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.

1. General Set Up and Tissue Preparation
<ol>
	<li>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 ...

<ol>
	<li>Robinson, H. P. C., Kawai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+digitally+synthesized+synaptic+conductance+transients+to+measure+the+integrative+properties+of+neurons.">Injection of digitally synthesized synaptic conductance transients to measure the integrative properties of neurons.</a> Journal of Neuroscience Methods. 49, 157-165 (1993).</li><li>Sharp, A. A., O'Neil, M. B., Abbott, L. F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+clamp:+artificial+conductances+in+biological+neurons.">The dynamic clamp: artificial conductances in biological neurons.</a> Trends in Neuroscience. 16, 389-394 (1993).</li><li>Sharp, A. A., O'Neil, M. B., Abbott, L. F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+clamp:+computer-generated+conductances+in+real+neurons.">Dynamic clamp: computer-generated conductances in real neurons.</a> Journal of Neurophysiology. 69, 992-995 (1993).</li><li>Johnson, D., Wu, S. M. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Foundations of cellular neurophysiology. , (1995).</li><li>Taylor, W. R., Vaney, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+synaptic+mechanisms+generate+direction+selectivity+in+the+rabbit+retina.">Diverse synaptic mechanisms generate direction selectivity in the rabbit retina.</a> Journal of Neuroscience. 22 (17), 7712-7720 (2002).</li><li>Jurkat-Rott, K., Lehmann-Horn, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+patch+clamp+technique+in+ion+channel+research.">The patch clamp technique in ion channel research.</a> Current Pharmaceutical Biotechnology. 4, 221-238 (2003).</li><li>Prinz, A. A., Abbott, L. F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+clamp+comes+of+age.">The dynamic clamp comes of age.</a> Trends in Neuroscience. 27, 218-224 (2004).</li><li>Williams, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+compartmentalization+and+functional+impact+of+conductance+in+pyramidal+neurons.">Spatial compartmentalization and functional impact of conductance in pyramidal neurons.</a> Nature Neuroscience. 7 (9), 961-967 (2004).</li><li>Perkins, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-attached+voltage-clamp+and+current-clamp+recording+and+stimulation+techniques+in+brain+slices.">Cell-attached voltage-clamp and current-clamp recording and stimulation techniques in brain slices.</a> Journal of Neuroscience Methods. 154 (1-2), 1-18 (2006).</li><li>Feng, S. S., Jaeger, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+SK+calcium-dependent+potassium+currents+in+regulating+the+activity+of+deep+cerebellar+nucleus+neurons:+a+dynamic+clamp+study.">The role of SK calcium-dependent potassium currents in regulating the activity of deep cerebellar nucleus neurons: a dynamic clamp study.</a> Cerebellum. 7 (4), 542-546 (2008).</li><li>Butera, R., Lin, R., Destexhe, A., Bal, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+factors+for+improving+dynamic+clamp+performance.+In:+Dynamic-Clamp+From+Principles+to+Applications+Series.">Key factors for improving dynamic clamp performance. In: Dynamic-Clamp From Principles to Applications Series.</a> Dynamic-Clamp From Principles to Applications Series: Springer Series in Computational Neuroscience. 1, (2009).</li><li>Marco, S. D. i., Nguyen, V. A., Bisti, S., Protti, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permanent+functional+reorganization+of+retinal+circuits+induced+by+early+long-term+visual+deprivation.">Permanent functional reorganization of retinal circuits induced by early long-term visual deprivation.</a> The Journal of Neuroscience. 29 (43), 13691-13701 (2009).</li><li>Zhao, Y., Inayat, S., Dikin, D. A., Singer, J. H., Ruoff, R. S., Troy, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+technique:+review+of+the+current+state+of+the+art+and+potential+contributions+from+nanoengineering.">Patch clamp technique: review of the current state of the art and potential contributions from nanoengineering.</a> Proceedings of the Institution of Mechanical Engineers, Part N: Journal of Nanoengineering and Nanosystems. 222 (1), 1-11 (2009).</li><li>Lobb, C. J., Paladini, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+NMDA+receptor+conductance+in+rat+midbrain+dopaminergic+neurons+using+the+dynamic+clamp+technique.">Application of a NMDA receptor conductance in rat midbrain dopaminergic neurons using the dynamic clamp technique.</a> J. Vis. Exp. (46), e2275 (2010).</li><li>Briggman, K. L., Euler, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+electroporation+and+population+calcium+imaging+in+the+adult+mammalian+retina.">Bulk electroporation and population calcium imaging in the adult mammalian retina.</a> Journal of Neurophysiology. 105 (5), 2601-2609 (2011).</li><li>Middleton, T. P., Protti, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoids+modulate+spontaneous+synaptic+activity+in+retinal+ganglion+cells.">Cannabinoids modulate spontaneous synaptic activity in retinal ganglion cells.</a> Visual Neuroscience. 28 (5), 393-402 (2011).</li><li>Pottek, M., Knop, G. C., Weiler, R., Dedek, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+characterization+of+GFP-expressing+cell+populations+in+the+intact+retina.">Electrophysiological characterization of GFP-expressing cell populations in the intact retina.</a> J. Vis. Exp. (57), e3457 (2011).</li></ol>

All procedures were first approved by the Animal Care Ethics Committee of The University of Sydney, and then performed in accordance with the guidelines of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (National Health and Medical Research Council of Australia).

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...

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 neurotransmitters to ganglion cell response, however, is usually hindered as pharmacological agents act on various sites.
Physiological responses of retinal neurons to light, the natural stimulus, can be recorded with glass pipettes filled with intracellular fluid. Using patch clamp techniques, neuronal responses to light stimulation can be recorded as membrane potential fluctuations (current clamp) or as currents (voltage clamp). By holding the membrane potential at different voltages and implementing a posteriori conductance analysis, it is possible to isolate inhibitory and excitatory synaptic inputs 5,12. This type of experiments can be carried out in normal bathing medium and in the presence of different pharmacological agents to isolate the contribution of different neurotransmitters and receptors to neuronal responses. A wealth of studies from many laboratories characterized the dependence of spiking output and excitatory and inhibitory inputs on stimulus properties such as size, contrast, spatial and temporal frequencies, direction, orientation and other stimulus variables. Although these experimental approaches provide information about the relationship between spike output and synaptic inputs as a function of stimulus properties, interpretation of the contribution of specific cell types and their synaptic inputs to cell excitability is not straightforward. This is due to the fact that typically both excitatory and inhibitory inputs vary with stimulus properties and thus, it is not possible to assess the precise impact that changes in either of these inputs has on neuronal spiking.
An alternative approach to circumvent these limitations is to carry out dynamic clamp recordings, which allow a critical evaluation of the contribution of individual synaptic inputs to spiking output. The dynamic clamp technique allows direct injection of current into the cell and the amount of current injected at a given time depends on the recorded membrane potential at that time 1,2,3 (for review, see 7,14). It is a modified current clamp set-up where a real-time, fast feedback interaction between the cell under recording and the equipment comprising specialized hardware, software and a computer is achieved. The amount of current injected into the cell is computed accordingly. Hence, the advantage of this method is that the cell can be stimulated with different combinations of conductance waveforms, and its response will mimic the activation of receptors that mediate synaptic inputs. For example, comparison of the response to injection of excitatory and inhibitory conductances for a small spot with the response to injection of excitatory conductance for a small spot only provides information about the impact of inhibition on cell response. Likewise, other combinations of physiologically recorded conductances can be co-injected to reveal how stimulus-dependent changes in excitatory and/or inhibitory conductances affect spike output.
In our study, the dynamic clamp technique is used to demonstrate the impact of the relative amplitude and timing of synaptic inputs on the firing properties of retinal ganglion cells. Various conductances obtained from physiological experiments in control conditions or in the presence of pharmacological agents were employed as inputs. In addition, artificial conductances based on alpha functions were also used in order to investigate how synaptic inputs are integrated by neurons. Thus this is a versatile technique that allows various types of conductance generated either physiologically, pharmacologically or computationally to be injected into the same ganglion cell, so comparison of responses to these inputs can be made.

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implementing dynamic clamp with synaptic and artificial conductances in mouse retinal ganglion cells

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 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...

Watch this Scientific Journal Video about Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells at JoVE.com

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

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 ...

implementing-dynamic-clamp-with-synaptic-artificial-conductances

Research

JoVE Journal

Neuroscience

12.2K Görüntüleme.  University of Sydney. Bu video makalede hücre gövdeleri yama kurulum, usul ve nasıl tüm montaj fare retinae ganglion hücrelerinden dinamik kelepçe kayıtları uygulamaya göstermektedir. Bu teknik, eksitatör ve inhibitör sinaptik girdi kesin katkısının incelenmesi ve nöronal spike için göreceli büyüklüğü ve zamanlaması sağlar.