Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

Summary

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Abstract

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.

With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.

Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

Introduction

Neurons in the central nervous system receive a variety of synaptic inputs on their thin and ramified dendritic processes¹. There, the majority of the excitatory dendritic inputs are mediated by glutamatergic synapses. These synapses can be activated in a spatially distributed way, resulting in postsynaptic linear integration of excitatory postsynaptic potentials (EPSPs). If the synapses are activated simultaneously and in spatial proximity on the dendrite, these excitatory inputs can be integrated supra-linearly and generate dendritic spikes^2-5.

Furthermore, the integration of excitatory inputs depends on the location....

Protocol

1. System Requirements

1. Microscope system: Good visualization of the dendrite is crucial. If available, use a two-photon laser scanning microscope system. In our experiments we used a TRIM Scope II, LaVision Biotec, Bielefeld, Germany or an Ultima IV system, Prairie Technologies, Middleton, Wisconsin equipped with a Ti:Sapphire ultrafast-pulsed laser (Chameleon Ultra II, Coherent) and a high NA objective (60X, 0.9 NA, Olympus) to visualize the dendrites which we had filled with a fluorescent dye via the patch pipette. Although photo-damage is thought to be less severe using 2-photon scanning, reduce the laser power (below 8 mW at the tissue) and dwell times....

Results

A simple approach to determine the spatial spread of iontophoresis is to retract the iontophoretic pipette stepwise from the dendrite, while keeping the ejected glutamate constant. We found that the spatial extent of a micro-iontophoretic stimulation had a diameter of approximately 12 μm (Figure 1 showing radius). How deep in the tissue the iontophoresis can be used depends on the rigidity of the pipette. However, the iontophoretic pipettes needed for experiments in slices (Figure 2), wh.......

Discussion

Here we explain how to apply fast micro-iontophoresis of neurotransmitters to investigate synaptic integration on dendrites. This technique has been successfully used to investigate glutamatergic and GABAergic synaptic transmission in different brain regions in vitro and in vivo^9,20-22. Micro-iontophoresis has been used for more than 60 years, but in early years it was mostly used to either locally apply neurotransmitters and drugs on slow or intermediate timescales²³ or for microi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
			Material
Two-photon laser scanning microscope (TRIM Scope II), and Ultima IV, Prairie Technologies, Middleton, Wisconsin)	LaVision Biotec, Bielefeld, Germany
Two-photon laser scanning microscope Ultima IV	Prairie Technologies, Middleton, Wisconsin, USA
Ti:Sapphire ultrafast-pulsed laser	Chameleon Ultra II, Coherent
60X Objective, NA 0.9	Olympus
Zeiss Axioskop 2 FS upright microscope	TILLPhotonics, Gräfelfing, Germany
Monochromator	TILLPhotonics, Gräfelfing, Germany
Micro-iontophoresis system MVCS-02	NPI Electronics, Tamm, Germany
Sutter puller P-97	Sutter Instrument Company, Novato, CA
Glass filaments (150 GB F 8P)	Science Products, Hofheim, Germany
			Reagent
Alexa Fluor 488 hydrazide	Molecular Probes life technologies	A-10436
Alexa Fluor 594	Molecular Probes life technologies	A-10438
NaCl	Sigma Aldrich	S7653
KCl	Sigma Aldrich	P9333
NaH2PO4	Sigma Aldrich	S8282
NaHCO3	Sigma Aldrich	S6297
Sucrose	Sigma Aldrich	S7903
CaCl2	Sigma Aldrich	C5080
MgCl2	Sigma Aldrich	M2670
Glucose	Sigma Aldrich	G7528
K-Gluconate	Sigma Aldrich	G4500
HEPES-acid	Sigma Aldrich	H4034
Phosphocreatin	Sigma Aldrich	P7936
EGTA	Sigma Aldrich	E3889
Glutamic acid	Sigma Aldrich	G8415
GABA	Sigma Aldrich	A5835
NaOH	Merck	1.09137.1000
HCl	Merck	1.09108.1000