Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Summary

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Abstract

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time¹. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)^2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning^4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.

First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs^6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode³. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings⁸. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Protocol

1. Odor Preparation and Delivery

1. Dilute odor solutions in mineral oil by volume to achieve the desired concentration level. Store a 20 ml mixture of mineral oil and the odorant in a 60 ml glass bottle. Insert two syringe needles into a rubber stopper (gauge 19), one from the bottom and the other from the top, to provide an inlet and an outlet line. Seal the glass bottle with this rubber stopper and attach a custom designed activated carbon filter to the inlet line (Figure 1A).
2. The carbon filter is made using two 6 ml syringes. Cut the syringes in half and discard the plunger end. Fill each of the remaining pieces with cotton and ....

Results

Odor-evoked responses of a single ORN to two different alcohols are shown in the Figure 3D. Depending on the recording location (sensilla type, placement of the electrode) multi-unit recordings can be achieved.

A raw extracellular waveform from an AL recording is shown in Figure 6A. Action potentials or spikes of varying amplitudes originating from different PNs can be observed in this voltage trace. Although the locust antennal lobe has excitatory projection .......

Discussion

Most sensory stimuli evoke combinatorial responses that are distributed across ensembles of neurons. Hence, simultaneous monitoring of multi-neuron activity is necessary to understand how stimulus-specific information is represented and processed by neural circuits in the brain. Here, we have demonstrated extracellular multi-unit recording techniques to characterize odor-evoked responses at the first three processing centers along the insect olfactory pathway. We note that the techniques presented here have been used in .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Electrophysiology Equipment
A.C. amplifier	GRASS	Model P55	for single sensillum recordings
Audio monitor (model 3300)	A-M Systems	940000
Custom-made 16 channel pre-amplifier and amplifier	Cal. Tech. Biology Electronics Shop		for AL and MB recordings
Data acquisition unit	National Instruments	BNC-2090
Fiber optic light	WPI	SI-72-8
Light source 115 V	WPI	NOVA
Manual micromanipulator	WPI	M3301R	for locust brain recordings
Stereomicroscope1 on boom stand	Leica	M80	for locust brain recordings
Stereomicroscope2	Leica	M205C	for single sensillum recordings
Vibration-isolation table	TMC	63-500 series
Motorized micromanipulator	Sutter Instruments	MP285/T
Oscilloscope	Tektronix	TD2014B
Electrodes/Construction Tools
16-channel electrode	NeuroNexus	A2x2-tet-3mm-150-121	for antennal lobe recordings
Borosilicate capillary tubes with filament, ID 0.69 mm	Sutter Instruments	BF120-69-10	for making glass electrodes
Micropipette puller	Sutter Instruments	P-1000
Function generator	Multimeter Warehouse	SG1639A	for gold-plating electrodes
Gold plating solution (non cyanide)	SIFCO Industries	NC SPS 5355
Impedance tester	BAK Electronics Inc.	IMP-2	for gold-plating electrodes
Switch rotary	Electroswitch	C7D0123N	for gold-plating electrodes
Pulse isolator	WPI	A365	for gold-plating electrodes
Q series electrode holder	Warner Instruments	64-1091
Silver wire 0.010" diameter	A-M Systems	782500	ground electrode
8 pin DIP IC socket	Digikey	ED90032-ND
Borosilicate capillary tubes with filament, ID 0.58 mm	Warner Instruments	64-0787
Heat gun	Weller	6966C
Rediohm-800 wire	Kanthal Precision Technologies	PF002005
Titer plate shaker	Thermo Scientific	4625Q	twisting wires
Carbide scissors, 4.5"	Biomedical Research Instr	25-1000	for cutting twisted tetrode wires
Fine point tweezers	HECO	91-EF5-SA	for teasing tetrode wires apart
Odor Delivery
6 ml syringe	Kendall	1180600777	for custom designed activated carbon filter
Brown odor bottles	Fisher	08-912-165
Charcoal	BuyActivatedCharcoal.com	GAC-48C
Desiccant	Drierite	23005
Drierite gas drying jar	Fischer Scientific	09-204
Heat shrink tubing	3M	EPS-200	odor filter preparation
Hypodermic needle aluminum hub, gauge 19	Kendall	8881-200136	for providing inlet and outlet lines for odor bottles
Mineral oil	Mallinckrodt Chemicals	6357-04	for odor dilution
Nalgene plastic tubing, 890 FEP	Thermo Scientific	8050-0310	for carrier gas delivery
Pneumatic picopump	WPI	sys-pv820	for odor delivery
Polyethylene tubing ID 0.86 mm	Intramedic	427421	for odor bottle outlet connections and saline profusion tubing
Stoppers	Lab Pure	97041	for sealing odor bottles
Time tape	PDC	T-534-RP
Tubing luer	Cole-Parmer	30600-66
Vacuum tube	McMaster-Carr	5488K66
Preparation/Dissection
100 x 15 mm petri dish	VWR International	89000-304
18 AWG copper stranded wire	Lapp Kabel	4510013
22 AWG stranded hookup wire	AlphaWire	1551	brain platform
Batik wax	Jacquard	7946000
Dental periphery Wax	Henry-Schein Dental	6652151
Electrowaxer	Almore International	66000
Epoxy, 5 min	Permatex	84101
Hypodermic needle aluminum hub	Kendall	8881-200136
Protease from Streptomyces griseus	Sigma-Aldrich	P5147	for desheathing locust brain
Suture thread non-sterile	Fisher	NC9087024	for tying the abdomen after gut removal
Vetbond	3M	1469SB	for sealing amputation sites
Dumont #1 forceps (coarse)	WPI	500335
Dumont #5 titanium forceps (fine)	WPI	14096
Dumont #5SF forceps (super-fine)	WPI	500085	desheathing locust brain
10 cm dissecting scissors	WPI	14393	for removing legs and wings
Vannas scissors (fine)	WPI	500086	for removing cuticle, cutting the foregut
Saline Profusion
Extension set with rate flow regulator	Moore Medical	69136	for regulating saline flow
IV administration set with Y injection site	Moore Medical	73190	for regulating saline flow