Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

Summary

This article features a method to test the monosynaptic connections between neurons by employing tetrodotoxin and the tetrodotoxin-resistant sodium channel, NaChBac.

Abstract

Here, a new technique termed Tetrotoxin (TTX) Engineered Resistance for Probing Synapses (TERPS) is applied to test for monosynaptic connections between target neurons. The method relies on co-expression of a transgenic activator with the tetrodotoxin-resistant sodium channel, NaChBac, in a specific presynaptic neuron. Connections with putative post-synaptic partners are determined by whole-cell recordings in the presence of TTX, which blocks electrical activity in neurons that do not express NaChBac. This approach can be modified to work with any activator or calcium imaging as a reporter of connections. TERPS adds to the growing set of tools available for determining connectivity within networks. However, TERPS is unique in that it also reliably reports bulk or volume transmission and spillover transmission.

Introduction

A major goal of neuroscience is to map the connections between neurons to understand how information flows through circuits. Numerous approaches and resources have become available to test for functional connectivity between neurons in a range of model systems^1,2. To generate the most accurate wiring diagrams using electrophysiology, it is important to resolve whether observed connections between two cells are direct and monosynaptic versus indirect and polysynaptic. A gold standard for making this distinction in mammalian neurons is to measure the latency between single action potentials in the presynaptic ce....

Protocol

1. Prepare Flies and Identify GAL4 promoter Lines

1. Select a GAL4 promoter line and cross it with flies carrying both the UAS-NaChBac transgene and an optogenetic transgene for activation.
   NOTE: UAS-CsChrimson¹¹ is selected due to its high conductance and the ease with which it activates NaChBac-mediated action potentials. Both UAS-NaChBac and UAS-CsChrimson are available from the Bloomington Stocks Repository.
2. Prepare all-trans retinal as a stock solution in ethanol (35 mM) and store the solution in freezer at -20 °C.
3. Mix 28 µL of this stock into approximately 5 mL of rehydrated potato flake food ....

Results

TERPS is used to distinguish between mono- and polysynaptic contributions in synaptic connections between neurons. While weak stimulation of a cell may be used to test direct connections, driving greater presynaptic activity often recruits polysynaptic connections (Figure 1A). TERPS works by co-expressing the TTX-insensitive sodium channel NaChBac and an optogenetic activator, and testing connections in the presence of TTX to eliminate polysynaptic connection.......

Discussion

TERPS analysis compliments current techniques used for circuit cracking by enabling the detection of synapses between identified neurons. Specifically, the approach reveals monosynaptic connections by broadly silencing action potentials with TTX while restoring excitability in a select population of neurons with the TTX-insensitive sodium channel NaChBac. Synaptic release is elicited by optogenetic stimulation while postsynaptic events are monitored with whole-cell recordings. TERPS distinguishes itself from other approa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
UAS-csChrimson	Bloomington Drosophila Stock Center	55135	Used as a neural activator
UAS-NaChBac	Bloomington Drosophila Stock Center	9466	Resotores excitibility in cells in TTX
Tetrodotoxin	Tochris	1078	Special permission may be needed to purchase TTX as it is a controlled substance
all trans-Retinal	Sigma-Aldrichall trans-Retinal	R2500	Require co-factor for channelrhodopsin
Weldable 321 Stainless Steel Sheet, 0.002" Thick, 10" Wide	McMaster Carr	3254K7	Used to make custom fly holder. Custom foil can be laser cut at pololu.com from the provided PDF file
Dissection Microscope	Zeiss	Stemi 2000-C	Used for dissection of preparation
Waxer	Almore	Eectra Waxer 66000	Used during dissection to secure fly in foil
Paraffin Wax	Joann	4917217	Used with waxer
Number 5 forceps	Fine Science Tools	#5CO	Used for dissection and desheathing
Dissection Scissors	Fine Science Tools	15001-08	Used to remove parts of the cuticle during dissection
Tungsten wire	A-M Systems	797500	Use with electrolysis to make sharpened needles for dissection
Reciculating Peristaltic Pump	Simply Pumps (Amazon)	PM200S	for recirculating TTX
Speed Controller for peristaltic pump	Zitrades (Amazon)	N/A	PWM Dimming Controller For LED Lights or Ribbon, 12 Volt 8 Amp,Adjustable Brightness Light Switch Dimmer Controller DC12V 8A 96W for Led Strip Light B
Versa-Mount Precision Compressed Air Regulator	McMaster Carr	1804T1	For applying positive pressure during patching
Glass capilaries	World Precision Instruments	TW150F-3	For patch pipettes
Multipurpose Gauge	McMaster Carr	3846K431	Gauge for pressure regulator
Electrophysiology Camera	Dage MTI	IR-1000	Any camera that works in the IR range (850 nm) will work. You do not want to use red illumination as this can activate csChrimson
IR LED	Thorlabs	M850F2	For oblique illumination
fiber optic for IR LED	Thorlabs	M89L01	Couples to IR LED
Objective lens	Olympus 40X	LUMPlanFLN	This can be used on most microscipes and works well for visualizing fly neurons.
Amber LED	Thorlabs	M590L3d	For visualizing RFP and mCherry
Blue LED	Thorlabs	M470L3d	For visualizing GFP
GFP filter set	Chroma	49011	For visualizing GFP or stimulating channel2rhodopsin
Custom mCherry Filter Set	Chroma	et580/25x and t600lpxr (from the 49306 set) but with an et610lp barrier/emission optic	Use only if you wish to patch identified neurons with channel2rhodopsin
Dichroic to combine Amber and blue LED	Thorlabs	DMLP550R	Use only patch under mCherry and excite channel2rhodopsin with blue light.
Red LED	LEDSupply	Cree XPE 620 - 630 nm	Used to drive csChrimson
LED Driver	LEDSupply	3021-D-E-1000	Used to drive LEDs for optogenetic stimulation
Manipulator	Sutter Instruments	MP-225	Used to position pipette during recordings
Patchclamp Amplifier	A-M Systems	Model 2400	An equivalent amplifier is suitable
Bessel Filter	Warner Instruments	LPF 202A	Auxillary filter used to filter current trace to oscilloscope during patching.
Data Acquisition System	National Instruments	NI PCIe-6321       781044-01	Used to record data from amplifier to computer
Connector Block - BNC Terminal BNC-2090A	National Instruments	779556-01	Used to connect amplifier to DAQ card.
Steel Foil	McMaster Carr	3254K7	Steel foil for custom recording chamber
Magnets	K&J Magnetics	D42	To secure recording chamber to ring stand
1/16" Cell Cast Acrylic Clear	Pololu		Used to make custom recording chamber. Acrylic can be laser cut at pololu.com from the provided PDF file