Biotelemetry system experimental setup
We developed a wireless biologging system that enables the monitoring of extracellular neuronal signals from the brains of free-swimming trout. Figure 1a shows the experimental setup for the simultaneous monitoring of neuronal activity and swimming trajectory. We used an array of extracellular tetrodes to record extracellular neural activities. This device is an electrode consisting of four twisted microwires; it has become a standard electrode widely used to simultaneously record multiple single-neuronal activities in rodent brains [12]. Extracellular neuronal signals were stored on an SD card using a commercially available neurologger (Mouselog-16B, Deuteron Technologies, Jerusalem, Israel) placed in a custom-made, 3D-printed, waterproof case (Fig. 1b). Using dedicated control software for MouseLog-16, we controlled the neurologger via a transceiver that transmitted radio signals on the 915-MHz frequency band. The neurologger also recorded external trigger signals to identify where the fish was located in the water tank when the action potential occurred from the neuron. These signals were recorded for each frame captured by a USB 3.0 video camera (ace acA3088-57uc, Basler AG, Ahrensburg, Germany) mounted 1.5 m above the water tank and transmitted from a personal computer via a radio link. As was the case in the previous study, the radio link was available within a few tens of centimeters below the water surface. The details are described in the following subsections.
Animals
The three fish (rainbow trout [Oncorhynchus mykiss]; body weights: 1,548–2,230 g) used in the experiment were purchased from the Fuji Trout farm in Fujinomiya City, Shizuoka Prefecture, Japan, where they were fed commercial pellets once a day. To facilitate the neurologger attachment, we conducted our experiments at the Fuji Nature Education Center of Nihon University. We chose our experimental fish without discriminating for sex. After purchase, they were transported by car to the Education Center, where they were acclimated in spring water at approximately 9 °C until required for experimentation.
The fish were not fed during the experiment, and all procedures were approved by the University of Tokyo Institutional Animal Care and Use Committee.
Surgery
The experimental fish were immersed in an anesthetic solution, prepared by adjusting FA 100 (eugenol; Tanabe Seiyaku Co. Ltd, Osaka, Japan) to a concentration of 0.5 mL/L, for 5–10 min, until their gill lids ceased moving. When the experimental fish were immobilized, they were fixed to an acrylic apparatus on a specific mounting table to facilitate the electrode implantation and neurologger attachment (Fig. 2a). The anesthesia, prepared with FA 100 at a concentration of 0.25 mL/L, was refluxed from the mouth to the gills by tube, using a peristaltic pump for circulation. The fish back was covered with a towel, kept wet during the experiment by pumping water onto it to prevent the body from drying. During the experiment, a bag of ice was added to the water tank every 30 min to prevent sudden increases in water temperature, maintaining it at approximately 9 °C.
With the help of an imaginary line connecting the back of the eyes as a starting point, a rectangular portion of the epidermis was removed with a scalpel for a length of 3–4 cm toward the caudal fin. A micro-drill was then used to prepare an oval hole in the exposed skull, followed by a meticulous procedure intended to avoid damage to the brain. After removing the excised skull, fat and tissue fluid were gently extracted with a Kimwipe (Kimtech® Science™ Kimwipes™) to expose the olfactory bulb, telencephalon, and optic tectum.
For the anchor screw, a maximum of 12 holes equally spaced were drilled along the edge of the oval hole. The number of holes was determined on a subject-by-subject basis to avoid scattered cartilaginous tissues that mainly reside in the rostral area. After the anchor was screwed into the holes, the sites were covered with dental acrylic resin (Unifast III, GC Inc., Japan). The ground wire was implanted into the medial optic tectum. The tetrode array described below was implanted into the targeted recording locations of the telencephalon with micromanipulators (SM-15L or SMM-200, Narishige Inc., Japan, respectively) (Fig. 2b). Since the brain depth was approximately 3–4 cm below the skin, the precise coordination of electrode locations was performed on a case-by-case basis.
The space above the brain was filled with Vaseline, while the space between the head and guide tubes was filled with small, 3D-printed pieces and then covered with dental acrylic resin. Finally, the neurologger case was attached to the electrodes, fixed, and covered with dental acrylic resin (Fig. 2c). All operations were completed within 90 min. After surgery, the fish were immediately moved to an outdoor experimental tank (145 cm × 105 cm) and acclimated until they were observed to swim normally.
Electrode fabrication
Based on the experience acquired in rodent studies [10, 11], we used tetrodes containing four tungsten microwires (12.5 μm, HML-coated, California Fine Wire, CA, USA), twisted and bundled with a heat gun, to record extracellular neuronal activity. Each tetrode was inserted into a polyimide tube (inner diameter: 0.04 mm, outer diameter: 0.20 mm). The tube array was constructed by gluing the tubes in a concentric circle arrangement, with a distance of approximately 0.2 mm between them (Fig. 3a). To stabilize the electrode drift, a flexible electrode tip (approximately 0.5 cm) was exposed from the tubes to absorb the torque caused by the trout’s powerful and fast swimming. Each microwire within a tetrode was crimped to the corresponding hole in the electrode interface board (EIB) using a gold pin (Fig. 3b, c). Immediately before surgery, the tip of the tetrode was cut at a right angle. The impedance was approximately 600 kΩ at a frequency of 1 kHz. A ground electrode, an insulated stainless-steel wire (diameter: 0.2 mm) implanted into the medial optic tectum, also served as a reference point for neuronal recordings (Fig. 3c, green wire).
Waterproof case
We manufactured a waterproof neurologger case using a pioneering study on goldfish as a reference [8]. The base, lid, and tetrode array housing were fabricated using a 3D printer (Fig. 3h, j) (Form 2 or 3, Formlabs, MA, USA). The base and tetrode array housing were designed to mesh with each other, and the tetrode array was connected to the neurologger through a hole in the base. The slight gap between the base and the socket was filled with an ultraviolet-curable adhesive (Bondic®, Bondic Co., NY, USA) (Fig. 4a, arrow). After the base and housing were covered with dental acrylic resin, the lid with a rubber seal (chloroprene, thickness: 2 mm) (Fig. 3k) was placed on the base and tightened using four screws (Figs. 3l, 4d). For logger protection against high torque, we added a lid, in addition to the base and supports, to close the case. The outer dimensions of the waterproof case were 5 cm × 5 cm × 3 cm. The total weight of the case was approximately 17 g. Before the initial recording session, the neurologger and battery were placed in the waterproof case, and the anesthetized fish was fixed to the mounting table (Fig. 4b, c).
Neurologger
Before the extracellular neuronal signals were stored in an SD card, they were unity-gain buffered, digitized, and continuously sampled at 31.25 kHz with an RHA2000 chip (Intan Technologies, CA, USA) in the MouseLog-16B, operating in either wide- (1–7000 Hz) or narrow-band (300–7000 Hz) mode. We simultaneously recorded up to four tetrodes (16 channels) for over one hour with a battery (3.7 V, 170 mAh). The weight of the neurologger, including the battery, was approximately 6 g. We used a magnetic switch to turn on the neurologger immediately before each recording session to reduce battery consumption.
Offline preprocessing
After the neuronal signals were downloaded from the SD card, action potential (spike) data were digitally filtered at 800–7500 Hz. The tetrode recording contained multiple single-neuronal activities. The spikes were isolated using spike-sorting software (KlustaKwik, open-source software by Harris Lab, UCL, London, UK, and available from https://sourceforge.net/projects/klustakwik) and manually verified [13] to extract individual neuronal activity. Cells with ≤ 99 spikes were excluded from the analysis. Cell type classification using the extracellular spike waveform feature was not performed, as the necessary criteria have not been established for fish neurobiology. The unit isolation quality was quantified for each cell based on the isolation distance index [14]. An example of the spike-sorting performance quality is shown in Fig. 5.
Video tracking
We used DeepLabCut™ (Mathis Lab, Cambridge, MA, USA) [15] to track the leading edge and fixed base of the 3D-printed neurologger case mounted on the skull through imagery captured at 30 frames per second. We used a USB 3.0 digital video camera with a non-distorting lens (C-Mount, Manual Iris, Wide Angle Lens, # 89–524, Edmund Optics, Japan) mounted 1.5 m above the water tank (Fig. 1a); then, by concatenating the tracked fixed base of the case, we were able to reconstruct swimming trajectories (Fig. 7a). The image definition was set to 800 × 800 pixels. We computed the head direction from the tracked leading edge and fixed base using the inverse of the tangent function.
Head direction cell analysis
The directional tuning function for each cell was obtained by plotting its firing rate as a function of the fish head direction, divided into bins of 0.5°, and smoothed using a 14.5° mean window filter.
The directional tuning strength was estimated by computing the mean vector length for the circular distribution of the firing rate. Head direction-modulated cells were defined as cells in the recorded data with mean vector lengths > 95th percentile of the shuffled data. For each permutation trial, the entire sequence of spikes fired by a cell was time-shifted along the fish swimming path with a random interval between 20 s and 20 s less than the trial length, with the end of the trial wrapped to the beginning. A head-direction-tuning function was then constructed, and the mean vector length was calculated. This procedure was repeated 100 times for each cell, the mean vector length distribution was computed for the entire set of permutations from all examined cells, and the 95th percentile was determined.
Analysis software
All analyses were performed using custom-made programs based on MATLAB functions (v9.6; MathWorks, Natick, MA, USA).
Histology
The fish were deeply anesthetized with 0.5 mL/L FA 100 and then transcardially perfused with 10% phosphate-buffered formalin fixative (3.5–3.8% formaldehyde). The extracted brains were post-fixed overnight with Davidson’s fixative (also known as Hartmann’s fixative) solution (22.2 mL 10% buffered formalin, 32.0 mL 99% ethanol, 11.1 mL acetic acid, and 100 mL distilled water) at 4 ℃. The brains were incubated in gelatin solution (10% gelatin in phosphate-buffered saline [PBS]) at 37 °C for 4 h and solidified at 4 °C. The brain embedded in the gelatin block was fixed in 10% phosphate-buffered formalin fixative (3.5–3.8% formaldehyde) at 4 °C and sank in 30% sucrose in PBS at 4 °C. The brains were cut coronally with a microtome (Ritratome REM-710, Yamato Koki Co., Saitama, Japan) set at 40 µm. The resulting brain slices were then stained with cresyl violet (Sigma-Aldrich, C5042-10G) to facilitate the examination of their cytoarchitecture.