Name: electrophysiological investigations of retinogeniculate and corticogeniculate synapse function
Uploaded: 2019-08-07T13:00:00
Description: Watch this Scientific Journal Video about Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function at JoVE.com

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.

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	<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>
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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...

Watch this Scientific Journal Video about Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function at JoVE.com

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 ...

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