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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 opposi....

Protocol

All the experiments were approved by the Governmental Supervisory Panel on Animal Experiments of Rhineland-Palatinate.

1. Solutions

  1. Dissection solution
    1. 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.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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

....

Materials

NameCompanyCatalog NumberComments
Amplifier HEKA ElektronikEPC 10 USB Double patch clamp amplifier
BiocytinSigma-AldrichB4261-250MG
CaCl2EMSURE1.02382.1000
choline chlorideSigma-AldrichC1879-1KG
Confocal Laser Scanning MicroscopeLeica MicrosystemsTCS SP5
CsClEMSURE1.02038.0100
Cs-gluconateSelf-preparedSince there was no commercial Cs-gluconate, we prepared it by ourselves 
D-600 Sigma-AldrichM5644-50MGmethoxyverapamil hydrochloride
D-APV Biotrend BN0085-100NMDA-receptor antagonist
Digital camera for microscopeOlympusXM10
EGTASERVA11290.02
ForeneAbbvie2594.00.00isoflurane
GlucoseSigma-Aldrich49159-1KG
HEPESROTH9105.2
High Current Stimulus IsolatorWorld Precision InstrumentsA385
KClEMSURE1.04936.1000
MgCl2EMSURE1.05833.0250
MicromanipulatorsLuigs & NeumannSM7
MiroscopeOlympusBX51
mounting medium ThermoFisher ScientificP36930Prolong Gold Invitrogen
NaClROTH3957.1
NaH2PO4EMSURE1.06346.1000
NaHCO3EMSURE1.06329.1000
PipetteHilgenberg1807502
PullerSutter P-1000
razor blade Personna 60-0138
Semiautomatic VibratomeLeica  BiosystemsVT1200S
SR 95531 hydrobromide Biotrend AOB5680-10GABAA-receptor antagonist 

References

  1. Guido, W. Development, form, and function of the mouse visual thalamus. Journal of Neurophysiology. 120, 211-225 (2018).
  2. Guillery, R. W., Feig, S. L., Lozsadi, D. A. Paying attention to the thalamic reticular nucleus.

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