This work has been funded by the German Research Foundation (DFG) within the Collaborative Research Center (SFB) 1134 &#34;Functional Ensembles&#34; (J.v.E. and X.C.) and the Research Grant EN948/1-2 (J.v.E.).

All the experiments were approved by the Governmental Supervisory Panel on Animal Experiments of Rhineland-Palatinate.
1. Solutions
<ol>
	<li>Dissection solution
	<ol>
		<li>To reduce excitotoxicity, prepare a choline-based solution to be used during the dissection as presented here (in mM): 87 NaCl, 2.5 KCl, 37.5 choline chloride, 25 NaHCO3, 1.25 NaH2PO4, 0.5 CaCl2, 7 MgCl2, and25 glucose. Prepare the dissection solution less...

<ol>
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We describe an improved protocol based on a previously published method3, which allows for the investigation of the high probability of release retinogeniculate synapses and low probability of release corticogeniculate synapses from the same slice. This is of great importance since these two inputs interact with each other to modulate the visual signal transmission: retinogeniculate inputs are the main excitatory drive of relay neurons, whereas corticothalamic inputs function as a modulator, which...

Relay neurons of the lateral geniculate nucleus integrate and relay visual information to the visual cortex. These neurons receive excitatory input from ganglion cells via retinogeniculate synapses, which provide the main excitatory drive for relay neurons. In addition, relay neurons receive excitatory inputs from cortical neurons via corticogeniculate synapses. Moreover, relay neurons receive inhibitory inputs from local interneurons and GABAergic neurons of the nucleus reticularis thalami1. The nucleus reticularis thalami is present like a shield between thalamus and cortex such that fibers projecting from cortex to thalamus and in the opposite direction must go through the nucleus reticularis thalami2.
Retinogeniculate inputs and corticogeniculate inputs display distinct synaptic properties3,4,5,6,7,8. Retinogeniculate inputs form large terminals with multiple release sites9,10. In contrast, corticogeniculate inputs display small terminals with single release sites7. In addition, retinogeniculate synapses efficiently drive action potentials of relay neurons despite constituting only 5&#8722;10% of all synapses on relay neurons3,8,11. Corticogeniculate synapses, on the other hand, serve as a modulator of retinogeniculate transmissions by controlling the membrane potential of relay neurons12,13.
These two main excitatory inputs to relay neurons are also functionally different. One prominent difference is the short-term depression of retinogeniculate synapses and the short-term facilitation of corticogeniculate synapses3,5,8. Short-term plasticity refers to a phenomenon in which synaptic strength changes when the synapse is repeatedly active within a time period of few milliseconds to several seconds. Synaptic release probability is an important factor underlying short-term plasticity. Synapses, with a low initial release probability, display short-term facilitation due to the buildup of Ca2+ in the presynapse and consequently an increase in the release probability is observed upon repeated activity. In contrast, synapses with high release probability usually display short-term depression due to the depletion of ready-releasable vesicles14. In addition, desensitization of postsynaptic receptors contributes to the short-term plasticity in some high-release probability synapses8,15. High release probability and desensitization of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors contribute to the prominent short-term depression of retinogeniculate synapses. In contrast, low-release probability underlies the short-term facilitation of corticogeniculate synapses.
In mice, the optic tract enters the dorsal lateral geniculate nucleus (dLGN) from the caudolateral site, whereas corticogeniculate fibers enter the dLGN rostroventrally. The distance between the two inputs allows for the investigation of the individual properties of two very different excitatory inputs impinging onto the same cell. Here, we build on and improve a previously described dissection method in which retinogeniculate and corticogeniculate fibers are preserved in acute brain slices3. We, then, describe the electrophysiological investigation of relay neurons and stimulation of retinogeniculate and corticogeniculate fibers with extracellular stimulation electrodes. Finally, we provide a protocol for the filling of relay neurons with biocytin and subsequent anatomical analysis.

<table border="0" cellpadding="0" cellspacing="0" style="width:615px;" width="614">
	<tbody>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">Amplifier&#160;</td>
			<td style="width:98px;">HEKA Elektronik</td>
			<td style="width:123px;">EPC 10 USB Double patch clamp amplifier</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Biocytin</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">B4261-250MG</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CaCl2</td>
			<td style="width:98px;">EMSURE</td>
			<td style="width:123px;">1.02382.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">choline chloride</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">C1879-1KG</td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;">Confocal Laser Scanning Microscope</td>
			<td style="width:98px;">Leica Microsystems</td>
			<td style="width:123px;">TCS SP5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CsCl</td>
			<td style="width:98px;">EMSURE</td>
			<td style="width:123px;">1.02038.0100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Cs-gluconate</td>
			<td style="width:98px;">Self-prepared</td>
			<td style="width:123px;"></td>
			<td>Since there was no commercial Cs-gluconate, we prepared it by ourselves&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-600&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">M5644-50MG</td>
			<td>methoxyverapamil hydrochloride</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">D-APV&#160;</td>
			<td style="width:98px;">Biotrend&#160;</td>
			<td style="width:123px;">BN0085-100</td>
			<td style="width:172px;">NMDA-receptor antagonist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Digital camera for microscope</td>
			<td style="width:98px;">Olympus</td>
			<td style="width:123px;">XM10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA</td>
			<td style="width:98px;">SERVA</td>
			<td style="width:123px;">11290.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Forene</td>
			<td style="width:98px;">Abbvie</td>
			<td style="width:123px;">2594.00.00</td>
			<td style="width:172px;">isoflurane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">49159-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td style="width:98px;">ROTH</td>
			<td style="width:123px;">9105.2</td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">High Current Stimulus Isolator</td>
			<td style="width:98px;">World Precision Instruments</td>
			<td style="width:123px;">A385</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:98px;">EMSURE</td>
			<td style="width:123px;">1.04936.1000</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MgCl2</td>
			<td style="width:98px;">EMSURE</td>
			<td style="width:123px;">1.05833.0250</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Micromanipulators</td>
			<td style="width:98px;">Luigs &#38; Neumann</td>
			<td style="width:123px;">SM7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Miroscope</td>
			<td style="width:98px;">Olympus</td>
			<td style="width:123px;">BX51</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mounting medium&#160;</td>
			<td style="width:98px;">ThermoFisher Scientific</td>
			<td>P36930</td>
			<td>Prolong Gold Invitrogen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:98px;">ROTH</td>
			<td align="right" style="width:123px;">3957.1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">NaH2PO4</td>
			<td style="width:98px;">EMSURE</td>
			<td style="width:123px;">1.06346.1000</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">NaHCO3</td>
			<td style="width:98px;">EMSURE</td>
			<td style="width:123px;">1.06329.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette</td>
			<td style="width:98px;">Hilgenberg</td>
			<td style="width:123px;">1807502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Puller</td>
			<td style="width:98px;">Sutter&#160;</td>
			<td style="width:123px;">P-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">razor blade&#160;</td>
			<td style="width:98px;">Personna&#160;</td>
			<td>60-0138</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Semiautomatic Vibratome</td>
			<td style="width:98px;">Leica&#160; Biosystems</td>
			<td style="width:123px;">VT1200S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">SR 95531 hydrobromide&#160;</td>
			<td style="width:98px;">Biotrend&#160;</td>
			<td style="width:123px;">AOB5680-10</td>
			<td style="width:172px;">GABAA-receptor antagonist&#160;</td>
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electrophysiological investigations of retinogeniculate and corticogeniculate synapse function

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ganglion cells and project it to the visual cortex. In addition, relay neurons receive top-down excitation from the cortex. The two main excitatory inputs to the relay neurons differ in several aspects. Each relay neuron receives input from only a few retinogeniculate synapses, which are large terminals with many release sites. This is reflected by the comparably strong excitation, the relay neurons receive, from retinal ganglion cells. Corticogeniculate synapses, in contrast, are simpler with few release sites and weaker synaptic strength. The two synapses also differ in their synaptic short-term plasticity. Retinogeniculate synapses have a high release probability and consequently display a short-term depression. In contrast, corticogeniculate synapses have a low release probability. Corticogeniculate fibers traverse the reticular thalamic nuclei before entering the lateral geniculate nucleus. The different locations of the reticular thalamic nucleus (rostrally from the lateral geniculate nucleus) and optic tract (ventro-laterally from the lateral geniculate nucleus) allow stimulating corticogeniculate or retinogeniculate synapses separately with extracellular stimulation electrodes. This makes the lateral geniculate nucleus an ideal brain area where two excitatory synapses with very different properties impinging onto the same cell type, can be studied simultaneously. Here, we describe a method to investigate the recording from relay neurons and to perform detailed analysis of the retinogeniculate and corticogeniculate synapse function in acute brain slices. The article contains a step-by-step protocol for the generation of acute brain slices of the lateral geniculate nucleus and steps for recording activity from relay neurons by stimulating the optic tract and corticogeniculate fibers separately.

The slice preparation of dLGN containing the retinogeniculate and corticogeniculate pathways is shown under a 4x objective (Figure 2). Axons of retinal ganglion cells bundle together in the optic tract (Figure 2). The stimulating pipette was placed on the optic tract to induce retinogeniculate synapse-mediated current (Figure 2A) and on nucleus reticularis thalami to induce corticogeniculate synapses...

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Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function

Here, we present protocols for the preparation of acute brain slices containing the lateral geniculate nucleus and the electrophysiological investigation of retinogeniculate and corticogeniculate synapses function. This protocol provides an efficient way to study synapses with the high-release and low-release probability in the same acute brain slices.

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ...

Here we present the protocols for the preparation of acute brain slices containing the lateral geniculate nucleus and electrophysiological investigation of retinal geniculate and cortical geniculate synapse function. Retinal ganglion cell axons enter the lateral geniculate nucleus far away from the cortical inputs. This allows the separate stimulations of retinal geniculate and cortical geniculate synapses, which have very different functional properties.To begin this procedure, prepare two slice chambers, one filled with 100 milliliters of the dissection solution and the other with 100 milliliters of recording solution. Place the two beakers into a water bath at 37 degrees Celsius, and bubble the solutions with carbogen for at least 15 minutes before the dissection. Next fill a plastic beaker with 250 milliliters of near-ice-cold dissection solution, and bubble it with carbogen for at least 15 minutes before use.Then fill a plastic Petri dish with the cold dissection solution for brain dissection. Place a piece of filter paper in the lid of the Petri dish, and fill it with a small amount of dissection solution. Subsequently cool down the dissection chamber of the vibratome.Cut the skin of the head from caudal to nasal, and keep it opened with fingers to expose the skull. To obtain the forebrain, first cut the skull along the posterior/anterior midline. Then cut two more times through the coronal suture and lambdoid suture.Remove the skull between the coronal suture and lambdoid suture to expose the cerebrum. Cut the olfactory bulb, and separate the forebrain from the midbrain with a fine blade. Remove the brain from the skull with a thin spatula, and place it on a filter paper in the lid of the Petri dish.To preserve the integrity of the sensory and cortical inputs to the dorsal lateral geniculate nucleus in the brain slice, separate the two hemispheres with a parasagittal cut at an angle of three to five degrees. Afterward dry the medial lateral planes of the two hemispheres by placing them on a filter paper. Then glue them onto the cutting stage with an angle of 10 to 25 degrees from the horizontal plane.Place the stage in the center of the metal buffer tray, and gently pour the rest of the ice-cold dissection solution into the buffer tray. Perform this step carefully, since pouring too hard might remove the pasted brain. Next keep the buffer tray in the ice-filled tray to maintain the low temperature during dissection.Section 250-micrometer slices with a razor blade at a speed of 0.1 millimeter per second and an amplitude of one millimeter. Transfer the slices to the holding chamber filled with oxygenated dissection solution at 34 degrees Celsius for around 30 minutes, and then allow the slices to recover in the recording solution at 34 degrees Celsius for another 30 minutes. After recovery, remove the slice-holding chamber from the water bath, and keep the slices at room temperature until used in experiments.In this procedure, pull the recording pipettes using borosilicate glass capillaries and a filament puller. Next pull the stimulating pipettes using the same protocol, but break the tip slightly after pulling to increase the diameter. Subsequently fill the recording pipettes with intracellular solution and biocytin, and fill the stimulating pipettes with recording solution.Then place the slices in the recording chamber, and continuously perfuse them with oxygenated recording solution at room temperature. Visualize the slices with an upright microscope equipped with IR-DIC video microscopy. Check all the slices, and select the ones that display intact optic tracts.Place the stimulation pipette on the slice before patching the cell with a recording pipette. To investigate the retinal geniculate synapses, place the stimulating pipette directly on the optic tract where the axon fibers from retinal ganglion cells are bundled. To analyze the cortical geniculate synapses, place the stimulating electrode on the nucleus reticularis thalami, which is rostroventrally adjacent to the dorsal lateral geniculate nucleus.Once the recording pipette is immersed in the recording solution, apply a five-millivolt step to monitor the pipette resistance. Set the holding potential to zero millivolt, and cancel the offset potential so that the holding current is zero picoampere. Approach the cell with a recording pipette while applying positive pressure.When the pipette is in direct contact with the cell membrane, release the positive pressure, and set the holding potential to minus 70 millivolts. Then apply slight negative pressure to allow the cell membrane to attach to the glass pipette such that a gigaohm seal forms. Compensate the pipette capacitance, and open the cell by applying negative pressure pulses.To investigate the synaptic function, apply 0.1-millisecond current pulses via the stimulation pipette. Monitor the series resistance continuously by applying a five-millivolt step. The series resistance can be estimated by dividing five millivolts by the peak amplitude of the evoked current.Use only the cells with a series resistance smaller than 20 megaohms for analysis. For biocytin labeling, maintain the whole-cell configuration for at least five minutes to allow the diffusion of biocytin into the distal dendrites. After recording, gently remove the pipette from the cell so that the soma is not destroyed.Prepare a 24-well cell culture plate. Fill each well with 300 microliters of 4%PFA. Take care to not contaminate anything with PFA that will be in contact with the slices to be recorded.Transfer the slices from the recording chamber to the PFA-containing plate with a rubber pipette, and fix them overnight. The next day, replace the PFA with PBS. Keep the slices in PBS at four degrees Celsius for future processing.To stain the cells, wash the slices with fresh PBS three times. Block nonspecific binding sites by incubating the slices in the blocking solution for two hours at room temperature on an orbital shaker. Then discard the blocking solution, and incubate the slices with streptavidin Alexa 568 diluted in the blocking solution at four degrees Celsius overnight on an orbital shaker.The next day, discard the antibody solution, and wash the slices three times with PBS for 10 minutes each at room temperature on an orbital shaker. Then wash the slices with tap water before mounting. Place the slices from one mouse on one glass slide.Ensure that the stained cells are present on the upper surface of the slices. Absorb the water around the slices with tissues, and then leave the slices to dry for approximately 15 minutes. Subsequently apply two drops of mounting medium on each slice, and mount a cover slip on the slide without producing air bubbles.Store the labeled slices at four degrees Celsius after viewing them with a confocal microscope. This IR-DIC image shows the soma of a dorsal lateral geniculate nucleus relay neuron with a tip of the patch pipette. Biocytin staining enables us to gain a view of the recorded neuron.Different from interneurons, which have bipolar morphology, relay neurons have multipolar dendritic arbors containing more than three primary dendrites. Three-dimensional reconstruction of a biocytin-labeled relay neuron was then generated, which shows a radially symmetric dendritic architecture. Preserve the retinal geniculate inputs and the cortical geniculate inputs in the same brain slices are most important in this protocol.Following this procedure, we can, for example, also investigate the inhibitor inputs from the nucleus reticularis thalami onto dorsal lateral geniculate nucleus neurons. Remember to wear gloves when fixing the brain slices with PFA, as PFA is hazardous.

electrophysiological-investigations-retinogeniculate

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JoVE Journal

Neuroscience

6.0K ビュー.  University Medical Center of the Johannes Gutenberg University Mainz. ここでは、側方のジェラジアル原核を含む急性脳スライスの調製のためのプロトコルと、レチナルジェノレートおよび皮質原性シナプス機能の電気生理学的調査を提示する。レチナルガンギ細胞軸索は、皮質入力から遠く離れた側側の原形核に入る。これは、非常に異なる機能的特性を有するレチナルジェノレートおよび皮質原性シナプスの別々の刺激を可能にする。