All experiments were performed in accordance with regulations of the Canadian Council and Animal Care. Mice in this study were heterozygous C57BL/6 BAC transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of the GAD65 promoter [14
]. Adult transgenic mice (3–5 months) were anaesthetized with ketamine/xylazine and perfused intracardially with ice-cold oxygenated (95% O2
, 5% CO2) sucrose substituted ACSF containing (mM) 252 sucrose, 2.5 KCl, 1.5 CaCl2
, 6 MgCl2
, 10 glucose, 26 NaHCO3
, 1.25 NaH2
and 5 kynurenic acid. Mice were decapitated, the spinal cord was removed by hydraulic extrusion and 250 μm thick parasagittal slices were cut from the lumbar portion [36
]. Slices were kept in normal oxygenated ACSF (126 NaCl, 2.5 KCl, 2 CaCl2
, 2 MgCl2
, 10 glucose, 26 NaHCO3
, 1.25 NaH2
) at room temperature until recording.
Slices were transferred in the recording chamber and continuously superfused at 2–3 ml/min with oxygenated ACSF at room temperature. Patch pipettes (7–8 MΩ) were filled with (in mM) 135 KCl, 10 HEPES, 2 MgCl2, 0.5 EGTA, 2 ATP, pH 7.2. For some recordings, KCl was substituted by 130 CsMeSO3/5 CsCl or by 135 CsCl. Whole cell patch clamp recordings were made using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, California, UK). Data were low pass filtered at 10 kHz, digitized at 20–30 kHz and acquired with the Strathclyde electrophysiology software (WinWCP and WinEDR courtesy of Dr. J. Dempster, University of Strathclyde, Glasgow, UK).
Epifluorescence was used to identify EGFP+ neurons. All recordings in this study are from neurons with their somata located in lamina II. Action potential firing was analysed as previously described [20
]. Only cells with resting membrane potential more negative than -50 mV were included in the study. Instantaneous firing frequency (f
) was calculated as the reciprocal of the interspike interval.
The mIPSCs were detected and analysed using Mini Analysis (Synaptosoft, Decatur, Georgia, UK) and a locally designed software (YDK). Decay time constants were fitted using automated least square algorithms. The necessity to introduce additional exponential components to the fits was first judged on the basis of visual inspection. When the merit of additional components was not obvious, further statistical analysis was applied as previously described [26
]. To simplify analysis, complex mIPSC that did not contain the strychnine sensitive, fast decay component (< 14 ms; i.e. mIPSCs that consisted of multiple GABAA
R-mediated decay components), were treated as monotonic.
Monosynaptic IPSCs were evoked focally by electrical stimulation (30–70 μA, 250 μs) via patch pipette filled with ACSF placed 50–100 μm from the soma of the recorded cell as described previously [26
]. Single stimuli or trains of stimuli (12 pulses at 20 Hz intraburst frequency) were delivered every 10 or 20 s, respectively. To isolate the complex decay components of the eIPSCs pharmacologically, all tested cells were treated with strychnine or SR95531. In all cases strychnine blocked only the fast component. Similarly, SR95531, blocked only the slow component in all cases. The relative contribution of each component to the peak current was calculated from the pharmacologically blocked portion (by subtraction) and the portion that was not blocked. In some experiments, strychnine and SR95531 were applied together and abolished all components of the evoked current. eIPSC amplitude measurements from train stimulations were measured from the point immediately before the stimulation artefact to take into account baseline changes due to IPSC summation.
In simultaneous recordings from pairs of neurons (located within < 250 μm of each other), one of the neurons, defined as presynaptic, was kept in current clamp mode and injected with a 100 ms-long depolarizing current pulse of 100 pA to elicit a train of 4–7 action potentials. The other cell, defined as postsynaptic, was held at -70 mV in voltage clamp mode and postsynaptic currents were recorded. These recordings were conducted using KCl filled pipettes to allow for action potential generation and maximum amplification of Cl--mediated IPSCs. All glutamatergic transmission was blocked by APV and CNQX, thus all synaptic connections were assumed to be monosynaptic and inhibitory. This protocol was repeated for 40–50 trials at 10 s intervals for all tested pairs.
For immunostainings, animals were anaesthetized and perfused intracardially with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. Spinal cord segments L4–L5 were collected, postfixed for 60 minutes in PFA and cryoprotected in 30% sucrose in phosphate buffer overnight at 4°C. Fifty μm-thick transverse sections were cut on a freezing microtome (Leica SM2000R). Sections were collected into tissue culture plates with 24 wells, washed with phosphate-buffered saline (pH = 7.4) and 0.2% Triton (PBST), and pre-treated with 5% donkey normal serum in PBST for 10 minutes.
The sections were washed twice in PBST and incubated overnight in a mixture of a mouse anti-parvalbumin antibody (1:4000, Sigma) and a rabbit anti-GFP antibody (1:500, Clontech Living Colors, Mountain View, CA) in PBST. After washing in PBST, the tissue was incubated for 2 hrs at room temperature in a Rhodamine Red-X-conjugated donkey anti-mouse IgG (H+L) antibody (1:500, Jackson, West Grove, PA) and in Alexa Fluor 488-conjugated IgG (H+L) donkey anti-rabbit antibody (Invitrogen, Carlsbad, California, USA) in PBST. Lastly, sections were washed for 15 minutes (3 × 5) with PBS, mounted on gelatin-subbed slides, allowed to dry overnight at 4°C and cover-slipped using Aquapolymount (Polysciences, Warrington, PA). The sections were observed with a Zeiss LSM 510 confocal microscope (Zeiss Canada). Images were obtained using a multi-track approach for the detection of two signals (Alexa Fluor 488 and Rhodamine Red-X), with the help of a 20 × water immersion objective. Data were collected with Metamorph 7 (Molecular Devices) from 1815 cells counted from 20 sections of three mice. Only cells with visible nuclei were counted.
For morphological classification of EGFP+ neurons, in a subset of electrophysiological experiments, neurons (n = 6) were recorded and filled with patch pipettes containing 0.5% neurobiotin. Slices were then fixed in 4% PFA in PB. In addition, 200 μm thick lumbar parasagittal slices taken from 4% PFA fixed spinal cords were used to inject EGFP+ neurons with Lucifer Yellow (LY) as described previously [37
]. LY-injected slices were then incubated with a rabbit anti-LY antibody (1:20000) and subsequently with biotinylated goat anti-rabbit (1:500) antibody. Biotinylated antibodies and Neurobiotin were visualized with a horseradish peroxidase reaction (ABC kit, Vector Laboratories) and nickel-diaminobenzidine as a substrate. Cells were reconstructed using a light microscope with a computerized tracing system (Neurolucida, MicroBrightField Inc). Cells were classified according to previous descriptions [21
]. As the distinction between islet and central cells is sometimes difficult [15
] and due to our small sample, they were treated as one group.
Drugs were obtained as follows: 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D(-)-2-amino-5-phosphonopentanoic acid (D-APV) from Tocris Cookson (Ballwin, Montana, USa), Gabazine (SR95531) and strychnine hydrochloride from Sigma (St. Louis, Missouri, USA), and tetrodotoxin (TTX) from Alomone Labs (Jerusalem, Israel).
Data are presented as means ± SEM, n numbers refer to number of cells tested unless otherwise indicated. Normality of the data was tested with the Shapiro-Wilk test. Paired t-tests were used to compare IPSC frequencies. χ2 tests for contingency tables were used to determine differences in release probabilities. Repeated measure comparison of non-parametric data were analysed using the non-parametric Friedman test and subsequently analysed with the Student-Newman-Keuls posthoc test.