This research is funded by The National Health and Medical Research Council Project Grant (Grant Number 1109056).

<ol>
	<li>Greenberg, R. J., Velte, T. J., Humayun, M. S., Scarlatis, G. N., de Juan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computational+model+of+electrical+stimulation+of+the+retinal+ganglion+cell.">A computational model of electrical stimulation of the retinal ganglion cell.</a> IEEE Transactions on Bio-medical Engineering. 46 (5), 505-514 (1999).</li><li>Guo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mediating+retinal+ganglion+cell+spike+rates+using+high-frequency+electrical+stimulation.">Mediating retinal ganglion cell spike rates using high-frequency electrical stimulation.</a> Frontiers in Neuroscience. 13, 413 (2019).</li><li>Loizos, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+electrical+stimulation+efficacy+in+degenerated+retina:+Stimulus+waveform+design+in+a+multiscale+computational+model.">Increasing electrical stimulation efficacy in degenerated retina: Stimulus waveform design in a multiscale computational model.</a> IEEE Transactions on Neural Systems and Rehabilitation Engineering. 26 (6), 1111-1120 (2018).</li><li>Cao, X., Sui, X., Lyu, Q., Li, L., Chai, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+different+three-dimensional+electrodes+on+epiretinal+electrical+stimulation+by+modeling+analysis.">Effects of different three-dimensional electrodes on epiretinal electrical stimulation by modeling analysis.</a> Journal of Neuroengineering and Rehabilitation. 12 (1), 73 (2015).</li><li>Wilke, R. G. H., Moghadam, G. K., Lovell, N. H., Suaning, G. J., Dokos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+crosstalk+impairs+spatial+resolution+of+multi-electrode+arrays+in+retinal+implants.">Electric crosstalk impairs spatial resolution of multi-electrode arrays in retinal implants.</a> Journal of Neural Engineering. 8 (4), 046016 (2011).</li><li>AC/DC module user's guide. COMSOL AB Available from: <a target="_blank" href="https://doc.comsol.com/5.4/doc/com.comsol.help.acdc/ACDCModuleUsersGuide.pdf">https://doc.comsol.com/5.4/doc/com.comsol.help.acdc/ACDCModuleUsersGuide.pdf</a> (2018)</li><li>Malmivuo, P., Malmivuo, J., Plonsey, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioelectromagnetism: Principles and Applications of Bioelectric and Biomagnetic Fields. , (1995).</li><li>Rall, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiology+of+a+dendritic+neuron+model.">Electrophysiology of a dendritic neuron model.</a> Biophysical Journal. 2, 145-167 (1962).</li><li>Carnevale, N. T., Hines, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Neuron Book. , (2006).</li><li>Rattay, 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+basic+mechanism+for+the+electrical+stimulation+of+the+nervous+system.">The basic mechanism for the electrical stimulation of the nervous system.</a> Neuroscience. 89 (2), 335-346 (1999).</li><li>Hodgkin, A. L., Huxley, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+description+of+membrane+current+and+its+application+to+conduction+and+excitation+in+nerve.">A quantitative description of membrane current and its application to conduction and excitation in nerve.</a> The Journal of Physiology. 117 (4), 500-544 (1952).</li><li>Liang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threshold+suprachoroidal-transretinal+stimulation+current+required+by+different-size+electrodes+in+rabbit+eyes.">Threshold suprachoroidal-transretinal stimulation current required by different-size electrodes in rabbit eyes.</a> Ophthalmic Research. 45 (3), 113-121 (2011).</li><li>Jensen, R. J., Rizzo, J. F., Ziv, O. R., Grumet, A., Wyatt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thresholds+for+activation+of+rabbit+retinal+ganglion+cells+with+an+ultrafine,+extracellular+microelectrode.">Thresholds for activation of rabbit retinal ganglion cells with an ultrafine, extracellular microelectrode.</a> Investigative Ophthalmology and Visual Science. 44 (8), 3533-3543 (2003).</li><li>Kim, W., Choi, M., Kim, S. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+normative+retinal+and+choroidal+thicknesses+of+the+rabbit+as+revealed+by+spectral+domain+optical+coherence+tomography.">The normative retinal and choroidal thicknesses of the rabbit as revealed by spectral domain optical coherence tomography.</a> Journal of the Korean Ophthalmological Society. 62 (3), 354-361 (2021).</li><li>Guo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+cell+morphology+in+a+computational+model+of+ON+and+OFF+retinal+ganglion+cells.">Influence of cell morphology in a computational model of ON and OFF retinal ganglion cells.</a> 35th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC). 2013, 4553-4556 (2013).</li><li>Haberbosch, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+aspects,+tolerability+and+modeling+of+retinofugal+alternating+current+stimulation.">Safety aspects, tolerability and modeling of retinofugal alternating current stimulation.</a> Frontiers in Neuroscience. 13, 783 (2019).</li><li>Sheasby, B. W., Fohlmeister, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impulse+encoding+across+the+dendritic+morphologies+of+retinal+ganglion+cells.">Impulse encoding across the dendritic morphologies of retinal ganglion cells.</a> Journal of Neurophysiology. 81 (4), 1685-1698 (1999).</li><li>Rockhill, R. L., Daly, F. J., MacNeil, M. A., Brown, S. P., Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diversity+of+ganglion+cells+in+a+mammalian+retina.">The diversity of ganglion cells in a mammalian retina.</a> Journal of Neuroscience. 22 (9), 3831-3843 (2002).</li><li>Lukasiewicz, P., Werblin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+slowly+inactivating+potassium+current+truncates+spike+activity+in+ganglion+cells+of+the+tiger+salamander+retina.">A slowly inactivating potassium current truncates spike activity in ganglion cells of the tiger salamander retina.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 8 (12), 4470-4481 (1988).</li><li>Van Rossum, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Python Reference Manual. , (1995).</li><li>. Welcome to Spyder's Documentation - Spyder 5 documentation Available from: <a target="_blank" href="https://docs.spyder-idle.org/current/index.html">https://docs.spyder-idle.org/current/index.html</a> (2022)</li><li>Rattay, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ways+to+approximate+current-distance+relations+for+electrically+stimulated+fibers.">Ways to approximate current-distance relations for electrically stimulated fibers.</a> Journal of Theoretical Biology. 125 (3), 339-349 (1987).</li><li>Tsai, D., Morley, J. W., Suaning, G. J., Lovell, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+activation+and+temporal+response+properties+of+rabbit+retinal+ganglion+cells+following+subretinal+stimulation.">Direct activation and temporal response properties of rabbit retinal ganglion cells following subretinal stimulation.</a> Journal of Neurophysiology. 102 (5), 2982-2993 (2009).</li><li>Tsai, D., Morley, J. W., Suaning, G. J., Lovell, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency-dependent+reduction+of+voltage-gated+sodium+current+modulates+retinal+ganglion+cell+response+rate+to+electrical+stimulation.">Frequency-dependent reduction of voltage-gated sodium current modulates retinal ganglion cell response rate to electrical stimulation.</a> Journal of Neural Engineering. 8 (6), 066007 (2011).</li><li>Joucla, S., Gli&#232;re, A., Yvert, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+approaches+to+model+extracellular+electrical+neural+microstimulation.">Current approaches to model extracellular electrical neural microstimulation.</a> Frontiers in Computational Neuroscience. 8, 13 (2014).</li><li>. OpenFOAM Available from: <a target="_blank" href="https://www.openfoam.com/">https://www.openfoam.com/</a> (2022)</li><li>Barba, L., Forsyth, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFD+Python:+The+12+steps+to+Navier-Stokes+equations.">CFD Python: The 12 steps to Navier-Stokes equations.</a> Journal of Open Source Education. 1 (9), 21 (2018).</li></ol>

The authors declare no competing interests.

In this paper, we have demonstrated a modeling workflow that combined finite element and biophysical neuron modeling. The model is highly flexible, as it can be modified in its complexity to fit different purposes, and it provides a way to validate the results against empirical findings. We also demonstrated how we parameterized the model to enable automation.
The two-step modeling method combines the advantages of using FEM and neuron computational suite to solve the neuron's cable equation i...

Visual neuroprostheses are a group of devices that deliver stimulations (electrical, light, etc.) to the neural cells in the visual pathway to create phosphenes or sensation of seeing the light. It is a treatment strategy that has been in clinical use for almost a decade for people with permanent blindness caused by degenerative retinal diseases. Typically, a complete system would include an external camera that captures the visual information around the user, a power supply and computing unit to process and translate the image to a series of electrical pulses, and an implanted electrode array that interfaces the neural tissue and deliver the electrical pulses to the neural cells. The working principle allows a visual neuroprosthesis to be placed in different sites along the visual pathway from the retina to the visual cortex, as long as it is downstream from the damaged tissue. A majority of current research in visual neuroprostheses focuses on increasing the efficacy of the stimulation and improving the spatial acuity to provide a more natural vision.
In the efforts to improve the efficacy of the stimulation, computational modeling has been a cost- and time-effective method to validate a prosthesis design and simulate its visual outcome. Computational modeling in this field gained popularity since 1999 as Greenberg1 modeled the response of a retinal ganglion cell to extracellular electrical stimuli. Since then, computational modeling has been used to optimize the parameters of the electrical pulse2,3 or the geometrical design of the electrode4,5. Despite the variation in complexity and research questions, these models work by determining the electrical voltage distribution in the medium (e.g., neural tissue) and estimating the electrical response that the neurons in the vicinity will produce due to the electrical voltage.
The electrical voltage distribution in a conductor can be found by solving the Poisson equations6 at all locations:
<img alt="Equation 1" src="/files/ftp_upload/63792/63792eq01.jpg" style="margin: auto;" />
<img alt="Equation 2" src="/files/ftp_upload/63792/63792eq02.jpg" style="margin: auto;" />
where E is the electric field, V the electric potential, J the current density, and&#160;&#963; is the electrical conductivity. The <img alt="Equation 12" src="/files/ftp_upload/63792/63792eq12.jpg" style="margin: auto;" /> in the equation indicates a gradient operator. In the case of stationary current, the following boundary conditions are imposed on the model:
<img alt="Equation 3" src="/files/ftp_upload/63792/63792eq3.jpg" style="margin: auto;" />
<img alt="Equation 4" src="/files/ftp_upload/63792/63792eq04.jpg" style="margin: auto;" />
where n is the normal to the surface, &#937; represents the boundary, and I0 represents the specific current. Together, they create electrical insulation at the external boundaries and create a current source for a selected boundary. If we assume a monopolar point source in a homogeneous medium with an isotropic conductivity, the extracellular electric potential at an arbitrary location can be calculated by7:
<img alt="Equation 5" src="/files/ftp_upload/63792/63792eq05.jpg" style="margin: auto;" />
where Ie&#160;is the current and is the distance between the electrode and the point of measurement. When the medium is inhomogeneous or anisotropic, or the electrode array has multiple electrodes, a computational suite to numerically solve the equations can be convenient. A finite-element modeling software6 breaks up the volume conductor into small sections known as &#39;elements&#39;. The elements are interconnected with each other such that the effects of change in one element influence change in others, and it solves the physical equations that serve to describe these elements. With the increasing computational speed of modern computers, this process can be completed within seconds. Once the electric potential is calculated, one can then estimate the electrical response of the neuron.
A neuron sends and receives information in the form of electrical signals. Such signals come in two forms - graded potentials and action potentials. Graded potentials are temporary changes to the membrane potential wherein the voltage across the membrane becomes more positive (depolarization) or negative (hyperpolarization). Graded potentials typically have localized effects. In cells that produce them, action potentials are all-or-nothing responses that can travel long distances along the length of an axon. Both graded and action potentials are sensitive to the electrical as well as the chemical environment. An action potential spike can be produced by various neuronal cell types, including the retinal ganglion cells, when a threshold transmembrane potential is crossed. The action potential spiking and propagation then trigger synaptic transmission of signals to downstream neurons. A neuron can be modeled as a cable that is divided into cylindrical segments, where each segment has capacitance and resistance due to the lipid bilayer membrane8. A neuron computational programme9 can estimate the electrical activity of an electrically-excitable cell by discretizing the cell into multiple compartments and solving the mathematical model10:
<img alt="Equation 6" src="/files/ftp_upload/63792/63792eq06.jpg" style="margin: auto;" />
In this equation, Cm is the membrane capacitance, Ve,n is the extracellular potential at node n, Vi,n the intracellular potential at node n, Rn the intracellular (longitudinal) resistance at node n, and Iion is the ionic current going through the ion channels at node n. The values of V from the FEM model are implemented as Ve,n for all nodes in the neuron when the stimulation is active.
The transmembrane currents from ion channels can be modeled by using Hodgkin-Huxley formulations11:
<img alt="Equation 7" src="/files/ftp_upload/63792/63792eq07.jpg" style="margin: auto;" />
where gi is the specific conductance of the channel, Vm the transmembrane potential (Vi,n - Ve,n) and Eion the reversal potential of the ion channel. For voltage-gated channels, such as Na channel, dimensionless parameters, m, and h, that describe the probability of opening or closing of the channels are introduced:
<img alt="Equation 8" src="/files/ftp_upload/63792/63792eq08.jpg" style="margin: auto;" />
where <img alt="Equation 9" src="/files/ftp_upload/63792/63792eq09.jpg" style="margin: auto;" /> is the maximum membrane conductance for the particular ion channel, and the values of parameters m and h are defined by differential equations:
<img alt="Equation 10" src="/files/ftp_upload/63792/63792eq10.jpg" style="margin: auto;" />
where &#945;x and&#160;&#946;x are voltage-dependent functions that define the rate constants of the ion channel. They generally take the form:
<img alt="Equation 11" src="/files/ftp_upload/63792/63792eq11v2.jpg" style="margin: auto;" />
The values of the parameters in these equations, including maximal conductance, as well as the constants A, B, C, and D, were typically found from empirical measurements.
With these building blocks, models of different complexities can be built by following the steps described. An FEM software is useful when the Poisson equation cannot be solved analytically, such as in the case of inhomogeneous or anisotropic conductance in the volume conductor or when the geometry of the electrode array is complex. After the extracellular potential values have been solved, the neuron cable model can then be numerically solved in the neuron computational software. Combining the two software enables the computation of a complex neuron cell or network to a non-uniform electric field.
A simple two-step model of a retinal ganglion cell under a suprachoroidal stimulation will be built using the aforementioned programs. In this study, the retinal ganglion cell will be subjected to a range of magnitudes of electrical current pulses. The location of the cell relative to the stimulus is also varied to show the distance-threshold relationship. Moreover, the study includes a validation of the computational result against an in vivo study of the cortical activation threshold using different sizes of stimulation electrode12, as well as an in vitro study showing the relationship between the electrode-neuron distance and the activation threshold13.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Computer workstation</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Windows 64-bit operating system, at least 4GB of RAM, at least 3 GB of disk space</td>
		</tr>
		<tr>
			<td>Anaconda Python</td>
			<td>Anaconda Inc.</td>
			<td>Version 3.9</td>
			<td>The open source Individual Edition containing Python 3.9 and preinstalled packages to perform data manipulation, as well as Spyder Integrated Development Environment. It could be used to control the simulation, as well as to display and analyse the simulation data.</td>
		</tr>
		<tr>
			<td>COMSOL Multiphysics</td>
			<td>COMSOL</td>
			<td>Version 5.6</td>
			<td>The simulation suite to perform finite element modelling. The licence for the AC/DC module should be purchased. The Application Builder capability should be included in the licence to follow the automation tutorial.</td>
		</tr>
		<tr>
			<td>NEURON</td>
			<td>NEURON</td>
			<td>Version 8.0</td>
			<td>A freely-distributed software to perform the computation of neuronal cells and/or neural networks.</td>
		</tr>
	</tbody>
</table>

computational modeling of retinal neurons for visual prosthesis research - fundamental approaches

Computational modeling has become an increasingly important method in neural engineering due to its capacity to predict behaviors of in vivo and in vitro systems. This has the key advantage of minimizing the number of animals required in a given study by providing an often very precise prediction of physiological outcomes. In the field of visual prosthesis, computational modeling has an array of practical applications, including informing the design of an implantable electrode array and prediction of visual percepts that may be elicited through the delivery of electrical impulses from the said array. Some models described in the literature combine a three-dimensional (3D) morphology to compute the electric field and a cable model of the neuron or neural network of interest. To increase the accessibility of this two-step method to researchers who may have limited prior experience in computational modeling, we provide a video of the fundamental approaches to be taken in order to construct a computational model and utilize it in predicting the physiological and psychophysical outcomes of stimulation protocols deployed via a visual prosthesis. The guide comprises the steps to build a 3D model in a finite element modeling (FEM) software, the construction of a retinal ganglion cell model in a multi-compartmental neuron computational software, followed by the amalgamation of the two. A finite element modeling software to numerically solve physical equations would be used to solve electric field distribution in the electrical stimulations of tissue. Then, specialized software to simulate the electrical activities of a neural cell or network was used. To follow this tutorial, familiarity with the working principle of a neuroprosthesis, as well as neurophysiological concepts (e.g., action potential mechanism and an understanding of the Hodgkin-Huxley model), would be required.

We conducted two simulation protocols to demonstrate the use of the model. The first protocol involved varying the electrode size while keeping the location of the neuron and the electrical pulse parameters the same. The second protocol involved shifting the neuron in the x-direction in 100 &#181;m steps, while the size of the electrode remained constant. For both protocols, the pulse used was a single cathodic-first biphasic pulse of 0.25 ms width with a 0.05 ms interphase gap. For the first protocol, the radius of the ...

Watch this Scientific Journal Video about Computational Modeling of Retinal Neurons for Visual Prosthesis Research - Fundamental Approaches at JoVE.com

Computational Modeling of Retinal Neurons for Visual Prosthesis Research - Fundamental Approaches

We summarize a workflow to computationally model a retinal neuron's behaviors in response to electrical stimulation. The computational model is versatile and includes automation steps that are useful in simulating a range of physiological scenarios and anticipating the outcomes of future in vivo/in vitro studies.

Computational modeling has become an increasingly important method in neural engineering due to its capacity to predict behaviors of in vivo and in vitro ...

computational-modeling-retinal-neurons-for-visual-prosthesis-research

Research

JoVE Journal

Neuroscience

1.6K Views.  The University of Sydney. We summarize a workflow to computationally model a retinal neuron's behaviors in response to electrical stimulation. The computational model is versatile and includes automation steps that are useful in simulating a range of physiological scenarios and anticipating the outcomes of future in vivo/in vitro studies.

Video: Computational Modeling of Retinal Neurons for Visual Prosthesis Research - Fundamental Approaches

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.
Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.
Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas7, 8,13, and can be used to study coupling between other retinal neurons, as described here.

An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators&#39; ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

Secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. This study describes an innovative operative approach for partial optic nerve transection. The use of this space-saving operative approach extends the model's application range, and allows exploration of secondary injury mechanisms in RGCs in a new way.

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with&#160;histological analysis are demonstrated.

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective models recapitulating the early-onset neuronal impairment seen in the patients. Advances in the technology of induced pluripotent stem cells (iPSCs) enable the generation of three-dimensional (3D) brain organoids that can be used to investigate the impact of diseases on the development and organization of the nervous system. Researchers, including these authors, have recently introduced human brain organoids to model mitochondrial disorders. This paper reports a detailed protocol for the robust generation of human iPSC-derived brain organoids and their use in mitochondrial bioenergetic profiling and imaging analyses. These experiments will allow the use of brain organoids to investigate metabolic and developmental dysfunctions and may provide crucial information to dissect the neuronal pathology of mitochondrial diseases.

We describe a detailed protocol for the generation of human induced pluripotent stem cell-derived brain organoids and their use in modeling mitochondrial diseases.

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental ...