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A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.
The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.
Fish are the largest and most diverse group of vertebrates, and like other vertebrates they exhibit complex cognitive abilities such as navigating, socializing, sleeping, hunting, etc. Nevertheless, the neural mechanisms governing fish behavior remain for the most part unknown.
In the past few decades, extracellular recordings from immobilized fish have primarily been implemented to investigate different aspects of the neural basis of behavior1,2. Although this technique is appropriate for some sensory systems, investigation of the full spectrum of the neural basis of behavior is difficult if not impossible in immobilized animals. The first advances involved recording from the Mauthner cells of tethered swimming fish3,4. However, Mauthner cells are disproportionately large and the recorded action potential amplitudes, which can go as high as a few mV, facilitate recording. Later, Canfield et al. described a proof of concept when using a tethered animal to record from the telencephalon of fish5. Another recent technique for recording neural activity from fish is calcium imaging (see reviews by Orger and de Polavieja6, and Vanwalleghem et al.7). This technique was developed for use with zebrafish larvae because the skin and skull are transparent during the larval stage. However, this technique cannot be used to study complex behaviors in later stages of development.
Here, we present a novel technique for recording extracellular neural activity from the brains of freely swimming fish. This is a modified version of the protocol described in Vinepinsky et al.8. The main innovation is the addition of a microdrive that makes it possible to control the position of the electrodes after surgery. The technique is designed for recording from the telencephalon of goldfish using a set of tetrodes that are connected to a neural data logger via a microdrive. The whole setup is wireless and anchored to the fish's skull. The specific weight of the system is equalized to the water-specific weight by adding a small float that allows the fish to swim freely.
The technique is based on the use of a neural data logger that amplifies, digitizes, and stores the signal in an onboard memory device. The logger telemetry system is used to start and stop the recordings, and for synchronization with the video camera. In this protocol, a 16-channel neural logger is used, embedded in a waterproof box together with the microdrive.
The microdrive assembly is fabricated from two main components: the microdrive itself and the microdrive housing (Figure 1A,B). The housing holds the microdrive and the tetrodes, and also acts as the anchor between the skull and the logger box (Figure 1C). The PVC logger box is fabricated using a machine process and is sealed using an O-ring (Figure 1E-G, see also Supplementary Figure 1, Supplementary Figure 2, and Supplementary Figure 3 for a three-dimensional [3D] diagram). At one end, a piece of polystyrene foam is attached to the logger box to compensate for the weight of the implant and provide the fish with a buoyancy-neutral implant. The construction of the microdrive described in the protocol follows the procedure presented by Vandecasteele et al.9 with a modification to attach the microdrive to the housing (Figure 1A). All major steps are presented.
The procedure described in the protocol to prepare the fish skull is similar to the one presented in Vinepinsky et al.8 and is described briefly in the protocol. One day after surgery, the fish are normally fully recovered from the effects of anesthesia and are ready for the behavioral experiments. Note that the tetrode location can be adjusted by turning the microdrive screw. The screw has a spacing of 300 µm per full rotation and an advancement of 75 µm is recommended until the target brain location is reached. An appropriate brain atlas should be consulted to target the specific brain region of interest. It is advisable to test the electrode impedance each time the fish is anesthetized for battery or memory card replacement.
All surgery procedures must be approved by the local ethics committees on animal welfare (e.g., IACUC).
1. Construction of the Microdrive Housing
2. Construction of the Microdrive
3. Preparing the Tetrode Array
4. Assembling the Implant
5. Anesthesia Preparation — 1% MS-222 Stock Solution
CAUTION: Anesthesia preparation includes the use of powdered MS-222, a carcinogen. Hence, steps 5.2 and 5.3 must be done in a chemical hood using gloves.
6. Preparing the Fish Skull
NOTE: At this stage, the fish is ready for implant surgery. Prior to surgery, make sure that all components and supplies have been sterilized by the appropriate procedures. For this step, an out of water U-shaped fish holder is needed. In this protocol, an aluminum holder that fits a 15 cm head to tail long goldfish is used. This system holds the fish out of water while perfusing the gills with oxygenated water. For details see Vinepinsky et al.8.
7. Implanting the Probe
NOTE: To complete the final step in the protocol, a manipulator that can hold the implant in place while it is inserted into the brain is needed.
During a recording session the goldfish swam freely in a square water tank while the neural activity in its telencephalon was recorded. The goal of these experiments was to study how the neural activity of single cells determines the fish's behavior. To do so, spiking activity needed to be identified in the recorded data. The brain activity, while being recorded, was digitized at 31,250 Hz and high-pass filtered at 300 Hz by the data logger. Then, offline, a band-pass filter (300ͨ...
This protocol details the steps involved in implanting a tetrode array into the telencephalon of freely swimming goldfish. This technique implements a neural logger that amplifies and records the signals acquired from up to 16 channels along with a microdrive that can adjust the tetrode position in the brain. The microdrive makes it possible to adjust the position in the brain to optimize the recording.
This protocol can easily be modified for recording from other brain regions (see Vinepinsky...
The authors have nothing to disclose.
We are grateful to Nachum Ulanovsky and the members of the Ulanovsky lab for all their help. In addition, we are grateful to Tal Novoplansky-Tzur for helpful technical assistance. We gratefully acknowledge financial support from THE ISRAEL SCIENCE FOUNDATION - FIRST Program (grant no. 281/15), and the Helmsley Charitable Trust through the Agricultural, Biological and Cognitive Robotics Initiative of Ben-Gurion University of the Negev.
Name | Company | Catalog Number | Comments |
0.7 mm round drill bits | Compatible with the drill. | ||
15-blade Scalpel | Sigma-Aldrich | ||
16 channel PCB board | Neurlynx | EIB-16 | |
1X3M phillips flat head screws | Stainless steel. Any type. | ||
1X3M phillips round head screws | Stainless steel. Any type. | ||
27 cm X 19 cm X 1 mm brass plate | See Figure 2 | ||
2X6M phillips flat head screws | Stainless steel. Any type. | ||
3140 RTV coating | Dow Crowning | 2767996 | |
75 µm Silver wire | A-M Systems | ||
Brass machine screws #00-90 | 947-1006 | ||
Brass plates 7.5mm X 2.5mm X 0.6mm | A 3D drawing is provided. See supplementary 1 | ||
Coated Tungsten wire 25µm | California Fine Wire Company | 5000160 | Depending on the appication the tetrodes can be fabricated from any type of wire. Popular wires are nicrome wires that can be found with lower diameters (eg. A-M systems, 762000) |
Coated Tungsten wire 50µm | A-M Systems | 795500 | Can be replaced with any other wire with low impedance |
Cyanoacrilic glue | |||
Dental Burnisher | ComDent UK | Any small sterille stainless-still tool will do. | |
Dental cement - GCFujiPLUS | GC | 431011 | Other dental cements would probably will work as well although we have never tried any other. |
Dental drill or nail polish drill | Dental drills are expensive, a nail polish drill can be a cheap replacement. | ||
Drill bit #65 | 947-65 | ||
Fast curing epoxy | Any 5 minutes curing epoxy can be used here. | ||
Logger box with O-ring sealing | A 3D drawing is provided. See supplementary 1-3. The box should be machine fabricated (do not use 3D printers). Use transperant material, to be able to see the indicator LEDs on the logger. | ||
Motorized turning device | Custom made as described in "open ephys" website. Can also be purchusaed from neurolynx ("Tetrode Spinner 2.0") or bulit by other means. | ||
Mouselog-16 Neural logger | Deuteron Technologies Ltd | There are several neural loggers available on the market, including: SpikeGadget (UH32 32channels) and Neurologger 2/2A/2B of Alexei Vyssotski. It should be noted that weight is not a major contraint since it can be counterbalanced with floating Styrofoam | |
MS-222 | Sigma Aldrich | E10521 | Ethtl 3-aminobenzoate methanesulfonate 98% |
Nano-Z plating | White Matter LLC | The nano-Z can be bought from several supllieres. Any impedance meter can be used, e.g. IMP-1 / 6662 / 2788, BAK Electronics. | |
PCB pins | Neurlynx | Neuralynx EIB Pins | |
Polymide tubing 250µm | A-M Systems | 822000 | |
Rechargable battery | 3.7 Lipo battery, 370 mAh. Holds about 6 hours of recording. Smaller or larger battries can be used to reduce the weight or extend recording time. | ||
Silicone tubing 0.64 mm | A-M Systems | 806100 | |
Stainless steel 1.5 mm | A-M Systems | 846000 | |
Sudium Bicarbonate | Sigma Aldrich | S9625 | |
Tap #00-90 | 947-1301 | ||
Vaseline | Any type of soft petroleum skin protectant can be used here. |
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