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 border="0" cellpadding="0" cellspacing="0" width="627">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ames&#39; Medium</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:119px;">A1420</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Bicarbonate</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:119px;">S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hepes</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Carbogen</td>
			<td style="width:125px;">Air Liquide</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:119px;">A9045</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">35-mm petri dish (plastic)</td>
			<td style="width:125px;">Nunc Thermofisher</td>
			<td style="width:119px;">150318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass petri dish</td>
			<td style="width:125px;">Chemoline</td>
			<td style="width:119px;">50-1470-40</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Isoflurane</td>
			<td style="width:125px;">Dispensary university hospital</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vibratome injector blade</td>
			<td style="width:125px;">Biosystems</td>
			<td style="width:119px;">39053250</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vibratome</td>
			<td style="width:125px;">Leica Microsystems</td>
			<td style="width:119px;">VT1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vibrocheck</td>
			<td style="width:125px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microwave</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water bath</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">20-gauge needles</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Spring scissors</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Curved scalpel blades</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereo microscope</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic Pasteur pipette</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass Pasteur pipette</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></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 ...

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Neuroscience

10.0K Views.  Friedrich-Alexander-University (FAU) Erlangen-Nürnberg. 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.

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

Organotypic Culture of Full-thickness Adult Porcine Retina

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human retina and is therefore a valuable species for studying mechanisms of human retinal injury and degenerative disease. Here we describe a cost-effective technique for organotypic culture of adult porcine retina isolated from eyes obtained from an abattoir. After removing the anterior segment, a trephine blade was used to create multiple neural retina-Bruch's membrane-RPE-choroid-sclera explants from the posterior segment of adult porcine eyes. A piece of sterile filter paper was used to lift the neural retina off from each explant. The filter paper-retina complex was cultured (photoreceptor side up) atop an insert, which was held away from the bottom of the culture dish by a custom-made stand. The stand allows for good circulation of the culture medium to both sides of the retina. Overall, this procedure is simple, reproducible, and permits preservation of native retinal structure for at least seven days, making it a useful model for a variety of morphological, pharmacological, and biochemical studies on mammalian retina.

Here we describe a cost-effective technique for organotypic culture of adult porcine retina for seven days. Briefly, a sterile filter paper was used to lift the neural retina off from the RPE and place photoreceptor side up on an insert raised by a custom-made stand.

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Healthy Brain-pituitary Slices for Electrophysiological Investigations of Pituitary Cells in Teleost Fish

Electrophysiological investigations of pituitary cells have been conducted in numerous vertebrate species, but very few in teleost fish. Among these, the clear majority have been performed on dissociated primary cells. To improve our understanding of how teleost pituitary cells, behave in a more biologically relevant environment, this protocol shows how to prepare viable brain-pituitary slices using the small freshwater fish medaka (Oryzias latipes). Making the brain-pituitary slices, pH and osmolality of all solutions were adjusted to values found in body fluids of freshwater fish living at 25 to 28 &#176;C. Following slice preparation, the protocol demonstrates how to conduct electrophysiological recordings using the perforated whole-cell patch-clamp technique. The patch-clamp technique is a powerful tool with unprecedented temporal resolution and sensitivity, allowing investigation of electrical properties from intact whole cells down to single ion channels. Perforated patch is unique in that it keeps the intracellular environment intact preventing regulatory elements in the cytosol from being diluted by the patch pipette electrode solution. In contrast, when performing traditional whole-cell recordings, it was observed that medaka pituitary cells quickly lose their ability to fire action potentials. Among the various perforation techniques available, this protocol demonstrates how to achieve perforation of the patched membrane using the fungicide Amphotericin B.

The article describes an optimized protocol for making viable brain-pituitary tissue slices, using the teleost fish medaka (Oryzias latipes), followed by electrophysiological recordings of pituitary cells using the patch-clamp technique with the perforated patch configuration.

Electrophysiological investigations of pituitary cells have been conducted in numerous vertebrate species, but very few in teleost fish. Among these, the ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Summary

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Chapters in this video

Organotypic Culture of Full-thickness Adult Porcine Retina

In vivo Electroporation of Developing Mouse Retina

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

Healthy Brain-pituitary Slices for Electrophysiological Investigations of Pituitary Cells in Teleost Fish

Assessment of Long-term Depression Induction in Adult Cerebellar Slices