were cut into 80-μm sections using a freezing microtome. Sections were mounted on double-subbed slides and dried overnight at room temperature. Slides were then placed for one hour in PFA at room temperature. Slides were rinsed with PB, transferred into cytochrome oxidase staining solution, containing 15 mg cytochrome c
(Sigma, Dorset, UK), 50 mg diaminobenzidine (Sigma), and 4 g sucrose per 100 mL of 0.1 M PB (Wong-Riley 1979
; Land and Simons 1985
), and incubated at 37 °C in the dark for 24 h. The reaction was stopped by placing the sections in PB. They were then rinsed in distilled water and dehydrated through graded alcohols (50%, 70%, 95%, and 100%), and finally cleared in xylene and mounted in DPX.
Immunohistochemistry for Biocytin-Filled Cells
Barrel cortex slices (400 μm) with biocytin-filled cells were first fixed with 4% PFA and 0.1% glutaraldehyde in PB and resectioned at 30–40 μm. They were then incubated overnight at 4–8 °C in Alexa Fluor–conjugated streptavidin (1:1000; Molecular Probes, Eugene, OR) in phosphate-buffered saline (PBS), washed with PBS, and mounted using Vectashield fluorescence mounting medium (Vector Laboratories, Burlingame, CA).