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Materials

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Neuroscience

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

Published: May 16th, 2013

DOI:

10.3791/50400

1Discipline of Biomedical Science, School of Medical Sciences, Sydney Medical School and Bosch Institute, University of Sydney , 2The MARCS Institute, University of Western Sydney, 3Discipline of Physiology, School of Medical Sciences, Sydney Medical School and Bosch Institute, University of Sydney

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.

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.

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....

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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 .......

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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.......

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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.......

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This work is supported by the Australian Research council (ARC DP0988227) and the Biomedical Science Research Initiative Grant from the Discipline of Biomedical Science, The University of Sydney. The equipment Patch Clamp Amplifier EPC 8 was funded by the Startup Fund from the Discipline of Biomedical Science, The University of Sydney. The equipment InstruTECH LIH 8+8 Data Acquisition System was purchased with the funds from Rebecca L. Cooper Foundation and Startup Fund from the Discipline of Biomedical Science, The University of Sydney. We would like to thank the anonymous reviewers for their insightful suggestions and comments.

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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-aminoethylether)-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 Microelectrode 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    

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