Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Summary

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

Abstract

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp ^{1, 2, 3} and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

Introduction

The retina is a near-transparent neural tissue lining the back of the eye. Many studies use the retina as the model to investigate the first steps in visual processing and mechanisms of synaptic signaling. Since the retinal network in the whole-mount preparation remains intact after dissection, it represents an ideal system to study synaptic interactions as its physiological responses are very similar to the in vivo conditions. Thus, using an isolated retina the properties of its neurons can be studied using patch clamp techniques (for reviews on the technique, see ^6,9,13). Identification of the exact contribution of specific circuits and neurotran....

Protocol

1. General Set Up and Tissue Preparation

1. Keep the mouse in darkness for 30 min (aim to reduce its stress level). While waiting, prepare 1 L of extracellular solution. First dissolve 1.9 g of sodium bicarbonate in half a liter of Milli-Q water. Its pH is maintained at 7.4 by bubbling with 95% O₂ and 5% CO₂. Five minutes later, dissolve 8.8 g of Ames Medium in 100 ml of Milli-Q water, add to the sodium bicarbonate solution, top up to 1 L with Milli-Q water and mix well. Keep this solution carboxygenated for the rest of the experiment.
2. Take 250 ml of the carboxygenated Ames Medium and set up the fluid reperfusion system in the ....

Results

The contribution of different sources of inhibitory inputs to ganglion cell responses is demonstrated through the application of various conductance waveforms. These waveforms were obtained with stimuli of different luminance in normal conditions and in the presence of TTX, a voltage-gated Na⁺ channel blocker that blocks action potential generation only in a subset of inhibitory retinal interneurons. Figure 2A shows a representative response to injection of excitatory and inhibitory conductanc.......

Discussion

Here we show the use of dynamic clamp to assess the influence of the ratio and relative timing of excitation and inhibition on retinal ganglion cell output. Dynamic clamp makes use of computer simulations to introduce physiologically recorded or artificial synaptic conductances into living neurons. This methodology provides an interactive tool by which conductances can be modified and injected into neurons for computing their influence on neuronal responses. Conductance waveforms can be obtained from experiments in which.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent
Isoflurane Inhalation Anaesthetic	Pharmachem
Ames Medium with L-Glutamate (Powder)	Sigma-Aldrich
Potassium Gluconate, Anhydrous	Sigma-Aldrich
HEPES Sodium salt	Sigma-Aldrich
Magnesium chloride solution (4.9 mol/l)	Sigma-Aldrich
Adenosine 5'-triphosphate (ATP) disodium salt hydrate	Sigma-Aldrich
Guanosine 5'-triphosphate sodium salt hydrate	Sigma-Aldrich
Ethylene glycol-bis(2-amin–thylether)-N,N,N',N'-tetraacetic acid	Sigma-Aldrich
Paraformaldehyde Powder, 95%	Sigma-Aldrich
Anti-Lucifer Yellow, Rabbit IgG Fraction (3 mg/ml)	Invitrogen
Alexa Fluor 594 Goat Anti-Rabbit IgG (H+L) 2 mg/ml	Invitrogen
Fluorescent Preserving Media	BioFX Laboratories Inc.
Equipment
Capillary Glass Tubing with flame polished ends (OD = 1.50 mm, ID = 0.86 mm, Length = 15 cm)	Warner Instruments	64-0794
Single Stage Glass Micr–lectrode Puller	Narishinge Japan	Model PP-830
Minipuls 2	Gilson
Millex-GV 0.22 μm Filter Unit	Millipore Corporation	SLGV004SL
Luer Lock Reusable Hypodermic Needle: 30 G	Smith Nephew (Australia)
Single Inline Solution Heater	Warner Instruments	Model SH-27B
Dual Automatic Temperature Controller	Warner Instruments	TC-344B
Olympus Stereomicroscope SZ61	Olympus Corporation
Olympus Microscope BX50WI: with 40X objective	Olympus Corporation
0-30 V 2.5 A DC Power Supply	Dick Smith Electronics	Q1770
Digital Microscopic Camera ProgResMF cool	Jenoptik
Micromanipulator MP-225	Sutter Instrument Company
Patch Clamp Amplifier EPC 8	HEKA Elektronik
InstruTECH LIH 8+8 Data Acquisition System	HEKA Elektronik
Computer: DELL	Dell Corporation