The work presented in the paper was supported by the National Science Foundation, Emerging Frontiers in Research and Innovation (NSF-EFRI) program grant number 0938072. The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views of the NSF. The authors also wish to thank Dr. Samsoon Inayat for his work designing and testing the initial experimental setup for chemical stimulation and Mr. Ashwin Raghunathan for his work designing, fabricating, and evaluating the multiport microfluidic device used in this study.

All animal experiments were conducted in accordance with the guidelines outlined by the National Research Council&#39;s Guide for the Care and Use of Laboratory Animals. Animal handling and euthanasia protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Illinois at Chicago.
1. Animal Models
<ol>
	<li>Wild-type Long-Evans rats
	<ol>
		<li>Procure a 24 - 32 day old wild-type Long Evans Hooded rat of either sex rai...

<ol>
	<li>Pascolini, D., Mariotti, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+estimates+of+visual+impairment:+2010.">Global estimates of visual impairment: 2010.</a> Br J Ophthalmol. , (2011).</li><li>Fritsche, L. G., Fariss, R. N., Stambolian, D., Abecasis, G. R., Curcio, C. A., Swaroop, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-Related+Macular+Degeneration:+Genetics+and+Biology+Coming+Together.">Age-Related Macular Degeneration: Genetics and Biology Coming Together.</a> Annu Rev Genomics Hum Genet. 15, 151-171 (2014).</li><li>Marc, R. E., 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=Neural+reprogramming+in+retinal+degeneration.">Neural reprogramming in retinal degeneration.</a> Invest Ophthalmol Vis Sci. 48, 3364-3371 (2007).</li><li>Jones, B. W., Kondo, M., Terasaki, H., Lin, Y., McCall, M., Marc, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+remodeling.">Retinal remodeling.</a> Jpn J Ophthalmol. 56, 289-306 (2012).</li><li>Soto, F., Kerschensteiner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+remodeling+of+neuronal+circuits+in+early+retinal+degeneration.">Synaptic remodeling of neuronal circuits in early retinal degeneration.</a> Front Cell Neurosci. 9, (2015).</li><li>Trenholm, S., Awatramani, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+of+spontaneous+activity+in+the+degenerating+retina.">Origins of spontaneous activity in the degenerating retina.</a> Front Cell Neurosci. 9, (2015).</li><li>Euler, T., Schubert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Independent+Oscillatory+Networks+in+the+Degenerating+Retina.">Multiple Independent Oscillatory Networks in the Degenerating Retina.</a> Front Cell Neurosci. 9, (2015).</li><li>Boye, S. E., Boye, S. L., Lewin, A. S., Hauswirth, W. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+Transplantation+in+Late+Stage+Retinal+Degeneration.">Photoreceptor Transplantation in Late Stage Retinal Degeneration.</a> Invest Ophthalmol Vis Sci. 57, (2016).</li><li>Zrenner, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fighting+blindness+with+microelectronics.">Fighting blindness with microelectronics.</a> Sci Transl Med. 5, (2013).</li><li>Humayun, M. S., de Juan, E., Dagnelie, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bionic+Eye:+A+Quarter+Century+of+Retinal+Prosthesis+Research+and+Development.">The Bionic Eye: A Quarter Century of Retinal Prosthesis Research and Development.</a> Ophthalmol. 123, S89-S97 (2016).</li><li>Cruz, 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=The+Argus+II+epiretinal+prosthesis+system+allows+letter+and+word+reading+and+long-term+function+in+patients+with+profound+vision+loss.">The Argus II epiretinal prosthesis system allows letter and word reading and long-term function in patients with profound vision loss.</a> Br J Ophthalmol. 97, 632-636 (2013).</li><li>Zrenner, E., 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=Subretinal+electronic+chips+allow+blind+patients+to+read+letters+and+combine+them+to+words.">Subretinal electronic chips allow blind patients to read letters and combine them to words.</a> P R Soc B. 278, 1489-1497 (2011).</li><li>Stronks, H. C., Dagnelie, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+performance+of+the+Argus+II+retinal+prosthesis.">The functional performance of the Argus II retinal prosthesis.</a> Expert Rev Med Devices. 11, 23-30 (2014).</li><li>Stingl, 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=Artificial+vision+with+wirelessly+powered+subretinal+electronic+implant+alpha-IMS.">Artificial vision with wirelessly powered subretinal electronic implant alpha-IMS.</a> P R Soc B. 280, (2013).</li><li>Rizzo, J. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+implants:+a+systematic+review.">Retinal implants: a systematic review.</a> Br J Ophthalmol. 98, 852-856 (2014).</li><li>Cai, C., Twyford, P., Fried, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+response+of+retinal+neurons+to+high-frequency+stimulation.">The response of retinal neurons to high-frequency stimulation.</a> J Neural Eng. 10, 036009 (2013).</li><li>Eiber, C. D., Lovell, N. H., Suaning, G. 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B., Humayun, M. S., Weiland, J. D., Suzuki, S., D'Anna, S. A., 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=Long-Term+Histological+and+Electrophysiological+Results+of+an+Inactive+Epiretinal+Electrode+Array+Implantation+in+Dogs.">Long-Term Histological and Electrophysiological Results of an Inactive Epiretinal Electrode Array Implantation in Dogs.</a> Invest Ophthalmol Vis Sci. 40, 2073-2081 (1999).</li><li>Peterman, M. C., Noolandi, J., Blumenkranz, M. S., Fishman, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+chemical+release+from+an+artificial+synapse+chip.">Localized chemical release from an artificial synapse chip.</a> PNAS. 101, 9951-9954 (2004).</li><li>Finlayson, P. 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The method presented here demonstrates a unique neural stimulation paradigm, wherein retinal neurons are chemically stimulated by injecting native neurotransmitter chemicals into the subsurface of the retina in vitro. This chemical stimulation technique offers several benefits over the conventional electrical stimulation technique, including selectivity and high focal specificity of target neurons. The protocol above details how small volume pneumatic injections of the neurotransmitter glutamate delivered near t...

Photoreceptor degenerative diseases, such as retinitis pigmentosa and age-related macular degeneration, are leading inheritable causes of vision loss and are currently incurable1,2. Although these diseases arise from a variety of specific genetic mutations, photoreceptor degenerative diseases are characterized as a group by the progressive loss of the photoreceptor cells in the retina, which eventually causes blindness. The loss of photoreceptors triggers widespread remodeling throughout the retina but surviving retinal neurons, including the bipolar cells and RGCs, remain intact and relatively functional even in advanced stages of photoreceptor degeneration3,4,5,6,7.
The mechanisms and pathologies of these diseases have been well characterized3,4,5,6,7 but an effective treatment remains elusive. Over the past three decades, researchers worldwide have investigated a variety of therapeutic treatments for restoring vision to those affected with photoreceptor degenerative diseases including gene therapy8, stem cell treatment9, retinal transplantation10, and artificial stimulation11,12 of the surviving retinal neurons. Of these, the most clinically available are retinal prostheses, which are artificial neurostimulation devices that have traditionally utilized an array of electrodes to electrically stimulate either the bipolar cells or RGCs in specific patterns with the goal of creating artificial visual perceptions in patients11. Current generation electrical prostheses, such as the Argus II13 and Alpha-IMS14 devices, have achieved clinical approval and preliminary studies have indicated that they can improve the quality of life for patients by restoring a measure of vision using both epiretinal (front of the retina) and subretinal (back of the retina) implanted devices15,16. Research groups around the world are working on advancing retinal prostheses beyond the successes of these first-generation devices17,18,19,20 but have faced difficulties designing an electrical prosthesis capable of restoring high acuity vision below the legal blindness level to patients. Recent studies have shown that achieving higher spatial resolution than that enabled by the current generation electrical-based prostheses is challenging because of the charge injection limit, which necessitates the use of large electrodes to safely stimulate retinal neurons at the cost of spatial resolution, i.e. visual acuity11,21. Moreover, electrical stimulation is further limited because it typically stimulates all nearby cells and therefore elicits unnatural and confusing perceptions in patients, largely because it is an inherently unnatural stimulation paradigm21. Nevertheless, the early successes of electrical stimulation have demonstrated that artificial neurostimulation can be an effective treatment for photoreceptor degenerative diseases. This leads one to hypothesize that an even more effective treatment might be achievable by stimulating the retina with neurotransmitter chemicals, the natural agents of communication at chemical synapses. The purpose of the method presented in this paper is to explore the therapeutic feasibility of chemical stimulation, which seeks to mimic the natural system of synaptic communication between retinal neurons, as a biomimetic alternative to electrical stimulation for a retinal prosthesis.
Translation of the concept of therapeutic chemical stimulation to a chemical retinal prosthesis relies on chemically activating target retinal neurons with small quantities of native neurotransmitters, such as glutamate, released through a microfluidic device comprising a large array of microports in response to visual stimulation. In this way, a chemical retinal prosthesis would essentially be a biomimetic artificial photoreceptor layer that translates photons naturally reaching the retina to chemical signals. Since these chemical signals use the same neurotransmitters utilized in normal retinal signaling and stimulate the surviving retinal neurons of a degenerated retina through the same synaptic pathways used by normal vision pathways, the resulting visual perception achieved through a chemical retinal prosthesis could be more natural and comprehensible compared to one evoked through an electrical prosthesis. Moreover, since the microports through which neurotransmitters are released can be made extremely small and arrayed in high density, unlike the electrodes, a potential chemical prosthetic might be able to achieve more focal stimulation and higher spatial resolution than an electrical prosthesis. Thus, based on these potential advantages, a chemical retinal prosthesis offers a highly promising alternative to electrical prostheses.
Chemical stimulation of the retina, however, has been relatively little explored until recently. While electrical stimulation of the retina has been well characterized over decades of work through in vitro22,23, in vivo23,24, and clinical studies13,14, studies on chemical stimulation have been limited exclusively to a few in vitro works25,26,27,28. Iezzi and Finlayson26 and Inayat et al.27 demonstrated epiretinal chemical stimulation of the retina in vitro using a single electrode and a multielectrode array (MEA), respectively, to record the glutamate evoked responses of retinal neurons. More recently, Rountree et al.28 demonstrated the differential stimulation of the OFF and ON retinal pathways using glutamate from the subretinal side and an MEA to record the neuronal responses from multiple sites on the retina. Although these works have preliminarily established the feasibility of chemical stimulation, further studies are essential to investigate many aspects of this approach beyond those addressed so far25,26,27,28, and fine-tune the therapeutic stimulation parameters in both in vitro and in vivo animal models before translating this concept to a chemical retinal prosthesis as discussed above. However, currently there is no established methodology for accomplishing chemical stimulation of the retina in the literature and the methods used in the previous works have not been described in such detail as would be essential for replicative studies. Therefore, the rationale for this methods paper is to provide a well-defined framework for conducting in vitro chemical stimulation of the retina for those investigators interested in either replicating our previous studies27,28 or further advancing this nascent concept of chemical neurostimulation.
Here we demonstrate a method for conducting in vitro chemical stimulation of retinal neurons in wholemount retinas of wild-type rats and a photoreceptor degenerated rat model that closely mimics the progression of photoreceptor degenerative diseases in humans. The rationale behind developing this stimulation method in in vitro models is to evaluate the therapeutic ranges of various stimulation parameters and study neural response characteristics that would be impossible or difficult to observe in in vivo models, especially during the initial studies focused on evaluating the feasibility of this approach. In this procedure, we show both single-site and simultaneous multi-site chemical stimulations of retinas by delivering small quantities of 1 mM glutamate near target retinal neurons via commercially available single-port glass micropipettes and a custom micromachined multi-port microfluidic device, respectively. While both single-site and multi-site stimulations accomplish the basic objective of investigating the therapeutic feasibility of chemical neuromodulation, each serves a distinct purpose with a unique advantage. The single-site stimulation, which may be accomplished with commercially available pre-pulled glass micropipettes, can be used to inject chemicals directly into the subsurface of the retina at a single site and serves to investigate if observable RGC spike rate responses that are similar to visually evoked light responses can be elicited focally under the injection site. On the other hand, multi-site stimulation, which requires a specially fabricated multiport microfluidic device, can be used to inject chemicals spatially at multiple sites over the surface of the retina and serves to investigate how well glutamate-evoked RCG response patterns correspond to the glutamate injection patterns in pattern stimulation studies.

<table>
	<tbody>
		<tr>
			<td>Microelectrode array, perforated layout</td>
			<td>Multi Channel Systems, GmbH</td>
			<td>60pMEA200/30iR-Ti-pr</td>
			<td>http://www.multichannelsystems.com/products/microelectrode-arrays/60pmea20030ir-ti</td>
		</tr>
		<tr>
			<td>MEA amplifier</td>
			<td>Multi Channel Systems, GmbH</td>
			<td>MEA1060-Inv</td>
			<td>http://www.multichannelsystems.com/products/mea1060-inv</td>
		</tr>
		<tr>
			<td>Bottom perfusion groundplate for pMEA</td>
			<td>Multi Channel Systems, GmbH</td>
			<td>MEA1060-Inv-(BC)-PGP</td>
			<td>http://www.multichannelsystems.com/products/mea1060-inv-bc-pgp</td>
		</tr>
		<tr>
			<td>3-axis Motorized Micromanipulator</td>
			<td>Sutter Instruments, Novato, CA</td>
			<td>MP-285</td>
			<td>https://www.sutter.com/MICROMANIPULATION/mp285.html</td>
		</tr>
		<tr>
			<td>Micromanipulator Control System</td>
			<td>Sutter Instruments, Novato, CA</td>
			<td>MPC-200</td>
			<td>https://www.sutter.com/MICROMANIPULATION/mpc200.html</td>
		</tr>
		<tr>
			<td>Gantry style micromanipulator stand with linear slide</td>
			<td>Sutter Instruments, Novato, CA</td>
			<td>MT-75/LS</td>
			<td>https://www.sutter.com/STAGES/mt75.html</td>
		</tr>
		<tr>
			<td>8-channel Programmable Multichannel Pressure Injector</td>
			<td>OEM: MicroData Instrument, S. Plainfield, NJ 
			Vendor: Harvard Apparatus UK</td>
			<td>PM-8000 or PM-8</td>
			<td>OEM: http://www.microdatamdi.com/pm8000.htm 
			Vendor: https://www.harvardapparatus.co.uk/webapp/wcs/stores/servlet/product_11555_10001_39808_-1_HAUK_ProductDetail</td>
		</tr>
		<tr>
			<td>Axopatch 200A Integrating Patch Clamp Amplifier</td>
			<td>Molecular Devices, Sunnyvale, CA</td>
			<td>Axopatch 200A</td>
			<td>Axopatch 200A has been replaced with a newer model Axopatch 200B: 
			https://www.moleculardevices.com/systems/axon-conventional-patch-clamp/axopatch-200b-amplifier</td>
		</tr>
		<tr>
			<td>Patch clamp headstage</td>
			<td>Molecular Devices, Sunnyvale, CA</td>
			<td>CV 201A</td>
			<td>http://mdc.custhelp.com/app/answers/detail/a_id/16554/~/axopatch-200a%3A-selection-cv-headstage</td>
		</tr>
		<tr>
			<td>Vacuum waste kit</td>
			<td>ALA Scientific Instruments, Farmingdale, NY</td>
			<td>VMK</td>
			<td>http://alascience.com/product/vacuum-waste-kit/</td>
		</tr>
		<tr>
			<td>Pipette holder</td>
			<td>Warner Instruments, Hamden, CT</td>
			<td>QSW-A10P</td>
			<td>https://www.warneronline.com/product_info.cfm?id=915</td>
		</tr>
		<tr>
			<td>Pre-pulled 10 &#956;m tip diameter glass micropipettes</td>
			<td>World Precision Instruments, Sarasota, FL</td>
			<td>TIP10TW1</td>
			<td>https://www.wpiinc.com/products/laboratory-supplies/make-selection-pre-pulled-glass-pipettes-plain/</td>
		</tr>
		<tr>
			<td>Zoom stereomicroscope</td>
			<td>Nikon, Tokyo, Japan</td>
			<td>SMZ-745T</td>
			<td>https://www.nikoninstruments.com/Products/Stereomicroscopes-and-Macroscopes/Stereomicroscopes/SMZ745</td>
		</tr>
		<tr>
			<td>Microscope boom stand with dual linear ball bearing arm</td>
			<td>Old School Industries, Inc., Dacono, CO</td>
			<td>OS1010H-16BB</td>
			<td>http://www.osi-incorp.com/productdisplay/dual-linear-ball-bearing-arm</td>
		</tr>
		<tr>
			<td>Zoom Stereo Microscope with C-LEDS Hybrid LED Stand</td>
			<td>Nikon, Tokyo, Japan</td>
			<td>SMZ-445</td>
			<td>https://www.nikoninstruments.com/Products/Stereomicroscopes-and-Macroscopes/Stereomicroscopes/SMZ445</td>
		</tr>
		<tr>
			<td>Inverted microscope system</td>
			<td>Nikon, Tokyo, Japan</td>
			<td>Eclipse Ti-E</td>
			<td>https://www.nikoninstruments.com/Products/Inverted-Microscopes/Eclipse-Ti-E</td>
		</tr>
		<tr>
			<td>Ames medium</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>A1420</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/a1420</td>
		</tr>
		<tr>
			<td>L-Glutamic Acid (Glutamate)</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>G5667</td>
			<td>http://www.sigmaaldrich.com/catalog/product/mm/100291</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>S8761</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/s8761</td>
		</tr>
		<tr>
			<td>60 mm Petri dish (10 mm tall)</td>
			<td>Fischer Scientific, Waltham, MA</td>
			<td>FB0875713A</td>
			<td>60 mm clear petri dish; https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/fb0875713a</td>
		</tr>
		<tr>
			<td>Jewelers #5 Forceps</td>
			<td>World Precision Instruments, Sarasota, FL</td>
			<td>555227F</td>
			<td>https://www.wpiinc.com/products/laboratory-supplies/555227f-jewelers-5-forceps-11cm-straight-titanium/</td>
		</tr>
		<tr>
			<td>Standard Scalpel Blad #24</td>
			<td>World Precision Instruments, Sarasota, FL</td>
			<td>500247</td>
			<td>https://www.wpiinc.com/products/laboratory-supplies/500247-standard-scalpel-blade-24/</td>
		</tr>
		<tr>
			<td>Scalpel Handle #4</td>
			<td>World Precision Instruments, Sarasota, FL</td>
			<td>500237</td>
			<td>https://www.wpiinc.com/products/laboratory-supplies/500237-scalpel-handle-4-14cm/</td>
		</tr>
		<tr>
			<td>Vannas Tubingen Dissection Scissors</td>
			<td>World Precision Instruments, Sarasota, FL</td>
			<td>503378</td>
			<td>https://www.wpiinc.com/products/laboratory-supplies/503378-vannas-tubingen-scissors-8cm-straight-german-steel/</td>
		</tr>
		<tr>
			<td>Nylon mesh kit</td>
			<td>Warner Instruments, Hamden, CT</td>
			<td>NYL/MESH</td>
			<td>https://www.warneronline.com/product_info.cfm?id=1173</td>
		</tr>
		<tr>
			<td>Harp slice grid</td>
			<td>ALA Scientific Instruments, Farmingdale, NY</td>
			<td>HSG-5AD</td>
			<td>http://alascience.com/product/standard-harp-slice-grids/</td>
		</tr>
		<tr>
			<td>Ag/AgCl reference electrode pellet</td>
			<td>Multi Channel Systems, GmbH</td>
			<td>P1060</td>
			<td>http://www.multichannelsystems.com/products/p1060</td>
		</tr>
		<tr>
			<td>4 Channel Valve Controlled Gravity Perfusion System</td>
			<td>ALA Scientific Instruments, Farmingdale, NY</td>
			<td>VC3-4xG</td>
			<td>http://alascience.com/product/4-channel-valve-controlled-gravity-perfusion-system/</td>
		</tr>
		<tr>
			<td>Zyla 5.5 sCMOS microscope camera</td>
			<td>Andor Technology, Belfast, UK</td>
			<td>Zyla 5.5 sCMOS</td>
			<td>http://www.andor.com/scientific-cameras/neo-and-zyla-scmos-cameras/zyla-55-scmos</td>
		</tr>
		<tr>
			<td>Silver wire (50 &#956;m diameter)</td>
			<td>Fischer Scientific, Waltham, MA</td>
			<td>AA44461G5</td>
			<td>https://www.fishersci.com/shop/products/silver-wire-0-05mm-0-002-in-dia-annealed-99-99-metals-basis-3/aa44461g5</td>
		</tr>
		<tr>
			<td>Tygon microbore tubing (1.6 mm diameter)</td>
			<td>Cole Parmer, Vernon Hills , IL</td>
			<td>EW-06419-01</td>
			<td>https://www.coleparmer.com/i/tygon-microbore-tubing-0-020-x-0-060-od-100-ft-roll/0641901</td>
		</tr>
		<tr>
			<td>Tilting Tool Holder with Steel Cannula</td>
			<td>ALA Scientific Instruments, Farmingdale, NY</td>
			<td>TILTPORT</td>
			<td>One each of these were utilized for top perfusion and suction; http://alascience.com/product/tilting-tool-holder-with-steel-cannula/</td>
		</tr>
		<tr>
			<td>Roscolux #26 Light Red Filter Sheet</td>
			<td>Rosco Laboratories Inc., 52 Harbor View, Stamford, CT</td>
			<td>R2611</td>
			<td>Manufacturer: http://us.rosco.com/en/products/catalog/roscolux 
			Vendor: https://www.bhphotovideo.com/c/product/43957-REG/Rosco_RS2611_26_Filter_Light.html</td>
		</tr>
		<tr>
			<td>Smith &#38; Wesson Galaxy Red Flashlight</td>
			<td>Smith &#38; Wesson, 2100 Roosevelt Avenue, Springfield, MA</td>
			<td>4588</td>
			<td>Manufacturer: https://www.smith-wesson.com/ 
			Vendor: http://www.mypilotstore.com/mypilotstore/sep/4588</td>
		</tr>
		<tr>
			<td>MC_Rack Software</td>
			<td>Multi Channel Systems, GmbH</td>
			<td>MC_Rack</td>
			<td>http://www.multichannelsystems.com/software/mc-rack</td>
		</tr>
		<tr>
			<td>Labview Software</td>
			<td>National Instruments, Austin, TX</td>
			<td>LabVIEW</td>
			<td>http://www.ni.com/labview/</td>
		</tr>
		<tr>
			<td>NIS-Elements: Basic Research Software</td>
			<td>Nikon, Tokyo, Japan</td>
			<td>NIS-Elements BR</td>
			<td>https://www.nikoninstruments.com/Products/Software/NIS-Elements-Basic-Research</td>
		</tr>
	</tbody>
</table>

methodology for biomimetic chemical neuromodulation of rat retinas with the neurotransmitter glutamate in vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

This protocol can be used to chemically stimulate both normal, wild-type retinas as well as photoreceptor degenerated retinas, despite the substantial cellular remodeling caused by the loss of the photoreceptors. Before beginning experiments with either photoreceptor degenerated or wild-type retinas, the recording and stimulation equipment (Figure 1 and Figure 2) need to be readied and the pMEA (Figure 5</str...

Watch this Scientific Journal Video about Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro at JoVE.com

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

The overall goal of this methodology is to enable a novel chemical-based neuromodulation of retinal neurons using neurotransmitters, which could potentially replace the functionality of defective retinal cells in patients with neurodegenerative blindness to restore vision. This method can help answer key questions in the field of artificial vision. Such as can glutamate, a primary retinal neurotransmitter, be used to therapeutically stimulate a photoreceptor degenerated retina?The main advantage of our technique is it is biomimetic, stimulating the retina this way offers the potential for higher visual acuity and more naturalistic vision than is possible with electrical stimulation. The implications of this technique extend toward the treatment of blindness, due to photoreceptor degenerative diseases because early therapeutic intervention of a degenerated retina could restore glutamate sensitivity in retinal neurons. To begin this procedure, place both enucleated eyes in a 60 milimeter diameter Petri dish with fresh oxygenated Ames'medium solution.Using a dissection stereo microscope, make a small incision in the corneal face using a scalpel or a pair of sharp scissors. Next, make a cut from this small incision to the edge of the cornea. And extend it in a circumferential section around the entire edge of the cornea.Then, remove the detached cornea along with the lens, translucent aqueous, and vitreous humours. While gently holding the eye cup with one pair of forceps, carefully make two small incisions on opposite sides of the eye cup. Next, use two pairs of forceps to gently pull apart the eye cup at each of the incisions.And separate the retina from the sclera. Slowly lift the entire retina from the sclera and the eye cup and cut the optic nerve if it is still attached. After that, make longitudinal cuts in the retina to obtain half or quarter sections.And then gently spread one retinal section onto a nylon mesh with the ganglion cells facing away from the mesh. Place the mesh and retina onto a perforated MultiElectrode Array with the ganglion cells in contact with a PMEA surface. And then place a weight on top of the mesh to keep the retina in place.In this procedure, under dim red illumination, place PMEA in the MEA amplifier and close the amplifier latches. Position the top perfusion outlet inside the PMEA chamber and turn on the top perfusion valve to achieve a perfusion rate of approximately three milliliters per minute. Next, position the top suction inlet at the desired perfuse eight level.Ensure that it is working. Afterward, open the data acquisition software and click the play button to start receiving data. Ensure that all the PMEA channels are noise free and recording neuro-signals.If not, reposition the PMEA within the amplifier to obtain better contact between the amplifier pins and the PMEA contacts. Then, ensure that the bottom perfusion line is clear of any air bubbles. Turn on the bottom perfusion valve to ensure the retina is supplied with oxygen and nutrients.Subsequently, turn on the high speed camera attached to the inverted optical microscope and open the imaging software. Once the bottom perfusion is confirmed to be flowing, slowly ramp up the bottom suction by manually turning the vacuum pressure knob on the vacuum waste kit while observing the retina through the inverted microscope. Once a suction force is observed to act on the retina, cease increasing the suction.After ensuring the retina has been held in place by the bottom suction, take the weight off the retina using forceps and gently remove the nylon mesh by peeling one corner carefully from the retina. Keep the perfusion running for approximately 30 minutes to allow the retina to stabilize from the surgical trauma. In this step, carefully insert a pre-pulled 10 micrometer diameter micropipette into a standard pipette holder containing a 50 micrometer diameter silver-silver chloride wire electrode.If using a multiport microfluidic device instead, insert the stainless steel rod connected to the device into a standard pipette holder without a wire electrode. If utilizing a glass micropipette, interface the pipette holder with a patch clamp headstage and connect the pressure port lower connection of the pipette holder to channel one of the pressure injection system. Or, if using the multiport device, connect the pressure port luer connections of each of the eight injection ports, with channel one through eight of the pressure injection system.Next, manually turn on the pressure injection system and turn on channel one. Ensure that the system is vented to atmosphere and set the injection pressure to 0.1 pounds per square inch. Afterward, turn on the micromanipulator and calibrate it by pressing the calibrate button on the manipulator controller.Position the micromanipulator so that the micropipette tip is approximately 30 millimeters above the MEA amplifier. Then, fill a small Petri dish with glutamate solution and place it underneath the micromanipulator. Lower the micropipette tip into the solution and fill the glass micropipette or the multiport device tubing with approximately 20 millimeters of solution.Subsequently, lift the micropipette tip, or device, out of solution. Remove the Petri dish and position the micromanipulator above the PMEA chamber. Using a boom stand mounted stereo microscope, align the micropipette tip, or the corners of the device, with a reference mark etched into the PMEA chamber ring.Then, store the manipulator positions into the control surface using the store reference A and store reference B buttons to map the coordinate system of the manipulator of the PMEA electrodes. Following that, use the manipulator control software. Select a target PMEA electrode with robust spontaneous activity and click the move to channel button to align the glass micropipette with the target electrode.If using the multiport device, align the device microports with target PMEA electrodes with robust spontaneous activity using the same process. If impedance measurement is available, turn on patch clamp amplifier and initiate the impedance visualization software by clicking the start button to visualize the impedance of the silver-silver chloride electrode inside the pipette holder. While observing the realtime impedance signals, slowly lower the micropipette, or device, until it contacts the retinal surface as indicated by a rapid increase in the impedance signal.For subsurface stimulation, lower the pipette 40 micrometers further for S334 tera three retinas, or 70 micrometers for wild-type retinas. Perform a few short duration injections using the pressure injection system. To determine if the cells near the micropipette tip, or device microports, are receptive to glutamate stimulation by observing the neurosignals.Now, orient the green LED towards the top surface of the retina. Begin recording using the data acquisition software by typing the file name and clicking the record button. Once recording has started, open the stimulus control program and load the default stimulus file by clicking the read stimulus file button.Next, click on the run stimulus file button to initiate the default stimulus file. After the stimulus file has been completed, stop the recording to save the file for future spike sorting and data analysis. Here are the representative recordings from nine PMEA electrodes, showing the high-pass filtered electrode data during visual light stimulation with a green LED, where each rectangle shows the neural data from a unique electrode.Each electrode recording illustrates data collected in the first second after turning on the green LED. Spikes were identified using a threshold voltage of negative 18 microvolts. Visual stimulation caused a burst of spikes in all electrodes except the top center one, which possessed an inhibitory response to light.A similar plot for the same electrodes, showing spontaneous neural activity without visual or injection stimulation. Although smaller bursts were present, the patterns of spikes were very different from those recorded in response to visual stimulation. Here are the representative recordings from the same subset of electrodes recorded immediately after a glutamate injection at the central electrode.The injected glutamate elicited a burst of spikes in the central electrode that was very similar to the visually evoked spike bursts. All other electrodes were unaffected by the glutamate injection which demonstrates the fine spatial resolution of the chemical stimulation technique. Once mastered, this technique can be done in four to six hours if it is performed properly.While attempting this procedure, it's important to remember to monitor the perfusion to ensure the retina is continuously supplied with oxygen and nutrients throughout the entire experiment. This technique could pave the way for researchers in the field of artificial vision and visual prosthesis development to explore chemical-based neuromodulation for treating blindness from photoreceptor degenerative diseases. After watching this video, you should have a good understanding of how to accomplish neuromodulation of retinal neurons using neurotransmitters.

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Research

JoVE Journal

Bioengineering

7.7K visualizações.  University of Illinois at Chicago. The overall goal of this methodology is to enable a novel chemical-based neuromodulation of retinal neurons using neurotransmitters, which could potentially replace the functionality of defective retinal cells in patients with neurodegenerative blindness to restore vision. This method can help answer key questions in the field of artificial vision. Such as can glutamate, a primary retinal neurotransmitter, be used to therapeutically stimulate a photoreceptor degenerated retina?The main...