Name: methodology for biomimetic chemical neuromodulation of rat retinas with the neurotransmitter glutamate in vitro
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Description: Watch this Scientific Journal Video about Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro at JoVE.com

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.

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The authors have nothing to disclose.

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&lt;br/&gt; Vendor: Harvard Apparatus UK</td><td>PM-8000 or PM-8</td><td>OEM: http://www.microdatamdi.com/pm8000.htm&lt;br/&gt; Vendor: https://www.harvardapparatus.co.uk/webapp/wcs/stores/servlet/product_11555_10001_39808_-1&lt;br /&gt;_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:&lt;br/&gt; 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 &amp;mu;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 &amp;mu;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&lt;br/&gt; Vendor: https://www.bhphotovideo.com/c/product/43957-REG/Rosco_RS2611_26_Filter_Light.html</td></tr><tr><td>Smith &amp;amp; Wesson Galaxy Red Flashlight</td><td>Smith &amp;amp; Wesson, 2100 Roosevelt Avenue, Springfield, MA</td><td>4588</td><td>Manufacturer: https://www.smith-wesson.com/&lt;br/&gt; 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...

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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 ...

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Research

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Bioengineering

7.7K Views.  University of Illinois at Chicago. 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.

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

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Medicine

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Neuroscience

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal vascular occlusion, retinal detachment, and diabetic retinopathy. It was the object of this study to introduce a standardized stress model for future preclinical therapeutic testing.
Bovine retinas were prepared and perfused with an oxygen saturated standard solution, and the ERG was recorded. After recording stable b-waves, hypoxia (pure N2) or glutamate stress (250 &#181;m glutamate) was exerted for 45 min. To investigate the effects on photoreceptor function alone, 1 mM aspartate was added to obtain a-waves. ERG-recovery was monitored for 75 min.
For hypoxia, a decrease in a-wave amplitude of 87.0% was noted (p &#60;0.01) after an exposition time of 45 min (decrease of 36.5% after the end of the washout p = 0.03). Additionally, an initial decrease in b-wave amplitudes of 87.23% was recorded, that reached statistical significance (p &#60;0.01, decrease of 25.5% at the end of the washout, p = 0.03).
For 250 &#181;m glutamate, an initial 7.8% reduction of a-wave amplitudes (p &#62;0.05) followed by a reduction of 1.9% (p &#62;0.05). A reduction of 83.7% of b-wave amplitudes (p &#60;0.01) was noted; after a washout of 75 min the reduction was 2.3% (p = 0.62). In this study, a standardized stress model is presented that may be useful to identify possible neuroprotective effects in the future.

With this study, we introduce a standardized stress model for the isolated superfused bovine retina for future preclinical therapeutic testing. The effect of either hypoxia (pure N2) or glutamate stress (250 &#181;M glutamate) on retinal function represented by a- and b-wave amplitudes was evaluated.

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Biology

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in retinal diseases, and testing therapeutic interventions. The ex vivo electroretinogram (ERG) allows the quantification of contributions from individual cell types in the isolated retina by addition of specific pharmacological agents and evaluation of tissue-intrinsic changes independently of systemic influences. Retinal light responses can be measured using a specialized ex vivo ERG specimen holder and recording setup, modified from existing patch clamp or microelectrode array equipment. Particularly, the study of ON-bipolar cells, but also of photoreceptors, has been hampered by the slow but progressive decline of light responses in the ex vivo ERG over time. Increased perfusion speed and adjustment of the perfusate temperature improve ex vivo retinal function and maximize response amplitude and stability. The ex vivo ERG uniquely allows the study of individual retinal neuronal cell types. In addition, improvements to maximize response amplitudes and stability allow the investigation of light responses in retina samples from large animals, as well as human donor eyes, making the ex vivo ERG a valuable addition to the repertoire of techniques used to investigate retinal function.

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Modification of existing multielectrode array or patch clamp equipment makes the ex vivo electroretinogram more widely accessible. Improved methods to record and maintain ex vivo light responses facilitate the study of photoreceptor and ON-bipolar cell function in the healthy retina, animal models of eye diseases, and human donor retinas.

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in ...

Evaluating Calcium Activity in Retinal Ganglion Cells Layer

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore vision by inducing visual percepts. One common avenue of investigation involves the design and production of implantable devices with a flexible physical structure, housing a high number of electrodes. This enables the efficient and precise generation of visual percepts. However, with each technological advancement, there arises a need for a reliable and manageable ex vivo method to verify the functionality of the device before progressing to in vivo experiments, where factors beyond the device's performance come into play. This article presents a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from individual cells within the GCL. By following this procedure, researchers can efficiently test new stimulation protocols prior to conducting in vivo experiments.

Author Spotlight: An Innovative Approach to Neural Electrical Stimulation Using Calcium Imaging 

Retinal prostheses have the ability to generate visual perceptions. To advance the development of new prostheses, ex vivo methods are needed to test devices before implantation. This article provides a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer when subjected to electrical stimulation.

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

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

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Explore More Videos

Chapters in this video

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Quantifying Spontaneous Ca²⁺ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases