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1Department of Biomedical Engineering, Washington University in St. Louis
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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.
Keywords: Neuroscience, Issue 71, Neurobiology, Biomedical Engineering, Bioengineering, Physiology, Anatomy, Cellular Biology, Molecular Biology, Entomology, Olfactory Receptor Neurons, Sensory Receptor Cells, Electrophysiology, Olfactory system, extracellular multi-unit recordings, first-order olfactory receptor neurons, second-order projection neurons, third-order Kenyon cells, neurons, sensilla, antenna, locust, Schistocerca Americana, animal model
Saha, D., Leong, K., Katta, N., Raman, B. Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits. J. Vis. Exp. (71), e50139, doi:10.3791/50139 (2013).
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 learning4,5. 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 ORNs6,7. 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.
1. Odor Preparation and Delivery
2. Preparing Locust Antenna for Single Sensillum Recording
3. Single Sensillum Recording to Monitor Odor-evoked Responses from Olfactory Receptor Neurons (ORNs)
4. Locust Dissection Procedure for Antennal Lobe and Mushroom Body Recordings
5. Multi-unit Recordings from the Antennal Lobe and the Mushroom Body
6. Procedures to Make Twisted Wire Electrode for KC Recordings
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 neurons and inhibitory local neurons, only PNs generate sodium spikes that can be detected extracellularly3. This observation suggests that the multi-unit recording technique presented here can be used to selectively monitor the output of the antennal lobe circuits, thereby making locusts an attractive invertebrate model for studying olfactory coding.
An example of a mushroom body recording is shown in Figure 6B. Unlike the ORNs and PNs, the KCs have lower baseline activity and respond to odors in a sparse and selective manner.
To isolate single unit responses from these multi-unit recordings, we performed off-line spike sorting (with the best four channels) using published software implemented in IGOR Pro (Wavemetrics)12. Examples of PN and KC spike sorting are shown in Figure 6C, and D, respectively.
Figure 1. Odor stimulation. (A) All components needed for preparing an odor bottle are shown. (B) The inlet connection from the pico-pump and the outlet connection from the odor bottle to the odor delivery tube are shown. A constant stream of desiccated air is used as the carrier gas stream and is directed at the antenna during experiments.
Figure 2. Preparation of a locust antenna for single sensillum recordings. (A) The locust is placed in a custom made chamber with a ground electrode placed in the gut. (B) A method to stabilize an antenna using a wax platform is shown.
Figure 3. Single sensillum recordings. (A) A typical recording set up. A mixture of carrier gas and odor vapor is supplied through a delivery tube. ORN action potentials are recorded using a glass electrode. Delivered odorants are removed using a vacuum funnel situated right behind the antenna. (B) Electrode placement as seen through the stereomicroscope. Arrows indicate the placement of the glass electrode tip at the base of a sensillum. (C) A schematic of the single sensillum recording approach. (D) Raw extracellular voltage traces showing responses of an ORN to two different odors (2-octanol and 1-hexanol).
Figure 4. Locust dissection procedure. (A) A locust is restrained and positioned in a custom-designed dissection setup as shown. (B) View of the locust head from above. Both compound eyes and antennae can be clearly seen (C) A wax cup is built around the dissection site to allow saline perfusion during and after the dissection process. (D) An exposed locust brain is shown (the yellow-pigmented neural tissue). A platform is placed beneath the brain as shown to stabilize the brain. A saline perfusion tube is attached to the wax cup. (E) A schematic of the locust brain. (F) A magnified image of the locust brain after the dissection clearly showing the regions of interest: antennal lobes (AL) and the mushroom bodies (MB). The antennal nerve (AN) contains axon bundles that transmit the ORN action potentials from the antenna to the antennal lobe.
Figure 5. Multi-unit recordings from the antennal lobe and the mushroom body. (A) A schematic showing the recording configuration and the odor delivery setup. (B) A 16-channel NeuroNexus recording electrode used for PN recordings is shown. (C) Left panel, a custom made 8-channel twisted wire electrode is shown. Right panel, the electrode tip and the connections of wires to the IC socket are shown. (D) Placement of the 16-channel recording electrode in the AL. Only the bottom four electrodes in each shank are inserted into the tissue. (F) Placement of the twisted wire electrode in the superficial MB layers for KC recordings is shown.
Figure 6. Representative results from an antennal lobe (AL) and a mushroom body (MB) recording. (A) A raw extracellular trace from a multi-unit AL recording is shown. A 4 s odor pulse was applied during the time period indicated by the gray box. (B) Similar plot but showing raw KC responses to an odor. (C) An example of PN spike sorting. Extracellular waveforms from four independent channels of a multi-channel electrode are shown for all spiking events arising from a single PN. Individual events (black), mean (red), and SDs (blue) are shown for both cells. Histograms obtained by projecting high-dimensional PN event representations (180 dimensional vector obtained by concatenating 3 ms signals from all four electrodes) onto the line connecting their means. To be considered a well-isolated unit, as in this case, a bimodal distribution with cluster centers at least five times the noise SD apart is expected for every pair of simultaneously recorded cells12. (D) An example of KC spike sorting is shown.
Figure 7. The electroplating set up: The circuit diagram showing connections between the different components are shown above a picture of the actual setup. Briefly, 3 Hz square pulses (5V amplitude) from a function generator (MCP, SG 1639A) are used to gate a stimulus isolator (WPI, A365) that then delivers 5 μA of current to an electrode impedance tester (BAK Electronics, IMP-2). The impedance tester can be operated to either test the electrode impedance or allow current pulses from the stimulus isolator to be applied to the electrode for gold plating. In both cases, the multi-unit electrode is kept immersed in an electroplating well containing gold solution. A switch allows selection of the electrode channel to be gold plated.
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 a number of previous studies on olfactory coding and are becoming a standard practice in this field3,6,13-17. Combining the techniques presented here one can develop a system's approach to investigating the design and computing principles of the invertebrate olfactory system. Here, we must recognize seminal contributions made by Gilles Laurent, Mark Stopfer, and their colleagues2,3,8,9,13,16,18-21, who pioneered these approaches to reveal and elucidate several fundamental principles of olfactory coding.
Finally, it is worth noting that optical techniques have also been successfully used to study ensemble activity in insect olfactory circuits22-27. While these optical techniques are advantageous when the goal is to simultaneously monitor neural activity across a large number of neurons, electrophysiology techniques are still the 'gold standard' when detection of individual action potentials is desired.
No conflicts of interest declared.
The authors would like to thank the following for funding this work: generous start-up funds from the Department of Biomedical Engineering in Washington University, a McDonnell Center for Systems Neuroscience grant, a Office of Naval Research grant (Grant#: N000141210089) to B.R.
|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|
|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|
|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|
|6 ml syringe||Kendall||1180600777||for custom designed activated carbon filter|
|Brown odor bottles||Fisher||08-912-165|
|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|
|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|
|Dental periphery Wax||Henry-Schein Dental||6652151|
|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|
|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|
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