Acute slice preparation
All procedures involving animals were performed according to methods approved by the UK Home Office and The Animals (Scientific Procedures) Act 1986. The authors have read, and the experiments comply with, the policies and regulations of The Journal of Physiology (Drummond, 2009). Acute coronal slices were prepared from C57BLn mice (postnatal day 16–25; Charles River, Margate, UK). Animals (n= 98) were decapitated under deep isoflurane anaesthesia (4% in O2), and their brains were rapidly removed and placed in ice-cold sucrose artificial cerebrospinal fluid (ACSF) cutting solution (containing (in mm): 75 sucrose, 87 NaCl, 25 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 7 MgCl2, 25 glucose, saturated with 95% O2–5% CO2, at pH 7.3–7.4). Slices (350 μm) containing amygdala (at approx. −0.9 mm Bregma level) were cut (Leica VT 1000S, Leica Microsystems GmbH, Nussloch, Germany) and transferred to a nylon mesh where they were maintained in a chamber initially containing sucrose ACSF cutting solution at 37°C for 30 min. During this period the cutting solution was replaced with normal ACSF (containing (in mm): 130 NaCl, 24 NaHCO3, 3.5 KCl, 1.25 NaH2PO4, 2.5 CaCl2, 1.5 MgSO4, 10 glucose saturated with 95% O2–5% CO2, at pH 7.3). Slices were then maintained at room temperature (20–22°C).
Electrophysiology and analysis
Acute slices were secured under a nylon mesh, submerged and superfused with ACSF in a 2 ml chamber mounted on the stage of an upright microscope (Axioskop, Zeiss, Jena, Germany). Slices were visualized with a 10×/0.3 NA or 40×/0.8 NA water-immersion objective (Zeiss, Oberkochen, Germany) coupled with infrared and differential interference contrast (DIC) optics linked to a video camera (Newvicon C2400, Hamamatsu, Hamamatsu City, Japan). Initially only cells with subsequent anatomical confirmation of their position (see below) were used for further analysis. However, in a minority of experiments the position of Im was identified online with infrared and DIC optics without further anatomical confirmation of the position of the recorded neuron within Im boundaries. Somatic whole-cell patch-clamp recordings (33–35°C) were made from visually identified cells using borosilicate glass capillaries (GC120F, 1.2 mm o.d., Clarke Electromedical Instruments, Reading, UK), pulled on a DMZ puller (Zeitz-instrumente GmbH, Munich, Germany) and filled with a filtered intracellular solution consisting of (in mm): 126 potassium gluconate, 4 KCl, 4 ATP-Mg, 0.3 GTP-Na2, 10 Na2-phosphocreatine, 10 Hepes and 0.5% w/v biocytin (all from Sigma-Aldrich Co. Ltd, Poole, UK), osmolarity 270–280 mosmol l−1 without biocytin, pH 7.3 with KOH. A caesium-based intracellular solution was used to isolate AMPA and NMDA receptor-mediated excitatory postsynaptic currents (AMPA-, NMDA-EPSCs) consisting of (in mm): 126 caesium methansulfonate, 4 CsCl, 10 Hepes, 10 Na2-phosphocreatine, 4 Mg-ATP, 0.3 Na-GTP and 0.5% w/v biocytin (all from Sigma-Aldrich), osmolarity 270–280 mosmol l−1 without biocytin, pH 7.3 with CsOH. Biocytin was added to allow post hoc visualization of the recorded neurons. Resistance of the patch pipettes was 5–6 MΩ, and recordings were accepted only if the initial seal resistance was greater than 1 GΩ. The series resistance (Rs) was compensated online by 60–70% in voltage-clamp mode to reduce voltage errors, and cells were only accepted for analysis if the initial Rs was less than or equal to 25 MΩ (range, 14–25 MΩ) and did not change by more than 20% throughout the recording period. Throughout the text and figures, membrane potentials for all whole-cell recordings have been corrected for an experimentally determined liquid junction potential of −12 mV for recordings with the potassium gluconate intracellular solution.
All electrophysiological signals were amplified (10 mV pA−1, EPC9/2 amplifier HEKA Electronik, Lambrecht, Germany; Pulse software), low-pass filtered at 2.9 kHz and digitized at 5 or 10 kHz. The amplifier was controlled from a personal computer running the Pulse data acquisition and analysis programme (HEKA). Currents/voltages were acquired online with Pulse software and analysed offline with Pulsefit (HEKA) and IGOR Pro5.05 (Wavemetrics Inc., OR, USA). The input resistance (Rin) was calculated from the slope of a line fitted to the subthreshold range on a plot of the injected current versus the steady-state membrane voltage when a family of hyperpolarising and depolarising current injections were applied (range, −30 to +120 pA). The apparent membrane time constant (τ) was calculated by fitting a single exponential to the response of the cell to a current injection of −50 pA in current-clamp mode. Membrane capacitance was calculated as τ/Rin. To study the kinetics of action potentials, a depolarising current step (3 ms, 100–150 pA) was applied. Action potential half-width, peak amplitude and membrane afterhyperpolarization (AHP) were measured from the initial point of the action potential raising phase by a user-defined program in IGOR. The sag ratio was calculated from the membrane potential at the end of a 1 s hyperpolarising pulse divided by the largest membrane potential change observed in response to a current step (range, −80 to –120 pA). The adaptation index was calculated as the ratio between the first and last interspike intervals evoked by a 1 s depolarising current pulse. The maximal (max.) firing rate was the number of action potentials elicited by a strong depolarising current pulse (range, 250–500 pA). Steps of depolarising current were injected into cells (200 ms, range, 50–100 pA) to evaluate the action of DA and DA receptor antagonists on Im neurons. The values of the resting membrane potential and number of action potentials were measured from the averaged last 10 trials of control and bath application of the drugs.
Extracellular stimulation was conducted by applying rectangular pulses of current (0.1 ms, range, 5–50 μA) delivered through an isolation unit (A360 Stimulus Isolator, World Precision Instruments, Stevenage, UK) to a monopolar patch pipette filled with ACSF. The stimulating electrode was placed in different sites of the amygdala to evoke inhibitory postsynaptic currents (eIPSCs) or excitatory postsynaptic currents (eEPSCs). Extracellular stimulation experiments were conducted in the presence of kynurenic acid (3 mm) or bicuculline metachloride (5 μm) during recordings of eIPSCs or eEPSCs, to block AMPA/kainate/NMDA or GABAA receptors, respectively. To determine the lowest intensity at which eIPSCs/eEPSCs could be detected, an input–output protocol was performed (range, 5–150 μA, step of 5–10 μA). The PSC kinetics analysis was performed on synaptic responses evoked by lowest possible stimulus intensities resulting in a detectable response. No response was judged by the absence of any detectable current in single sweeps following (2–100 ms) the stimulus artefact. The values of peak amplitude, latency, 20–80% rise time, decay time and area of events were measured from the average of three consecutive sweeps. The values of synaptic latency jitter were calculated from the standard deviation of the synaptic latency of 10 consecutive eEPSCs. The event peak amplitude was visually delimited and then measured using Pulsefit. The paired-pulse ratio was calculated as the mean peak amplitude of the response to the second stimulus divided by the mean peak amplitude of the response to the first stimulus, measured from the average of 10 consecutive sweeps. The latency of events was determined as the time between the onset of stimulation artefact and the onset of the postsynaptic response. The 20–80% rise time, decay time (fitted with a single exponential), jitter and area of the synaptic currents were analysed with a user-defined program in IGOR.
In the occlusion test, stimulating electrodes were placed within Im and IC or EC. The actual and predicted sums of values of peak amplitude eEPSCs elicited by two stimuli after independent and simultaneous stimulation were compared. If the latency of two events were different we corrected the onset of stimulation online in a way that the two stimulus artefacts appeared simultaneously. The values of peak amplitude of events recorded during the occlusion test were measured from 10 trials average.
To isolate AMPA- and NMDA-EPSCs we placed the stimulating electrode in Im (stimulating intensity range, 5–50 μA) and recorded in voltage-clamp mode at a holding potential (VH) range of −70 to +40 mV. AMPA-EPSCs were recorded in the presence of bicuculline (30 μm) and NMDA-EPSCs in the presence of 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX; 50 μm) and bicuculline (30 μm). The peak amplitude of the eEPSCs was measured using Pulsefit from the average of three sweeps for each VH. The reversal potential was calculated from the slope of a line fitted to a plot of values of VH versus the peak amplitude of recorded currents.
Histological processing of recorded cells
Immediately after electrophysiological recordings, slices were immersed overnight at 4°C in fixative composed of 4% paraformaldehyde, 15% (v/v) saturated picric acid and 0.1 m phosphate buffer (PB; pH 7.2–7.4). Gelatine-embedded slices were re-sectioned into 60 μm thick sections. To confirm the location of the recorded cells in Im, we processed the sections containing biocytin-loaded cells with the horseradish peroxidase method and counterstained the tissue with Nissl staining or immunohistochemistry reaction anti-NeuN. Briefly, biocytin-filled cells were incubated in an avidin–horseradish peroxidase complex (1:100 dilution; ABC Kit, Vector Laboratories, Burlingame, CA, USA) followed by a peroxidase reaction using diaminobenzidine (DAB, Sigma-Aldrich; 0.05%) as the chromogen and H2O2 (1%) as the substrate. Nissl staining was performed by dehydrating–rehydrating the sections and immersing them into a cresyl violet acetate solution (1 g per 400 ml H2O; pH 3.6), dehydrating sections were then immersed in xylene-based mounting medium (Entellan; Merck, Damstadt, Germany) and coverslipped. Alternatively to Nissl staining, anti-NeuN immunoperoxidase was performed to visualise the amygdala nuclei boundaries. In this case, only neuronal cell nuclei and somata were stained, resulting in a more accurate determination of central nucleus (CE) subdivision boundaries (McDonald, 1982). Non-specific antibody binding was blocked by incubation in 20% normal goat serum (NGS; Vector Laboratories Ltd, UK) diluted in Tris-buffered saline (TBS; 0.9% NaCl, 0.05 m Tris, pH 7.4 containing 0.2% Triton X-100 for 45 min). Mouse monoclonal antibody anti-NeuN (1:3000, Chemicon, Millipore; MAB377) was diluted in TBS, containing 1% NGS and 0.2% Triton X-100, and applied overnight (at 4°C). The sections were subsequently washed in TBS and incubated overnight (at 4°C) in biotinylated, secondary goat anti-mouse IgG (diluted 1:200, Chemicon, Millipore, MA, USA), diluted in TBS, containing 1% NGS and 0.2% Triton X-100. The sections were washed in PB and processed for peroxidase-based visualisation of the amygdala nuclei boundaries and mounted on slides for permanent storage (see above). A Neurolucida system (MicroBrightField, Inc., Magdeburg, Germany) was used to reconstruct the neurons (100× or 63× oil immersion objective).
Statistical analyses were carried out using SPSS 17.0 (SPSS, Chicago, IL, USA). All values are expressed as means ±s.e.m. Statistical comparisons were made using a Student's paired t test. For all analyses, statistical significance was set at P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***). Unsupervised, hierarchical cluster analysis using Ward's method (Ward, 1963) and Euclidian distance was performed to classify subtypes among Im cells. Ward's method minimizes the error sum of squares of any pair of clusters formed at a given step; this maximizes between-group differences and minimizes within-group differences. Before clustering, each electrophysiological property was converted into standardized z-scores. This achieves a normal distribution and prevents variables with larger ranges from having a greater influence on the cluster solution than variables with small ranges. Statistical differences between three groups were estimated based on one-way ANOVA with post hoc Bonferroni test, P < 0.05.
Chemicals and drugs
All drugs were applied to the slice via the bath solution. Salts used for the patch pipette and ACSF were obtained either from VWR International (Lutterworth, UK) or Sigma-Aldrich. Drugs obtained from Tocris (Cookson Inc., Avonmouth, UK) were added at the following concentrations: (–)-bicuculline metachloride, 5 or 30 μm; NBQX, 50 μm; d-AP5, 100 μm, SR95531, 5 μm, SCH 23390 hydrochloride, 5 μm; (S)-(–)-sulpiride, 5 μm. Drugs obtained from Sigma-Aldrich were added at the following concentrations: kynurenic acid, 3 mm; DA, 30 μm.