In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Summary

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

Abstract

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously naïve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

Introduction

Deciphering where and how the information is acquired in the brain through learning and subsequently stored as memory constitutes one of the most challenging tasks in neuroscience¹. Neuroscientific research has led to the concept of a change in synaptic transmission as the neuronal substrate that underlies learning and memory formation^2,3. It is hypothesized that, during learning, synaptic connections between neuronal ensembles that are active during the perception of a stimulus become modified such that their combined activity pattern can be retrieved during memory recall, thereby inst....

Protocol

1. Transgenic fruit flies, Drosophila melanogaster

1. Cross female virgin and male flies (raised at 25 °C in 60% relative humidity on a 12 h light/dark cycle) carrying the desired Gal4 and UAS constructs²⁵, respectively, to produce flies in which specific neurons of interest express a genetically encoded calcium indicator.
2. Age the female progeny of the above cross until they are in the range of 3-6 days post-eclosion. Female flies are preferable because of their slightly larger size.

2. Preparation of the fruit fly for in vivo calcium imaging

1. Select a single female ....

Results

An example of images acquired with the above protocol can be seen in Figure 2. dHomer-GCaMP3 is expressed in an MB output neuron whose dendrites innervate the compartment 1 of the MB γ-lobe (the neuron is termed MVP2^28,29) and is genetically targeted using the split-Gal4 line MB112C¹⁶. Also, demonstrated is the difference in the subcellular localization of a cytosolic .......

Discussion

The dissection of the neural circuitry underlying learning and memory is a prominent goal in the field of neuroscience. The genetic accessibility of Drosophila and the breadth and ease of behavioral testing makes this an ideal tool to investigate such phenomena. Here, a method is presented with which it is possible to visualize, within individual flies, the modulation that occurs at a subcellular level as a result of olfactory conditioning. By carrying out both pre-training and post-training visualization of odo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-Octen-3-ol	Sigma-Aldrich, St. Louis, MO, USA	O5284	Chemical used as odorant
3-Octanol	Sigma-Aldrich, St. Louis, MO, USA	218405	Chemical used as odorant
4-Methylcyclohexanol	Sigma-Aldrich, St. Louis, MO, USA	153095	Chemical used as odorant
Bandpass filter for EGFP (525/50 nm)	Carl Zeiss Microscopy GmbH, Jena, Germany
Clear adhesive tape	Tesa SE, Norderstedt, Germany		Standard claer adhesive tape
Concave-convex jaws	Fine Science Tools, North Vancouver, Canada	10053-09	Blade Holders with concave-convex jaws
Fine forceps	Fine Science Tools, North Vancouver, Canada	11412-11	Forceps with tip 0.1 x 0.06mm
Hypodermic needle	Sterican - B. Braun, Melsungenk, Germany	4665120	1.20x40mm
Insect Minutien pins	Fine Science Tools, North Vancouver, Canada	26002-10	Diameter 0.1mm, tip 0.0125mm
Kentoflow	Kent Express Dental Supplies, Gillingham, UK	953683	Blue light-curing glue
Microscope slide	Carl Roth GmbH & Co. KG, Karlsruhe, Germany	0656.1	Standard objective slide 76 x 26 mm
Mineral oil	Sigma-Aldrich, St. Louis, MO, USA	M8410	Used as diluent for odorants
Mode-locked Ti-Sapphire laser Chameleon Vision 2	Coherent Inc., Santa Clara, CA, USA		Tunable infrared femtosecond laser
Multiphoton Microscope LSM 7MP equipped with BiG detectors	Carl Zeiss Microscopy GmbH, Jena, Germany		Multiphoton microscope, multiple companies provide similar devices.
Plan-Apochromat 20x (NA = 1.0) water immersion objective	Carl Zeiss Microscopy GmbH, Jena, Germany	421452-9900-000	Objective W "Plan-Apochromat" 20x/1.0 DIC M27 70mm
Ringer's solution	n.a.	n.a.	5mM KCl, 130mM NaCl, 2mM MgCl2, 2mM CaCl2, 5mM Hepes-NaOH, 36mM sucrose, pH = 7.4
Stab knife	Sharpoint, Surgical Specialties Corporation, Reading, PA, USA	72-1551	5.0mm Straight restricted blade depth
Surgical scalpel blade	Swann-Morton, Sheffield, UK	0303	Product No. 11
Surgical scalpel handle	Swann-Morton, Sheffield, UK	0907	Product No. 7S/S
Visual Basics of Applicatons (VBA) software to receive a trigger from the odor-delivery device and the electric shock application device (power supply) to interact with the ZEN software from Zeiss that controls the microscope.	Custom-written and available upon request	n.a.	n.a.