We used an auditory cortex slice preparation similar to the one previously described (Atzori et al. 2001
). Sprague–Dawley rats, 23–35 days old (Charles River), were anesthetized with isoflurane (Baxter) and sacrificed according to the National Institutes of Health Guidelines (UTD IACUC number 04-04) and their brains sliced with a vibrotome (VT1000, Leica) in a cold solution (0–4 °C) containing (mM) 126 NaCl, 3.5 KCl, 10 glucose, 25 NaHCO3
, 1.25 NaH2
, 1.5 CaCl2
, 1.5 MgCl2
and 0.2 ascorbic acid, at pH 7.4 and saturated with a mixture of 95% O2
and 5% CO2
(ACSF). Coronal slices (270 μm thick) from the most caudal fourth of the brain were retained after removing the occipital convexity (caudal end of the brain after removal of the cerebellum) and subsequently incubated in ACSF at 32 °C before being placed in the recording chamber. The recording area was selected dorsally to the rhinal fissure corresponding to the auditory cortex (Rutkowski et al. 2003
). The recording solution also contained 6,7-dinitroquinoxaline-2,3-dione (10 μM) and kynurenate (2 mM) or (2R
)-amino-5-phosphonovaleric acid; (2R
)-amino-5-phosphonopentanoate (100 μM) for blocking α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor– and N
-aspartate receptor–mediated currents, respectively.
Slices were placed in an immersion chamber, where cells with a prominent apical dendrite, suggestive of pyramidal morphology, were visually selected using an upright microscope (BX51, Olympus) with a ×60 objective and an infrared camera system (DAGE-MTI). Whole-cell voltage-clamp recordings from layer II/III pyramidal neurons of the auditory cortex were performed under visual guidance. Neurons were selected by their pyramidal shape and by their pronounced apical dendrite. Inhibitory postsynaptic currents (IPSCs) were recorded in the whole-cell configuration, in voltage-clamp mode, at a holding membrane potential Vh = −60 mV, with 3–5 MΩ electrodes filled with a solution containing (mM) 100 CsCl, 5 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid K, 1 lidocaine N-ethyl bromide (QX314), 1 MgCl2, 10 N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid), 4 glutathione, 3 ATPMg2, 0.3 GTPNa2, 8 biocytin, and 20 phosphocreatine. The holding voltage was not corrected for the junction potential (<4 mV). The intracellular recording solution was titrated to pH 7.2 and had an osmolarity of 275 mOsm.
Electrically evoked IPSCs (eIPSCs) were measured by delivering 2 electric stimuli (90–180 μs, 10–50 μA) 50 ms apart every 10 s, with an isolation unit, through a glass stimulation monopolar electrode filled with ACSF, or with a concentric bipolar electrode (FHC Inc.), placed at about 100–200 μm from the perpendicular axis connecting the recorded neuron to the cortical neuropil, and layer II/III, lateral from the recorded cell. Synaptic responses were monitored at different stimulation intensities prior to baseline recording. “Normal” stimulation was defined as a stimulation reliably evoking a synaptic current in the range 100 pA to 1 nA. “Minimal” stimulation was defined by a percentage of failures in the range between 15% and 30% and a correspondingly lower response amplitude compared with “normal” stimulation. For each recording, a detection threshold was set at 150% of the standard deviation of the noise (typically around 4–5 pA, threshold around 7–8 pA). Evoked responses lower than the threshold level were counted as failures.
A 2-mV voltage step was applied at the beginning of every episode in order to monitor the quality of the recording. Access resistance (10–20 MΩ) was monitored throughout the experiment. Recordings displaying >20% change in input or access resistance were discarded from the analysis. All signals were filtered at 2 kHz and sampled at 10 kHz. We calculated the reversal potential for our postsynaptic currents through current–voltage (I-V) relationships for the eIPSCs (peak amplitude of 20 events at each of 5 holding potentials Vh in the range from Vh = −60 mV to Vh = +60 mV). The eIPSCs reversed polarity near 0 mV (−2.4 ± 0.3 mV, n = 3, data not shown), near the theoretical reversal potential of −4.6 mV. All experiments were performed at room temperature (22 °C).
Recorded neurons were injected with 8 mM biocytin in the intracellular solution for post hoc identification. Following recording, slices were immediately transferred to a 24-well plate and fixed in a phosphate buffer containing 80 mM Na2HPO4, 80 mM NaH2PO4, and 4% paraformaldehyde. Biocytin staining was then processed using diaminobenzidine as chromogen, using a standard ABC kit (Vector Labs). A light cresyl violet Nissl counterstain was used to identify the cortical layers.
Drugs and solutions
All drugs were purchased from Sigma or Tocris. After recording an initial baseline for 10–15 min, drugs were bath-applied for 10 min or longer, until reaching a stable condition (as defined below in Statistical Analysis). NE, isoproterenol, clonidine, and phenylephrine were prepared immediately before experiments and their exposure to intense light was avoided to prevent oxidation.
We defined a statistically stable period as a time interval (5–8 min) along which the IPSC mean amplitude measured during any 1-min assessment did not vary according to Mann–Whitney U
test. All data are expressed as mean ± standard error of the mean. Pair pulse ratio (PPR) was calculated as the mean of the second response divided by the mean of the first response, according to Kim and Alger (2001)
. The effects of drug application on the IPSC amplitude changes were reported as R
100 × (1 − Atreat
), where Atreat
are the mean IPSC amplitude (including failures) in treatment and control, respectively, or simply as percentage change between Atreat
. Drug effects were assessed by measuring and comparing the different parameters (R
, IPSC mean amplitude, or other parameters as indicated) of baseline (control) versus treatment with a Mann–Whitney U
test. One-way analysis of variance (ANOVA) with Tukey’s post hoc test was used for comparisons between different groups of cells. Wilcoxon test was used for comparing between PPRs, and paired Student’s t
-test was used to compare slow and fast kinetics from eIPSCs. Data were reported as different only if P
< 0.05 unless indicated otherwise. Single asterisk (*) indicates P
< 0.05 and double asterisk (**) indicates P