In vitro slice physiology and intracellular filling
To examine the synaptic responses evoked by optic tract stimulation, we adopted an acute thalamic slice preparation, which preserves retinal connections and intrinsic circuitry in dLGN [1–3, 10, 23]. Mice were deeply anesthetized with isoflurane vapors and decapitated. Individual (300 μm thick) sections were cut in the parasagittal plane using methods described elsewhere [1, 3, 23]. Sections containing dLGN were placed into a recording chamber and maintained at 32°C and perfused continuously at a rate of 2.0 ml/min with oxygenated artificial cerebrospinal fluid (ACSF; 124 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2.0 mM MgSO4, 26 mM NaHCO3, 10 mM glucose, and 2 mM CaCl2 (saturated with 95% O2/5% CO2), pH 7.4).
In vitro recordings were performed in the whole-cell current-clamp configuration with the aid of DIC and fluorescence optics on a fixed-stage, visualized recording apparatus (Olympus, EX51WI, Shinjuku, Tokyo, JP). Patch electrodes (3 to 7 MΩ) made of borosilicate glass were filled with a solution containing: 140 mM K-gluconate, 10 mM hydroxyethyl piperazine-ethanesulfonic acid (HEPES), 0.3 mM NaCl, 2 mM MgATP, 0.1 mM NaGTP, pH 7.25. Neuronal activity was digitized (10 to 20 kHz) through an interface unit (National Instruments), acquired and stored directly on the computer, and analyzed by using commercial software (Strathclyde Electrophysiology Software, Whole Cell Analysis Program, WinWCP V3.8.2, Glasgow, Scotland, UK). In some cases, the membrane properties and firing characteristics of interneurons were examined by recording the voltage responses to intracellular injections of square-wave current pulses.
To evoke synaptic activity in dLGN, square-wave pulses (0.1 to 0.3 ms, 0.1 to 1 mA) were delivered once every 20 s through a pair of thin-gauge tungsten wires (0.5 MΩ) positioned in optic tract. Stimulating electrodes were connected to a stimulus isolation unit (World Precision Instruments, A360) that received input from a computer controlled, multichannel pulse generator (World Precision Instruments, PulseMaster A300, Sarasota, FL, USA). Estimates of retinal convergence were determined by EPSP amplitude by stimulus intensity plots [2, 3]. These were constructed by first determining the minimum stimulus intensity needed to evoke a postsynaptic response. Once the single fiber response was determined, current intensity was increased in small increments (0.5 to 1.0 μA) until a response of maximal amplitude was consistently reached . A change in amplitude that was equal to or exceeded the value that corresponded to the single fiber response was used to distinguish one input from another. For each intensity value a minimum of five responses were obtained. It is important to note that we saw no evidence of retinally evoked inhibition in our recordings (however, see [14, 15]), nor did we see a change in resting membrane levels even when the highest stimulus intensities were used. To further verify this we compared our recordings performed in normal ACSF with some performed in the presence of 20 μM bicuculine and 10 μM 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP) to block GABAA-mediated and GABAB-mediated activity. There was no significant difference in the number of retinal inputs between cells recorded in the presence or absence of these GABA blockers (t test, P >0.5; mean retinal inputs ± SEM; P11 normal ACSF, 8 ± 1 vs P11 ACSF with GABA antagonists, 7 ± 1; n = 6 cells for both groups).
During some of the recordings a 0.1% to 0.2% biocytin solution containing (in mM): 130 K-gluconate, 10 HEPES, 8 NaCl, 2 MgATP, 0.1 NaGTP, pH 7.25 was included in the patch pipette and neurons were filled by passing alternating positive and negative current pulses (± 0.5 nA, 200 ms) through the recording electrode. After recording, these slices were fixed overnight with 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.2 and then incubated for 24 h in a 0.1% solution of Alexa Fluor 647 conjugated to streptavidin (Invitrogen, Carlsbad, CA, USA) dissolved in PBS with 0.1% Triton X-100. Slices were washed with PBS and then mounted with ProLong Gold antifade reagent (Invitrogen).