This work was supported by the Deutsche Forschungsgemeinschaft DFG FOR701 and FE464/11-1 to A.F. The authors want to acknowledge the excellent technical assistance of Andrea Nerz.

All procedures were carried out in accordance with the guidelines for animal experiments issued by the Federal Republic of Germany.
NOTE: Because the retinal tissue will be devoid of oxygen during prolonged parts of this procedure, all steps should be carried out as fast as possible. Mice should be dark-adapted at least 3 hr before dissection. To avoid light adaptation of the retina, preparation and slicing should be carried out under dim red light.
1. Preparation of Media and Other Chemicals
<ol>
	<li>Prepare Ames&#39; Medium6. After dissection, immerse retinal tissue in Ames&#39; Medium at all times.
	<ol>
		<li>Dissolve 2.2 g of Ames&#39; Medium and 298 mg HEPES in 250 ml distilled water. The concentration of HEPES will be 5 mM. Stir gently until all particles are dissolved.</li>
		<li>Bubble Ames&#39; Medium at room temperature with carbogen (95% oxygen, 5% carbon dioxide) for at least 5 min. Supply carbogen with a glass Pasteur pipette to the solution. Connect the Pasteur pipette via appropriate tubing to the outlet of the gas regulator.</li>
		<li>Add 475 mg sodium-bicarbonate to the solution. The addition of bicarbonate after bubbling prevents precipitation of calcium carbonate.</li>
	</ol>
	</li>
	<li>Prepare low gelling temperature agarose. Agarose is required for embedding of retinal tissue during vibratome sectioning.
	<ol>
		<li>Dissolve 180 mg agarose in 9 ml Ames&#39; Medium and 1 ml distilled water. Use a microwave to slowly heat the agarose solution until all solids are dissolved. Heat in 5 - 10 sec increments to avoid overflow. Solution should be clear and transparent.</li>
		<li>Store dissolved agarose in water bath at 37 &#176;C for duration of the procedure.</li>
	</ol>
	</li>
</ol>
2. Setting up the Vibratome
NOTE: All sectioning has been carried out with the vibratome listed in the Materials Table. The given settings refer to this type of vibratome only. For further details on vibratome sectioning of neural tissue please refer to reference7.
<ol>
	<li>Place vibratome injector blade into blade holder.</li>
	<li>Measure vertical deflection of the blade with the &#34;vibrocheck&#34; device. Use the adjustment screw on the blade holder to tilt the blade until it is parallel to the cutting plane. The exact positioning of the blade is necessary to prevent venetian blind effects on the section surface and to produce a flat section that contains living cells on its surface.</li>
	<li>Set vibratome to the following settings: amplitude, 1.00; speed, 0.20; thickness, 180 - 200 &#181;m.</li>
</ol>
3. Dissection of Mouse Retina
<ol>
	<li>Anaesthetize adult C57BL/6 mouse by vaporization of about 1 ml isoflurane in an air-tight container. When anesthesia is complete, euthanize animal by cervical dislocation. The mouse should not be older than 3 months, otherwise the quality of the slices will deteriorate.</li>
	<li>Remove eye ball by placing a curved forceps under the eye. Gently lifting up the eye ball will expose the optic nerve. Cut the optic nerve with a spring scissor and transfer the eye ball to a 35 mm petri dish containing 2 ml Ames&#39; Medium.</li>
	<li>Carry out all subsequent steps on the stage of a stereo microscope. Adjust the magnification to a value that allows optimal visual control of the following procedures (steps 3.4 - 3.9).</li>
	<li>Puncture the cornea at the transition to the sclera with pointed forceps or a 20 G&#160;needle. During this step, steady the eye ball with another forceps. Be careful not to damage the retina with the puncturing device.</li>
	<li>Insert the spring scissors into the small hole made in step 3.3 and cut a circle around the cornea. Again, steady the eye ball with forceps while cutting. Keep the incision very close to the surface in order not to cut into the retina.</li>
	<li>Carefully remove cornea, lens, and vitreous body with forceps. The vitreous body comprises only a thin transparent layer in the back of the eye. However, it is essential for vibratome sectioning that the vitreous body is fully removed.</li>
	<li>In the petri dish, position the eyecup in a way that one looks through the hole down onto the retina. The rim of the opening should consist of scleral tissue only. If the circular cut (step 3.4) has been carried out too close to the equator of the eyecup, retinal tissue will also be present close to the incision. Be careful then not to damage the retina in step 3.8.</li>
	<li>Grab the sclera with forceps and carefully tear the tissue apart. It is essential not to rupture the retina during this step. If necessary, detach the retina from the ora serrata by steadying the scleral part of the tissue with the forceps and carefully peeling off the retina with the other forceps. Keep the forceps closed; never handle the retina with forceps since the tissue is very soft and will give way immediately. 
	NOTE: At this step, the retina and the rest of the eyecup will be connected only at the optic nerve head.</li>
	<li>Cut the attachment site at the optic nerve head with the spring scissors. The retina should now be freely floating as a single piece of tissue in Ames&#39; Medium.</li>
	<li>Cut the retina into 4 - 6 small rectangles of approximately 23 mm using a curved scalpel blade by holding the retina along the edges with the forceps, to manipulate it into position. Avoid pinching the retinal tissue within the rectangles. Take pieces from the central parts of the retina, not the far periphery.</li>
	<li>Transfer retinal pieces with a large-diameter plastic Pasteur pipette to a 35 mm plastic petri dish, the bottom of which has been replaced by a mesh with grid size less than 1 mm. Fix the Petri dish within the upper third of a beaker or conical flask and submerge retinal pieces in Ames&#39; Medium bubbled with carbogen.</li>
</ol>
4. Embedding of Retinal Pieces in Agarose
<ol>
	<li>Carefully transfer 2 - 3 retinal pieces into a 35 mm plastic Petri dish together with some of the Ames&#39; Medium. It is most convenient to use a large-diameter plastic Pasteur pipette for transfer. In this step, the exact orientation of the retinal pieces does not matter.</li>
	<li>Remove as much as possible of the remaining Ames&#39; Medium with a glass Pasteur pipette. As soon as the fluid has been removed proceed with step 4.3. Do not let the retinal tissue dry.</li>
	<li>Immediately cover retinal pieces with 2 ml of dissolved agarose (37 &#176;C). Given the size mentioned in step 3.10, the pieces will not curl up within the agarose.</li>
	<li>Within the liquid agarose, gently push retinal pieces to the bottom of the petri dish. The ganglion cell layer should be facing down. Use a stereo microscope for judgement of exact position. For positioning, handle retinal pieces with a small spatula. Position the retina as fast and precise as possible since the agarose will solidify quickly.
	<ol>
		<li>Arrange retinal pieces parallel to the bottom of the glass dish; otherwise sections will be tangential or oblique. Individual pieces should not touch each other, but also should not be too far apart. Try to place retinal pieces more or less on the same horizontal level.</li>
	</ol>
	</li>
	<li>Put glass petri dish with embedded retinal pieces into the refrigerator (4 - 6 &#176;C) for at least 2 min. This will quickly harden the agarose for further processing. As soon as the agarose has polymerized proceed with the next step.</li>
</ol>
5. Mounting of Retinal Pieces
<ol>
	<li>Carefully remove the agarose block containing retinal pieces from the petri dish. Use a spatula or a similar device to lift the agarose block from the glass. Due to adhesive forces, this can turn out to be quite difficult. However, avoid breaking the agarose block. Carry out this and all subsequent steps at room temperature.</li>
	<li>Place the agarose block on a glass coverslip and adjust its size with a scalpel blade. Leave 1 - 2 mm of agarose on each side of the retinal pieces. The final size of the block should be around 1,066 mm (10 mm refers to the height of the block, 6 mm to its edge length).</li>
	<li>Glue the agarose block to the specimen holder of the vibratome using an instant adhesive. Retinal pieces should be located at the top of the 10 mm high block and oriented parallel to its surface, while the bottom is affixed to the holder.</li>
	<li>Cover the block immediately by pouring the previously bubbled Ames&#39; Medium into the buffer tray of the vibratome.</li>
</ol>
6. Preparation of Horizontal Vibratome Sections
<ol>
	<li>Start sectioning. At first, set the thickness of slicing manually until fragments of retinal tissue appear in the slice. Then change to the settings in step 2.3. Since the mouse retina is only about 200 &#181;m thick, there will be no more than 2 - 3 slices per agarose block.</li>
	<li>Remove sections from the buffer tray with a glass rod and transfer them to 2 ml Ames&#39; Medium in a 35 mm Petri dish.</li>
	<li>Store sections immediately in an incubator at 37 &#176;C (5% CO2/55% O2). The agarose surrounding the sections will stay solid in the incubator and can be used to handle the sections without damaging the retina. Better slice quality is obtained when storing at 37 &#176;C within the respective atmosphere compared to oxygenated Ames&#39; Medium at RT. Sections can be used immediately or can be stored up to 4 hr in the incubator with good physiological results.</li>
	<li>Embed next retinal pieces in agarose and repeat Sections 4 - 6.</li>
</ol>

<ol>
	<li>DeVries, S. H., Li, W., Saszik, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+processing+in+two+transmitter+microenvironments+at+the+cone+photoreceptor+synapse.">Parallel processing in two transmitter microenvironments at the cone photoreceptor synapse.</a> Neuron. 50, 735-748 (2006).</li><li>Jackman, S. 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=Role+of+the+synaptic+ribbon+in+transmitting+the+cone+light+response.">Role of the synaptic ribbon in transmitting the cone light response.</a> Nature Neuroscience. 12 (3), 303-310 (2009).</li><li>Rabl, K., Cadetti, L., Thoreson, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+exocytosis+is+faster+in+cones+than+in+rods.">Kinetics of exocytosis is faster in cones than in rods.</a> J. Neurosci. 25 (18), 4633-4640 (2005).</li><li>Singer, J. H., Lassov&#225;, L., Vardi, N., Diamond, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+multivesicular+release+at+a+mammalian+ribbon+synapse.">Coordinated multivesicular release at a mammalian ribbon synapse.</a> Nature Neuroscience. 7 (8), 826-833 (2004).</li><li>Peichl, L., Gonz&#225;lez-Soriano, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+types+of+horizontal+cell+in+rodent+retinae:+a+comparison+of+rat,+mouse,+gerbil,+and+guinea+pig.">Morphological types of horizontal cell in rodent retinae: a comparison of rat, mouse, gerbil, and guinea pig.</a> Vis. Neurosci. 11, 501-517 (1994).</li><li>Ames, A., Nesbett, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+retina+as+an+experimental+model+of+the+central+nervous+system.">In vitro retina as an experimental model of the central nervous system.</a> J. Neurochem. 37 (4), 867-877 (1981).</li><li>Mathis, D. M., Furman, J. J., Norris, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+acute+hippocampal+slices+from+rats+and+transgenic+mice+for+the+study+of+synaptic+alterations+during+aging+and+amyloid+pathology.">Preparation of acute hippocampal slices from rats and transgenic mice for the study of synaptic alterations during aging and amyloid pathology.</a> J. Vis. Exp. (49), (2011).</li><li>Peichl, L., Sandmann, D., Boycott, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+anatomy+and+function+of+mammalian+horizontal+cells.">Comparative anatomy and function of mammalian horizontal cells.</a> Development of the Retina. , (1998).</li><li>Feigenspan, A., Babai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+of+spontaneous+excitatory+currents+and+encoding+of+light/dark+transitions+in+horizontal+cells+of+the+mouse+retina.">Functional properties of spontaneous excitatory currents and encoding of light/dark transitions in horizontal cells of the mouse retina.</a> Eur. J. Neurosci. 42 (9), 2615-2632 (2015).</li><li>Enoki, R., Jakobs, T. C., Koizumi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horizontal+slice+preparation+of+the+retina.">Horizontal slice preparation of the retina.</a> J. Vis. Exp. (1), e108 (2006).</li><li>Babai, N., 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=Functional+roles+of+complexin3+and+complexin4+at+mouse+photoreceptor+ribbon+synapses.">Functional roles of complexin3 and complexin4 at mouse photoreceptor ribbon synapses.</a> J. Neurosci. 36, 6651-6667 (2016).</li><li>Thoreson, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horizontal+slices+of+mouse+retina+expose+horizontal+cells+and+their+properties+(Commentary+on+Feigenspan+&amp;+Babai).">Horizontal slices of mouse retina expose horizontal cells and their properties (Commentary on Feigenspan &amp; Babai).</a> Eur. J. Neurosci. 42 (9), 2613-2614 (2015).</li></ol>

The authors have nothing to disclose.

In contrast to vertical slices, the dendritic morphology of radially oriented neurons like horizontal cells and amacrine cells is well preserved. Compared to whole retina preparations, these cell types are easily accessible for microelectrodes and can be studied by both electrophysiological and imaging techniques. Here, we focused on the outer retina; however, a similar approach has been reported especially for amacrine cells of the inner retina10.
The properties of the horizontal slice preparation have several implications for functional studies. The number of synaptic contacts between cone photoreceptors and postsynaptic horizontal cells was nearly indistinguishable from the intact retina. Synaptic contacts were functional, showing tonic release of neurotransmitter in the dark. The synapse could be activated by either endogenous stimuli like light/dark transitions or by external bipolar stimulation.
One critical step of the technique is the isolation of the retina itself, which should be carried out as fast as possible without damaging the tissue. In addition, the presence of remnants of the vitreous body negatively affects the quality of the slices. Another critical step is the correct positioning of the retinal pieces. Although utmost care is taken to locate the retinal pieces parallel to the bottom of the glass dish, the plane of sectioning might still run slightly tangentially through the retina. Depending on the angle, the dendritic field of radially oriented neurons will be more or less truncated.
The technique presented here is mostly limited to horizontally oriented neurons. Therefore, it is not possible to record from photoreceptors and intact bipolar cells in a horizontal slice, since the bipolar cell axons will be truncated. In addition, it is impossible to perfectly control the level of sectioning. A single retinal piece might thus be cut at an undesired level, for instance between outer and inner segments of photoreceptors. Increasing the number of retinal pieces within one block of agarose increases chances of a correct plane of sectioning at least in one piece.
The horizontal slice preparation is well suited to studying the unique properties of ribbon synapses of rod and cone photoreceptors. So far, we have used an animal knock-out system to investigate the role of presynaptic proteins complexin 3 and complexin 4 on synaptic transmission between cone photoreceptors and horizontal cells11. It is also possible to study the properties of postsynaptic neurotransmitter receptors, as shown previously9.
Future applications will include the study of synaptic proteins based on transgenic mouse lines with electrophysiological and imaging techniques. It will also be possible to investigate the biophysical properties of gap junctions between horizontal cells. Since light responses can be measured, the technique will also be used to investigate the encoding of endogenous stimuli at the first synapse of the visual system. It is possible to carry out the preparation in darkness to prevent light adaptation of the tissue, otherwise the light responses will be largely cone-driven.
In summary, the horizontal slice preparation represents a novel approach to increase the accessibility of individual synapses as a prerequisite to studying their functional properties in the mammalian retina12.

Visual information is coded as a dynamic spatio-temporal pattern of photoreceptor activation, and the ribbon synapse between photoreceptors and second-order neurons determines the downstream transfer of visual information. The vertical slice preparation of the mammalian retina is a highly valuable tool to study signal processing in vertical pathways, i.e. between photoreceptors and bipolar cells, and between bipolar cells and some types of amacrine cell1,2,3,4.
However, vertical sectioning always causes severe truncation of the dendritic fields of many retinal neurons, leading to considerable loss of synaptic contacts. Especially in the outer retina, horizontal cells are affected due to their laterally spread extended dendritic fields and axon terminal systems5. In a vertical slice preparation, the synaptic input of hundreds of cone photoreceptor terminals to the dendrites of a horizontal cell is thus reduced to a few sparse contacts. This might suffice to study the properties of individual synapses, but it does by no means represent the signal processing capabilities underlying the synchronized activation of photoreceptor ribbon synapses.
We therefore developed a horizontal slice preparation, which leaves almost all of the cone photoreceptor synaptic input to horizontal cell dendrites intact and functional. The plane of sectioning runs parallel to the surface of the retina, ideally cutting through the inner nuclear layer between horizontal cells and amacrine cells. Thus, horizontal slices of the outer retina are created containing, on the presynaptic site, photoreceptors with inner and outer segments as well as synaptic terminals, and horizontal cells and bipolar cells on the postsynaptic site. This preparation is well suited to investigate coordinated synaptic inputs into horizontal cell dendrites by all cone photoreceptors within its dendritic field. It thus might prove useful to better understand the workings of the photoreceptor ribbon synapse, which is crucial for the transfer of visual information from the matrix of photoreceptor activation into a postsynaptic response.

<table><tbody><tr><td>Ames&#039; Medium</td><td>Sigma</td><td>A1420</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Hepes</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>Carbogen</td><td>Air Liquide</td><td></td><td></td></tr><tr><td>Agarose</td><td>Sigma</td><td>A9045</td><td></td></tr><tr><td>35 mm Petri dish (plastic)</td><td>Nunc Thermofisher</td><td>150318</td><td></td></tr><tr><td>Glass Petri dish</td><td>Chemoline</td><td>50-1470-40</td><td></td></tr><tr><td>Isoflurane</td><td>Dispensary university hospital</td><td></td><td></td></tr><tr><td>Vibratome injector blade</td><td>Biosystems</td><td>39053250</td><td></td></tr><tr><td>Vibratome</td><td>Leica Microsystems</td><td>VT1200</td><td></td></tr><tr><td>Vibrocheck</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Microwave</td><td></td><td></td><td></td></tr><tr><td>Water bath</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>20 G needles</td><td></td><td></td><td></td></tr><tr><td>Spring scissors</td><td></td><td></td><td></td></tr><tr><td>Curved scalpel blades</td><td></td><td></td><td></td></tr><tr><td>Stereo microscope</td><td></td><td></td><td></td></tr><tr><td>Plastic Pasteur pipette</td><td></td><td></td><td></td></tr><tr><td>Glass Pasteur pipette</td><td></td><td></td><td></td></tr></tbody></table>

preparation of horizontal slices of adult mouse retina for electrophysiological studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

In horizontal slices of the mouse retina, horizontal cell bodies could be easily identified according to their unique morphology with several primary dendrites emerging from a polygonal cell body (Figure 1A). Horizontal cell bodies located near the surface of the slice were routinely accessible for patch-clamp electrodes. During recordings, cells were filled with a fluorescent dye, revealing their intricate dendritic arborization, which closely resembled the morphology of axon-bearing horizontal cells of the intact mouse retina8 (Figure 1B). Labeling of cone pedicles with fluorescein-coupled peanut agglutinin showed a close spatial relationship between cone pedicles and horizontal cell dendrites (Figure 1C). With few exceptions, all cone pedicles within the horizontal cell&#39;s dendritic field were contacted by dendritic processes.
To prove that the contacts between cones and horizontal cells were functional, patch-clamp recordings were carried out from horizontal cell bodies. Under dark-adapted conditions, current responses were characterized by a large inward current accompanied by moderate to high-frequency synaptic activity (Figure 2A). The deviation from baseline and the excitatory postsynaptic currents were caused by the constant release of glutamate in the dark, activating postsynaptic AMPA- and kainate-type glutamate receptors9. Individual synaptic events were characterized by a monophasic waveform or more complex current trajectories (Figure 2B). The presence of tonic postsynaptic activity suggests that glutamate was released at the synaptic contacts between cone photoreceptors and horizontal cell dendrites shown in Figure 1C.
Horizontal cells recorded responded to changes in light intensity, indicating that the signal transduction cascade in cone photoreceptors was also intact and functional in a horizontal slice preparation. Due to the reduction of transmitter release, the tonic inward current of dark-adapted horizontal cells was inhibited, and synaptic activity appeared clearly diminished during the presence of a full-field light stimulus (1,300 W/cm2, 470 40 nm) (Figure 3A). Likewise, a switch to darkness from a light-adapted state caused an inward current and increased synaptic activity (Figure 3B). In addition, brief electrical pulses (1-15 V, 1 msec) were applied through a platinum-iridium bipolar stimulation electrode to depolarize all cone photoreceptors in the vicinity of a horizontal cell body. The stimulation-induced release of glutamate evoked large amplitude excitatory postsynaptic currents in the recorded horizontal cell (Figure 3C). In summary, these findings show convincingly that the synapse between cone photoreceptors and horizontal cells can be manipulated experimentally by either the endogenous stimulus light or by controlled depolarization of presynaptic elements.
<img alt="Figure 1" src="/files/ftp_upload/55173/55173fig1.jpg" /> 
Figure 1: Morphology and Synaptic Contacts of Horizontal Cells in a Horizontal Slice Preparation. A horizontal cell (arrow) located near the surface of the slice was visualized with Dodt contrast optics. Scale bar = 10 &#181;m (A). For imaging experiments, a horizontal cell was filled with Oregon Green 488 BAPTA-1 (100 &#181;M) via a patch-electrode, which is still attached to the cell. Scale bar = 10 &#181;m (B). Overlay of a dye-injected horizontal cell and the array of cone pedicles labeled with fluorescein-coupled peanut agglutinin reveals putative synaptic contacts. Scale bar = 10 &#181;m (C). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55173/55173fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55173/55173fig2.jpg" /> 
Figure 2: Synaptic Activity Recorded from a Horizontal Cell Body. Whole-cell voltage-clamp recording at a holding potential of -60 mV shows high-frequency tonic synaptic activity (A). At higher temporal resolution (B), synaptic events display monophasic waveforms (arrow) or more complex current trajectories (arrowheads). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55173/55173fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55173/55173fig3.jpg" /> 
Figure 3: Current Responses of Horizontal Cell Bodies Evoked by Light and Electrical Stimulation. Light response of a dark-adapted horizontal cell body recorded at -60 mV holding potential. Full-field illumination inhibits the tonic inward current and reduces synaptic activity (A). A light-adapted horizontal cell body recorded under the same conditions displays an inward current following a dark flash accompanied by increased synaptic activity during darkness (B). Horizontal lines in A and B represent the timing of the stimulus. Extracellular stimulation by brief electrical pulses induces large-amplitude excitatory postsynaptic currents due to the synchronized release of glutamate from cone photoreceptors (C). Holding potential: -60 mV. The arrow indicates the timing of the stimulation pulse. Truncated currents at the beginning of the trace represent stimulation artefacts. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55173/55173fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies at JoVE.com

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

preparation-horizontal-slices-adult-mouse-retina-for

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.0K Views.  Friedrich-Alexander-University (FAU) Erlangen-Nürnberg.  We ontwikkelden een horizontale slice voorbereiding van de zoogdierretina volwassene met het vlak van snijden parallel aan het netvliesoppervlak. Dendritische gebied van nonradially georiënteerde retinale neuronen werden niet afgekapt, waardoor de studie van de signaalverwerking met patch-klem en beeldvormende technieken. 

Video: Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Horizontal Slice Preparation of the Retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of retinal neurons, such as amacrine cells, expand their dendrites horizontally, so that the morphology of the cells is supposed to be severely damaged in the vertical slices. In the whole-mount preparation, especially for patch-clamp recordings, retinal neurons in the middle layer are not easily accessible due to the extensive coverage of glial cell (Mueller cell) s endfeets. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. The slice was made horizontally at the inner layer of the retina using a vibratome slicer after the retina was embedded in the low-temperature melting agarose gel. In this horizontal slice preparation of the retina, we studied the function of retinal neurons compared with their morphology, by using patch-clamp recording, calcium imaging technique, immunocytochemistry, and single-cell RT-PCR.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

Biology

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light whereas cones operate continuously under rapidly changing bright light conditions. Absorption of light by rod- and cone-specific visual pigments in the outer segments of photoreceptors triggers a phototransduction cascade that eventually leads to closure of cyclic nucleotide-gated channels on the plasma membrane and cell hyperpolarization. This light-induced change in membrane current and potential can be registered as a photoresponse, by either classical suction electrode recording technique1,2 or by transretinal electroretinogram recordings (ERG) from isolated retinas with pharmacologically blocked postsynaptic response components3-5. The latter method allows drug-accessible long-lasting recordings from mouse photoreceptors and is particularly useful for obtaining stable photoresponses from the scarce and fragile mouse cones. In the case of cones, such experiments can be performed both in dark-adapted conditions and following intense illumination that bleaches essentially all visual pigment, to monitor the process of cone photosensitivity recovery during dark adaptation6,7. In this video, we will show how to perform rod- and M/L-cone-driven transretinal recordings from dark-adapted mouse retina. Rod recordings will be carried out using retina of wild type (C57Bl/6) mice. For simplicity, cone recordings will be obtained from genetically modified rod transducin &alpha;-subunit knockout (T&alpha;-/-) mice which lack rod signaling8.

We describe a relatively simple method of transretinal electroretinogram (ERG) recordings for obtaining rod and cone photoresponses from intact mouse retina. This approach takes advantage of the block of synaptic transmission from photoreceptors to isolate their light responses and record them using field electrodes placed across the isolated flat-mounted retina.

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Studying Bipolar and Horizontal Neuronal Cells in the Murine Split Retina

Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina

Bipolar cells and horizontal cells of the vertebrate retina are the first neurons to process visual information after photons are detected by photoreceptors. They perform fundamental operations such as light adaptation, contrast sensitivity, and spatial and color opponency. A complete understanding of the precise circuitry and biochemical mechanisms that govern their behavior will advance visual neuroscience research and ophthalmological medicine. However, current preparations for examining bipolar and horizontal cells (retinal whole mounts and vertical slices) are limited in their capacity to capture the anatomy and physiology of these cells. In this work, we present a method for removing photoreceptor cell bodies from live, flatmount mouse retinas, providing enhanced access to bipolar and horizontal cells for efficient patch clamping and rapid immunolabeling. Split retinas are prepared by sandwiching an isolated mouse retina between two pieces of nitrocellulose, then gently peeling them apart. The separation splits the retina just above the outer plexiform layer to yield two pieces of nitrocellulose, one containing the photoreceptor cell bodies and another containing the remaining inner retina. Unlike vertical retina slices, the split retina preparation does not sever the dendritic processes of inner retinal neurons, allowing for recordings from bipolar and horizontal cells that integrate the contributions of gap junction-coupled networks and wide-field amacrine cells. This work demonstrates the versatility of this preparation for the study of horizontal and bipolar cells in electrophysiology, immunohistochemistry, and in situ hybridization experiments.

Author Spotlight: Using the Split Retina Technique for Enhanced Access and Accelerated Experiments 

This work presents an alternative flatmount retina preparation in which the removal of photoreceptor cell bodies enables faster antibody diffusion and improved patch pipette access to inner retinal neurons for immunohistochemistry, in situ hybridization, and electrophysiology experiments.

Bipolar cells and horizontal cells of the vertebrate retina are the first neurons to process visual information after photons are detected by ...

Establishing a Whole-Cell Patch Clamp for Electrophysiological Recording from a Flat-Mount Mouse Retina

Source: Tu, H., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54608">Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina.</a> J. Vis. Exp. (2016).
This video demonstrates a whole-cell patch-clamp technique for electrophysiological recording from a flat-mount mouse retina. A thin glass pipette with an electrode is used to approach starburst amacrine cells (SACs) within the retina. After forming a seal, suction is applied to rupture the membrane and form the whole-cell patch-clamp configuration. Finally, the flow of ions through the SAC synaptic receptors is measured using the configuration.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. External and Internal Solutions

	...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video