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Wireless logging of extracellular neuronal activity in the telencephalon of free-swimming salmonids

Wireless logging of extracellular neuronal activity in the telencephalon of free-swimming salmonids Background: Salmonids return to the river where they were born in a phenomenon known as mother-river migra- tion. The underpinning of migration has been extensively examined, particularly regarding the behavioral correlations of external environmental cues such as the scent of the mother-river and geomagnetic compass. However, neuronal underpinning remains elusive, as there have been no biologging techniques suited to monitor neuronal activity in the brain of large free-swimming fish. In this study, we developed a wireless biologging system to record extracellular neuronal activity in the brains of free-swimming salmonids. Results: Using this system, we recorded multiple neuronal activities from the telencephalon of trout swimming in a rectangular water tank. As proof of principle, we examined the activity statistics for extracellular spike waveforms and timing. We found cells firing maximally in response to a specific head direction, similar to the head direction cells found in the rodent brain. The results of our study suggest that the recorded signals originate from neurons. Conclusions: We anticipate that our biologging system will facilitate a more detailed investigation into the neural underpinning of fish movement using internally generated information, including responses to external cues. Keywords: Fish biotelemetry, Extracellular neuronal recording, Salmonids Background locations, and cells that fire maximally in response to Some fish, birds, and mammals exhibit outstanding navi - specific heading directions [3]. These internally gener - gational abilities, such as mother-river homing, seem- ated maps and compasses are thought to contribute to ingly possessing cognitive maps. Current self-location spatial navigation [4]. and compass bearings are required for such spatial navi- Until recently, there have been few reports of space- gation. In mammals, extracellular electrophysiology for responsive cells in fish brains. This lack of information freely navigating animals has led to the discovery of a is mainly due to the difficulties of underwater neuronal variety of space-responsive cells, including place cells recording. Zebrafish, a small, laboratory-bred, and non- [1] and grid cells [2], which fire maximally at specific migrant fish widely used as an animal model in neuro - science, can be genetically modified to record neuronal activity through optical imaging of intracellular calcium *Correspondence: stakahas@mail.doshisha.ac.jp; yuya.makiguchi@gmail.com dynamics. However, head clamping and genetic engi- Laboratory of Cognitive and Behavioral Neuroscience, Graduate School neering are prerequisites for such neuronal recordings. of Brain Science, Doshisha University, Kyotanabe City, Kyoto 610-0394, Japan While state-of-the-art technology has enabled us to College of Bioresource Sciences, Nihon University, Kanagawa 252-0813, image neuronal activity in the brains of free-swimming Japan Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Takahashi et al. Anim Biotelemetry (2021) 9:9 Page 2 of 12 zebrafish, they must be kept in a small restricted area under a microscope [5]. Therefore, the neural underpinning of fish movement cannot be examined in the field using the aforemen - tioned methods. For medium-sized fish, tethering cables are a prerequisite for recording extracellular neuronal activity from the brain; however, the apparatus generates significant torque in the water, severely inhibiting vol - untary movement. Despite such restrictive limitations, there have been reports of the presence of space-respon- sive cells in the brains of non-migrant fish [6]. Recent remarkable advances in microelectromechani- cal systems have led to the rapid evolution of biologging techniques. For instance, a lightweight logger for extra- cellular neuronal activity, called a neurologger, helped to discover place cells and head direction cells in the brains of flying bats [7]. Furthermore, a neurologger stored in a waterproof case [8] led to the discovery of neurons in the telencephalon of goldfish (Carassius auratus), whose activity was found to be correlated with swimming speed and a specific heading vector as they swam along a wall [9]. Theoretically, this method, pioneered on goldfish, could be applied to larger fish weighing several kilograms. However, the electrode implantation procedure devel- oped for medium-sized goldfish is not suitable for larger fish, as their brains are located deeper beneath the head surface. For example, the trout brain is located 3–4  cm Fig. 1 Experimental setup for the wireless neurologging system. a from the head surface, whereas the goldfish brain lies Head-mounted neurologger in a waterproof case records multiple only a few millimeters underneath [9]. In addition, pow- neuronal activities from the telencephalon of a free-swimming trout in a water tank. The recording parameters, including start erful (weight: approximately 2  kg trout vs. 100  g gold- and stop times, and the neuronal signal frequency bands were fish) and fast (average speed of approximately 20  cm/s) wirelessly controlled from a personal computer via a radio transceiver. swimming may affect logger protection and electrode Two-dimensional swimming trajectories were captured using a USB stabilization. 3.0 camera mounted 1.5 m above the tank. The frame timings were In the present study, combining the pioneering wireless also transmitted to the logger via the radio link (left, blue box) to synchronize the spike timing. Two pipes for water supply (top) and logging study developed in goldfish [8] with our extracel - drainage (bottom) are illustrated. b Free-swimming trout with and lular recording techniques for rodents [10, 11], we devel- without loggers. The logger was placed in a case with the base fixed oped a system for recording space-responsive cells in the to the skull, while for waterproofing, a rubber seal inserted between brains of large migrant fish. To verify the system validity, the lid and base was screwed into place. The logger recorded we recorded multiple neuronal activities from the brains neuronal activity from four tetrodes implanted in the brains of the test fish of trout swimming freely in a rectangular tank. Then, we examined the correlations between swimming behaviors and single neuron firing rates. of four twisted microwires; it has become a standard electrode widely used to simultaneously record multiple Methods single-neuronal activities in rodent brains [12]. Extracel- Biotelemetry system experimental setup lular neuronal signals were stored on an SD card using We developed a wireless biologging system that enables a commercially available neurologger (Mouselog-16B, the monitoring of extracellular neuronal signals from Deuteron Technologies, Jerusalem, Israel) placed in a the brains of free-swimming trout. Figure  1a shows the custom-made, 3D-printed, waterproof case (Fig.  1b). experimental setup for the simultaneous monitoring Using dedicated control software for MouseLog-16, we of neuronal activity and swimming trajectory. We used controlled the neurologger via a transceiver that trans- an array of extracellular tetrodes to record extracellular mitted radio signals on the 915-MHz frequency band. neural activities. This device is an electrode consisting T akahashi et al. Anim Biotelemetry (2021) 9:9 Page 3 of 12 The neurologger also recorded external trigger signals skull, fat and tissue fluid were gently extracted with a ® ™ ™ to identify where the fish was located in the water tank Kimwipe (Kimtech Science Kimwipes ) to expose when the action potential occurred from the neuron. the olfactory bulb, telencephalon, and optic tectum. These signals were recorded for each frame captured by For the anchor screw, a maximum of 12 holes equally a USB 3.0 video camera (ace acA3088-57uc, Basler AG, spaced were drilled along the edge of the oval hole. Ahrensburg,  Germany) mounted 1.5  m above the water The number of holes was determined on a subject-by- tank and transmitted from a personal computer via subject basis to avoid scattered cartilaginous tissues a radio link. As was the case in the previous study, the that mainly reside in the rostral area. After the anchor radio link was available within a few tens of centimeters was screwed into the holes, the sites were covered with below the water surface. The details are described in the dental acrylic resin (Unifast III, GC Inc., Japan). The following subsections. ground wire was implanted into the medial optic tec- tum. The tetrode array described below was implanted into the targeted recording locations of the telencepha- Animals lon with micromanipulators (SM-15L or SMM-200, The three fish (rainbow trout [Oncorhynchus mykiss]; Narishige Inc., Japan, respectively) (Fig.  2b). Since the body weights: 1,548–2,230  g) used in the experiment brain depth was approximately 3–4 cm below the skin, were purchased from the Fuji Trout farm in Fujinomiya the precise coordination of electrode locations was per- City, Shizuoka Prefecture, Japan, where they were fed formed on a case-by-case basis. commercial pellets once a day. To facilitate the neu- The space above the brain was filled with Vaseline, rologger attachment, we conducted our experiments at while the space between the head and guide tubes the Fuji Nature Education Center of Nihon University. was filled with small, 3D-printed pieces and then cov - We chose our experimental fish without discriminat - ered with dental acrylic resin. Finally, the neurologger ing for sex. After purchase, they were transported by case was attached to the electrodes, fixed, and cov - car to the Education Center, where they were acclimated ered with dental acrylic resin (Fig.  2c). All operations in spring water at approximately 9  °C until required for were completed within 90  min. After surgery, the fish experimentation. were immediately moved to an outdoor experimental The fish were not fed during the experiment, and all tank (145 cm × 105 cm) and acclimated until they were procedures were approved by the University of Tokyo observed to swim normally. Institutional Animal Care and Use Committee. Surgery Electrode fabrication The experimental fish were immersed in an anesthetic Based on the experience acquired in rodent studies solution, prepared by adjusting FA 100 (eugenol; Tan- [10, 11], we used tetrodes containing four tungsten abe Seiyaku Co. Ltd, Osaka, Japan) to a concentration of microwires (12.5  μm, HML-coated, California Fine 0.5  mL/L, for 5–10  min, until their gill lids ceased mov- Wire, CA, USA), twisted and bundled with a heat gun, ing. When the experimental fish were immobilized, they to record extracellular neuronal activity. Each tetrode were fixed to an acrylic apparatus on a specific mount - was inserted into a polyimide tube (inner diameter: ing table to facilitate the electrode implantation and 0.04 mm, outer diameter: 0.20 mm). The tube array was neurologger attachment (Fig.  2a). The anesthesia, pre - constructed by gluing the tubes in a concentric circle pared with FA 100 at a concentration of 0.25  mL/L, was arrangement, with a distance of approximately 0.2  mm refluxed from the mouth to the gills by tube, using a peri - between them (Fig.  3a). To stabilize the electrode drift, staltic pump for circulation. The fish back was covered a flexible electrode tip (approximately 0.5  cm) was with a towel, kept wet during the experiment by pump- exposed from the tubes to absorb the torque caused by ing water onto it to prevent the body from drying. During the trout’s powerful and fast swimming. Each microw- the experiment, a bag of ice was added to the water tank ire within a tetrode was crimped to the corresponding every 30  min to prevent sudden increases in water tem- hole in the electrode interface board (EIB) using a gold perature, maintaining it at approximately 9 °C. pin (Fig.  3b, c). Immediately before surgery, the tip of With the help of an imaginary line connecting the the tetrode was cut at a right angle. The impedance back of the eyes as a starting point, a rectangular por- was approximately 600 kΩ at a frequency of 1  kHz. tion of the epidermis was removed with a scalpel for a A ground electrode, an insulated stainless-steel wire length of 3–4  cm toward the caudal fin. A micro-drill (diameter: 0.2  mm) implanted into the medial optic was then used to prepare an oval hole in the exposed tectum, also served as a reference point for neuronal skull, followed by a meticulous procedure intended to recordings (Fig. 3c, green wire). avoid damage to the brain. After removing the excised Takahashi et al. Anim Biotelemetry (2021) 9:9 Page 4 of 12 Fig. 2 Surgery for tetrode implantation and case fixation. a To implant the tetrodes into the brain, each anesthetized fish was fixed to an acrylic fixture on a custom-made mounting table (arrow). The anesthetic solution was circulated using a peristaltic pump. The fish back was covered with a wet towel (pink) to prevent the body from drying, and water was poured onto it with a small pump during the experiments. A bag of ice was added to the water tank every 30 min during the experiments to prevent sudden water temperature increases. b Oval hole drilled into the exposed fish skull (white arrow). A scalpel was used to excise a rectangle of epidermis in the direction of the caudal fin, starting from an imaginary guideline between each fish’s eyes. To expose the olfactory bulb, the telencephalon, optic tectum, fat, and tissue fluid were carefully removed. Anchors were screwed into holes equally spaced along the edge of the oval hole and covered with dental acrylic resin. c A ground wire and tetrode array were implanted into the medial optic tectum and target recording locations of the telencephalon using micromanipulators. The space above the brain was then filled with Vaseline, while the space between the head and electrode interface board (EIB) was filled with small, 3D-printed pieces and then covered with a dental acrylic resin. The neurologger case was attached to the EIB and covered with dental acrylic resin (See figure on next page.) Fig. 3 Electrode array and waterproof case for neurologger. a An array of tetrodes; each tetrode was inserted into a polyimide tube, and the tubes were glued together to construct an array with constant internal spacing. b Housing for connecting the tetrode array to the custom-made electrode interface board (EIB). c Each tetrode microwire was crimped into a corresponding contact hole using a gold pin. d Cover to attach the EIB to the tetrode array. e Tetrode array assembly. f–g Tetrode array with an undercover to connect to the logger case (f). h–j Base (h), support (i), and lid (j) for the custom-made, 3D-printed, waterproof case. k Lid with rubber seal. l The base was attached to the housing and covered with dental acrylic resin (scale bar = 1 cm) T akahashi et al. Anim Biotelemetry (2021) 9:9 Page 5 of 12 Takahashi et al. Anim Biotelemetry (2021) 9:9 Page 6 of 12 Fig. 4 Attaching a logger. a To prevent water ingress, the slight gap between the base and the socket of the neurologger (white arrow) was filled with an ultraviolet-curable adhesive immediately after the electrode was implanted. b–c Each day, before the initial recording session, the neurologger, including its SD card (b) and battery (c), was placed in the waterproof case, while the anesthetized fish was fixed to the mounting table. d After the neurologger and the battery were attached to the base, the lid, complete with its rubber seal, was screwed into the base using four screws Waterproof case Neurologger We manufactured a waterproof neurologger case using Before the extracellular neuronal signals were stored in a pioneering study on goldfish as a reference [8 ]. The an SD card, they were unity-gain buffered, digitized, and base, lid, and tetrode array housing were fabricated continuously sampled at 31.25  kHz with an RHA2000 using a 3D printer (Fig.  3h, j) (Form 2 or 3, Formlabs, chip (Intan Technologies, CA, USA) in the MouseLog- MA, USA). The base and tetrode array housing were 16B, operating in either wide- (1–7000  Hz) or narrow- designed to mesh with each other, and the tetrode band (300–7000 Hz) mode. We simultaneously recorded array was connected to the neurologger through a hole up to four tetrodes (16 channels) for over one hour with a in the base. The slight gap between the base and the battery (3.7 V, 170 mAh). The weight of the neurologger, socket was filled with an ultraviolet-curable adhesive including the battery, was approximately 6  g. We used (Bondic , Bondic Co., NY, USA) (Fig.  4a, arrow). After a magnetic switch to turn on the neurologger imme- the base and housing were covered with dental acrylic diately before each recording session to reduce battery resin, the lid with a rubber seal (chloroprene, thickness: consumption. 2  mm) (Fig.  3k) was placed on the base and tightened using four screws (Figs.  3l, 4d). For logger protection Offline preprocessing against high torque, we added a lid, in addition to the After the neuronal signals were downloaded from the SD base and supports, to close the case. The outer dimen - card, action potential (spike) data were digitally filtered sions of the waterproof case were 5  cm × 5  cm × 3  cm. at 800–7500  Hz. The tetrode recording contained mul - The total weight of the case was approximately 17  g. tiple single-neuronal activities. The spikes were isolated Before the initial recording session, the neurologger using spike-sorting software (KlustaKwik, open-source and battery were placed in the waterproof case, and software by Harris Lab, UCL, London, UK, and avail- the anesthetized fish was fixed to the mounting table able from https ://sourc eforg e.net/proje cts/klust akwik ) (Fig. 4b, c). and manually verified [13] to extract individual neuronal T akahashi et al. Anim Biotelemetry (2021) 9:9 Page 7 of 12 Fig. 5 Histological identification of recording locations in the brain. a Illustrations showing the positions of the brains of trout (top) and goldfish (bottom). Unlike goldfish, the trout brain is located far below the head surface. b Sample coronal section stained to show Nissl material from a trout telencephalon ( Trout B). The arrowheads depict the estimated recording locations. For reference, a mirror image outline of the brain section was drawn on the left. c All estimated locations for the three trout (color-coded dots) are depicted on the brain section outlines (Dm = pars medialis; Dl = pars lateralis, in the dorsal pallium). d Color-coded planes indicate the locations of the coronal brain sections where the recording locations were estimated for the three trout shown in c through imagery captured at 30 frames per second. We activity. Cells with ≤ 99 spikes were excluded from the used a USB 3.0 digital video camera with a non-distorting analysis. Cell type classification using the extracellular lens (C-Mount, Manual Iris, Wide Angle Lens, # 89–524, spike waveform feature was not performed, as the neces- Edmund Optics, Japan) mounted 1.5  m above the water sary criteria have not been established for fish neurobiol - tank (Fig.  1a); then, by concatenating the tracked fixed ogy. The unit isolation quality was quantified for each cell base of the case, we were able to reconstruct swimming based on the isolation distance index [14]. An example of trajectories (Fig.  7a). The image definition was set to the spike-sorting performance quality is shown in Fig. 5. 800 × 800 pixels. We computed the head direction from the tracked leading edge and fixed base using the inverse Video tracking of the tangent function. 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 Takahashi et al. Anim Biotelemetry (2021) 9:9 Page 8 of 12 Fig. 6 Neuronal activities extracellularly recorded from free-swimming trout telencephalons with the wireless neurologging system. a Examples of well-isolated neuronal activities. Spikes recorded from a tetrode were plotted as dots in four selected 2D feature spaces (1st to 3rd principal components [PCs], or energies). Spikes were color-coded to indicate two different neurons. b-d Corresponding superimposed spike waveforms with averaged spike waveforms (solid black line) and spike-timing auto-correlograms [bin widths = 1 ms (c) and 0.1 ms (d)]. The superimposed spike waveform exhibits a signal-to-noise ratio. The spike auto-correlograms, especially those shown in d, indicate that the spike-timing intervals had a 1–2 ms refractory period. e Mean firing rate distributions for the entire suite of neurons observed in the test trout dorsal palliums T akahashi et al. Anim Biotelemetry (2021) 9:9 Page 9 of 12 Fig. 7 Trout head direction cells. a Representative trajectory (gray line) with positions showing neuron-generated spikes (red dots) as recorded from the telencephalon of trout swimming in a water tank (scale bar = 10 cm). Remarkable visible objects (pipe for water supply [black filled rectangle], hole for water sink [black filled circle], and radio transceiver [blue filled rectangle]), and compass bearing (bottom, left) are illustrated. b Polar plots of firing rate as a function of head direction, using examples from two representative neurons. The mean vector length (Mv) and peak firing rate (P) are indicated in the images. Visible objects are illustrated as in a. c–d Distribution of mean vector length for randomly shuffled data (c) and for the entire suite of neurons observed in our test trout dorsal palliums (d). The red line and the number indicate the 95th percentile for the shuffled data. e Circular distribution of the number of cells on the head orientation for seven individual head direction cells identified from trout G and #2. Head direction cells exist in the brains of trout G (red) and #2 (blue). In contrast, trout B had no head direction cells. Visible objects are illustrated as in a Table 1 Identified cells from the telencephalon of trout Trout G #2 B # of isolated cells 3 6 14 # of head direction cells 3 4 0 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 begin- ning. A head-direction-tuning function was then con- structed, 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 th set of permutations from all examined cells, and the 95 percentile was determined. Analysis software All analyses were performed using custom-made pro- grams 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- Head direction cell analysis buffered formalin fixative (3.5–3.8% formaldehyde). The The directional tuning function for each cell was obtained extracted brains were post-fixed overnight with David - by plotting its firing rate as a function of the fish head son’s fixative (also known as Hartmann’s fixative) solution direction, divided into bins of 0.5°, and smoothed using a (22.2  mL 10% buffered formalin, 32.0  mL 99% ethanol, 14.5° mean window filter. 11.1  mL acetic acid, and 100  mL distilled water) at 4 ℃. The directional tuning strength was estimated by com - The brains were incubated in gelatin solution (10% gela - puting the mean vector length for the circular distribu- tin in phosphate-buffered saline [PBS]) at 37  °C for 4  h tion of the firing rate. Head direction-modulated cells and solidified at 4  °C. The brain embedded in the gela - were defined as cells in the recorded data with mean vec - tin block was fixed in 10% phosphate-buffered forma - tor lengths > 95th percentile of the shuffled data. For each lin fixative (3.5–3.8% formaldehyde) at 4  °C and sank in permutation trial, the entire sequence of spikes fired by a ▸ Takahashi et al. Anim Biotelemetry (2021) 9:9 Page 10 of 12 30% sucrose in PBS at 4  °C. The brains were cut coro - found and called head direction cells [3]. Recently, a pio- nally with a microtome (Ritratome REM-710, Yamato neering study also reported the presence of head direc- Koki Co., Saitama, Japan) set at 40  µm. The resulting tion cells in the telencephalon of goldfish [9]. Similarly, brain slices were then stained with cresyl violet (Sigma- the neuronal activity of some cells in the trout telenceph- Aldrich, C5042-10G) to facilitate the examination of their alons exhibited head direction preferences, becoming cytoarchitecture. more active when their heads were oriented in a specific direction (Fig. 7b). Seven (30.4%) of the 23 cells recorded Results passed the criterion to be classified as head direction cells As a proof of principle, we recorded the activity of 23 (the mean vector length exceeded the 95th percentile of cells from the telencephalons of three trout using a wire- the mean vector lengths distribution in shuffled data). less biologging system with a lightweight neurologger This number was significantly larger than expected for a enclosed in a 3D-printed, waterproof case. All recordings random selection from the distribution (P < 0.001, bino- were made while the trout were voluntarily swimming mial test with an expected P of 0.05). Whereas cells in in a rectangular water tank, and the fish did not exhibit one trout did not exhibit head direction preferences, the reward-seeking behaviors during the recording periods. head direction cells were found in the telencephalon of Nissl staining showed that the electrode tracks were dis- two of the three trout (Table 1). The heading orientations tributed around the dorsal pallium of the telencepha- covered a wide span (Rayleigh’s test for nonuniform- lon (Fig.  5). Previous fish studies have reported that the ity, P > 0.05, z = − 4.68; Fig.  7c), suggesting that the head dorsal pallium is deeply involved in spatial learning and direction preference was not due to artifacts. Overall, the memory [9, 16–19]. head direction cells were tuned to neither a specific com - pass bearing nor visible objects, including pipes for water Neuronal activity recorded by the wireless logging system sink and source, and radio transceiver (Fig. 7e). However, To confirm whether the signals recorded from the test at least one cell from trout G or #2 mainly fired whenever trout brains originated from neurons, we examined spike the head pointed south or to the water supply. Further- statistics, including spike shape, firing rate, and spike- more, neighboring head direction cells recorded from the timing intervals. The recorded neurons typically exhib - same electrode exhibited a similar heading trigger (trout ited a narrow spike shape [mean peak-to-trough spike G: two of three neighboring head direction cells; trout #2: width: 0.18 ± 0.11 ms (mean ± SD)]. However, cells show- two of two neighboring head direction cells). ing a wider spike shape were also observed (widest spike width: 0.44  ms). In the telencephalons of rodents, cells Discussion exhibiting a narrow spike shape and high firing rates are We demonstrated the capacity of our novel wireless log- categorized as fast-spiking cells [20]. Although the neces- ging system to record neuronal activity in the brains of sary criteria have not been established for fish neurobiol - large salmonids. A previous pioneering study devel- ogy, these results appeared to show that our biologging oped a wireless logging system for goldfish [8]; however, system could record different neuron types. unlike goldfish, salmonids exhibit remarkable mother- The mean firing rate distributions were skewed river homing on a global scale. While swimming tra- (median: 1.45 Hz, Fig. 6e). The typical spike-timing auto- jectories and accompanying external cues have been correlogram for identified single neurons demonstrated researched previously [21], neuronal activity in the that the spike-timing intervals had a clear refractory brains of free-swimming salmonids has not been stud- period (1–2  ms) (Fig.  6d), suggesting that the identified ied before because of the difficult access to the salmonid neurons belonged physiologically to a single type. These brain, located deep in the head, and the trout weight of results suggest that our system was capable of simultane- several kilograms generating high torque. Thus, our sys - ously recording multiple neuronal activities in the brains tem extends the wireless logging in goldfish, providing of free-swimming trout. the possibility to examine the neuronal underpinning of underwater behavior in large salmonids. Trout head direction cells In a previous study on the telencephalon of goldfish Finally, to demonstrate our logging system capacity to [9], we also found head direction cells in large salmo- examine neuronal correlation with fish movement, we nids. However, we could not investigate whether the head examined the neuron firing rates as a function of fish direction preference was tuned to specific cues, including swimming trajectory and heading (Fig. 7a). In some brain geomagnetic compass bearing and landmarks, because of regions of rodents, including the anterior thalamus, sub- the lack of environmental manipulation. Further studies iculum, retrosplenial cortex, and entorhinal cortex, cells will be required to elucidate the neuronal underpinning that maximally fire at a specific heading direction were of spatial information processing in large salmonids. T akahashi et al. Anim Biotelemetry (2021) 9:9 Page 11 of 12 Availability of data and materials Despite its exceptional potential in the field of ani - The 3D CAD designs for the waterproof case and tetrode housing are available mal biotelemetry, we could not test the ability to record at https ://githu b.com/Takah ashiL ab/Trout . The datasets used and/or analyzed neuronal activity in natural rivers where unpredictable during the current study are available from the corresponding author upon reasonable request. events occur, such as higher water pressure, unexpected rocks, and animal recapture. Therefore, the question Ethics approval and consent to participate of whether our method can be used for mother-river All procedures were approved by the University of Tokyo Institutional Animal Care and Use Committee (#A-19–8). homing remains unanswered. Technical improvements are required to address this question. For instance, the Consent for publication waterproof case square box shape must be formed with Not applicable. a streamlined shape against hydraulic resistance in riv- Competing interests ers. Furthermore, since our system cannot synchronize The authors declare no competing interests. external event signals in deeper water and only record Author details neuronal activity for a few hours, neuronal recordings Laboratory of Cognitive and Behavioral Neuroscience, Graduate School need to be operated automatically and locally above the of Brain Science, Doshisha University, Kyotanabe City, Kyoto 610-0394, Japan. fish’s head. A state-of-the-art AI-assisted biologger [22] Graduate School of Environmental Studies, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan. Department of Cell Biology can accomplish such tasks, detect interest behaviors in and Neuroscience, Graduate School of Medicine, Juntendo University, Bun- real time, and activate sensors such as gyroscopes, accel- kyo-ku, Tokyo 113-8421, Japan. International Coastal Research Center, The eration, and water depth. In a natural environment, it is Atmosphere and Ocean Research Institute, The University of Tokyo, 1–19–8, Akahama, Otsuchi, Iwate 028–1102, Japan. College of Bioresource Sciences, desirable to run this system with an AI-assisted biologger Nihon University, Kanagawa 252-0813, Japan. to start recording neuronal activity at the time of interest. Received: 19 October 2020 Accepted: 27 January 2021 Conclusions In the present study, we developed a wireless logging References system capable of recording multiple neuronal activities 1. O’Keefe J, Nadel L. The hippocampus as a cognitive map. New York: from the brains of free-swimming trout, a large salmo- Oxford University Press; 1978. nid. The spike-timing intervals of the recorded neurons 2. Hafting T, Fyhn M, Molden S, Moser M, Moser EI. Microstructure of a spatial map in the entorhinal cortex. Nature. 2005;436(7052):801–6. with a specific refractory period suggested that the sys - 3. Taube JS, Muller RU, Ranck JB. Head-direction cells recorded from the tem could precisely record multiple neuronal activities. postsubiculum in freely moving rats. I. Description and quantitative Using the system on trout swimming in a water tank, we analysis. J Neurosci. 1990;10:420–35. 4. Buzsáki G. Neuroscience: neurons and navigation. Nature. found head direction cells in their telencephalons, firing 2005;436(7052):781–2. maximally in specific head directions, suggesting that the 5. Kim DH, Kim J, Marques JC, Grama A, Hildebrand DGC, Gu W, et al. Pan- system has the potential to examine the space-responsive neuronal calcium imaging with cellular resolution in freely swimming zebrafish. Nat Methods. 2017;14(11):1107–14. properties of neurons in fish brains. We anticipate that 6. Canfield JG, Mizumori SJY. Methods for chronic neural recording our system will stimulate the process of examining spatial in the telencephalon of freely behaving fish. J Neurosci Methods. cognition mechanisms in salmonid brains. 2004;133(1–2):127–34. 7. Yartsev MM, Ulanovsky N. Representation of three-dimensional space in the hippocampus of flying bats. Science. 2013;340(6130):367–72. 8. Vinepinsky E, Donchin O, Segev R. Wireless electrophysiology of the brain Abbreviations of freely swimming goldfish. J Neurosci Methods. 2017;278:76–86. EIB: Electrode interface board; PBS: Phosphate-buffered saline. 9. Vinepinsky E, Cohen L, Perchik S, Ben-Shahar O, Donchin O, Segev R. Representation of edges, head direction, and swimming kinematics in Acknowledgments the brain of freely—navigating fish. Sci Rep. 2020;10(1):1–16. We thank Kazuma Hase and Yojiro Yokomori for their helpful support in the 10. Takahashi S. Hierarchical organization of context in the hippocampal surgical preparations. episodic code. Elife. 2013;2013(2):e00321. 11. Takahashi S. Episodic-like memory trace in awake replay of hippocampal Authors’ contributions place cell activity sequences. Elife. 2015. https ://doi.org/10.7554/eLife ST, KY, TK, and YM conceived the project, while KI, SO, and ST performed the .08105 . histological verification. KI made tetrodes and waterproof cases designed by 12. Gray CM, Maldonado PE, Wilson M, McNaughton B. Tetrodes mark- ST, and ST, YM, TH, and RT performed the electrophysiological experiments edly improve the reliability and yield of multiple single-unit isolation and surgery. ST performed data analyses, and ST and YM wrote the manu- from multi-unit recordings in cat striate cortex. J Neurosci Methods. script, with inputs from all other authors. All authors read and approved the 1995;63(1–2):43–54. final manuscript. 13. Harris KD, Henze DA, Csicsvari J, Hirase H, Buzsaki G. Accuracy of tetrode spike separation as determined by simultaneous intracellular and extra- Funding cellular measurements. J Neurophysiol. 2000;84(1):401–14. This work was supported by the JSPS KAKENHI (Grant numbers 16H06543 and 14. Harris KD, Hirase H, Leinekugel X, Henze DA, Buzsaki G, Buzsáki G. 19H01131 to S.T., 15K07229 to Y.M., and 16H06541 to K.Y.). Temporal interaction between single spikes and complex spike bursts in hippocampal pyramidal cells. Neuron. 2001;32(1):141–9. Takahashi et al. Anim Biotelemetry (2021) 9:9 Page 12 of 12 15. Mathis A, Mamidanna P, Cury KM, Abe T, Murthy VN, Mathis MW, et al. interactions and extracellular features. J Neurophysiol. 2004;92(1):600–8. DeepLabCut: markerless pose estimation of user-defined body parts with https ://doi.org/10.1152/jn.01170 .2003. deep learning. Nat Neurosci. 2018;21(9):1281–9. 21. Putman NF, Lohmann KJ, Putman EM, Quinn TP, Klimley AP, Noakes DLG. 16. Saito K, Watanabe S. Spatial learning deficits after the development of Evidence for geomagnetic imprinting as a homing mechanism in pacific dorsomedial telencephalon lesions in goldfish. Neuroreport. 2004; salmon. Curr Biol. 2013;23(4):312–6. 17. Saito K, Watanabe S. Deficits in acquisition of spatial learning after 22. Korpela J, Suzuki H, Matsumoto S, Mizutani Y, Samejima M, Maekawa T, dorsomedial telencephalon lesions in goldfish. Behav Brain Res. et al. Machine learning enables improved runtime and precision for bio- 2006;172(2):187–94. loggers on seabirds. Commun Biol. 2020. https ://doi.org/10.1038/s4200 18. Rodríguez F, López JC, Vargas JP, Gómez Y, Broglio C, Salas C. Conserva-3-020-01356 -8. tion of spatial memory function in the pallial forebrain of reptiles and ray-finned fishes. J Neurosci. 2002;22(7):2894–903. Publisher’s Note 19. Salas C, Broglio C, Rodríguez F. Evolution of forebrain and spatial cogni- Springer Nature remains neutral with regard to jurisdictional claims in pub- tion in vertebrates: conservation across diversity. Brain Behav Evol. lished maps and institutional affiliations. 2003;62(2):82–92. 20. Bartho P, Hirase H, Monconduit L, Zugaro M, Harris KD, Buzsaki G. Char- acterization of neocortical principal cells and interneurons by network Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Animal Biotelemetry Springer Journals

Wireless logging of extracellular neuronal activity in the telencephalon of free-swimming salmonids

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Abstract

Background: Salmonids return to the river where they were born in a phenomenon known as mother-river migra- tion. The underpinning of migration has been extensively examined, particularly regarding the behavioral correlations of external environmental cues such as the scent of the mother-river and geomagnetic compass. However, neuronal underpinning remains elusive, as there have been no biologging techniques suited to monitor neuronal activity in the brain of large free-swimming fish. In this study, we developed a wireless biologging system to record extracellular neuronal activity in the brains of free-swimming salmonids. Results: Using this system, we recorded multiple neuronal activities from the telencephalon of trout swimming in a rectangular water tank. As proof of principle, we examined the activity statistics for extracellular spike waveforms and timing. We found cells firing maximally in response to a specific head direction, similar to the head direction cells found in the rodent brain. The results of our study suggest that the recorded signals originate from neurons. Conclusions: We anticipate that our biologging system will facilitate a more detailed investigation into the neural underpinning of fish movement using internally generated information, including responses to external cues. Keywords: Fish biotelemetry, Extracellular neuronal recording, Salmonids Background locations, and cells that fire maximally in response to Some fish, birds, and mammals exhibit outstanding navi - specific heading directions [3]. These internally gener - gational abilities, such as mother-river homing, seem- ated maps and compasses are thought to contribute to ingly possessing cognitive maps. Current self-location spatial navigation [4]. and compass bearings are required for such spatial navi- Until recently, there have been few reports of space- gation. In mammals, extracellular electrophysiology for responsive cells in fish brains. This lack of information freely navigating animals has led to the discovery of a is mainly due to the difficulties of underwater neuronal variety of space-responsive cells, including place cells recording. Zebrafish, a small, laboratory-bred, and non- [1] and grid cells [2], which fire maximally at specific migrant fish widely used as an animal model in neuro - science, can be genetically modified to record neuronal activity through optical imaging of intracellular calcium *Correspondence: stakahas@mail.doshisha.ac.jp; yuya.makiguchi@gmail.com dynamics. However, head clamping and genetic engi- Laboratory of Cognitive and Behavioral Neuroscience, Graduate School neering are prerequisites for such neuronal recordings. of Brain Science, Doshisha University, Kyotanabe City, Kyoto 610-0394, Japan While state-of-the-art technology has enabled us to College of Bioresource Sciences, Nihon University, Kanagawa 252-0813, image neuronal activity in the brains of free-swimming Japan Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Takahashi et al. Anim Biotelemetry (2021) 9:9 Page 2 of 12 zebrafish, they must be kept in a small restricted area under a microscope [5]. Therefore, the neural underpinning of fish movement cannot be examined in the field using the aforemen - tioned methods. For medium-sized fish, tethering cables are a prerequisite for recording extracellular neuronal activity from the brain; however, the apparatus generates significant torque in the water, severely inhibiting vol - untary movement. Despite such restrictive limitations, there have been reports of the presence of space-respon- sive cells in the brains of non-migrant fish [6]. Recent remarkable advances in microelectromechani- cal systems have led to the rapid evolution of biologging techniques. For instance, a lightweight logger for extra- cellular neuronal activity, called a neurologger, helped to discover place cells and head direction cells in the brains of flying bats [7]. Furthermore, a neurologger stored in a waterproof case [8] led to the discovery of neurons in the telencephalon of goldfish (Carassius auratus), whose activity was found to be correlated with swimming speed and a specific heading vector as they swam along a wall [9]. Theoretically, this method, pioneered on goldfish, could be applied to larger fish weighing several kilograms. However, the electrode implantation procedure devel- oped for medium-sized goldfish is not suitable for larger fish, as their brains are located deeper beneath the head surface. For example, the trout brain is located 3–4  cm Fig. 1 Experimental setup for the wireless neurologging system. a from the head surface, whereas the goldfish brain lies Head-mounted neurologger in a waterproof case records multiple only a few millimeters underneath [9]. In addition, pow- neuronal activities from the telencephalon of a free-swimming trout in a water tank. The recording parameters, including start erful (weight: approximately 2  kg trout vs. 100  g gold- and stop times, and the neuronal signal frequency bands were fish) and fast (average speed of approximately 20  cm/s) wirelessly controlled from a personal computer via a radio transceiver. swimming may affect logger protection and electrode Two-dimensional swimming trajectories were captured using a USB stabilization. 3.0 camera mounted 1.5 m above the tank. The frame timings were In the present study, combining the pioneering wireless also transmitted to the logger via the radio link (left, blue box) to synchronize the spike timing. Two pipes for water supply (top) and logging study developed in goldfish [8] with our extracel - drainage (bottom) are illustrated. b Free-swimming trout with and lular recording techniques for rodents [10, 11], we devel- without loggers. The logger was placed in a case with the base fixed oped a system for recording space-responsive cells in the to the skull, while for waterproofing, a rubber seal inserted between brains of large migrant fish. To verify the system validity, the lid and base was screwed into place. The logger recorded we recorded multiple neuronal activities from the brains neuronal activity from four tetrodes implanted in the brains of the test fish of trout swimming freely in a rectangular tank. Then, we examined the correlations between swimming behaviors and single neuron firing rates. of four twisted microwires; it has become a standard electrode widely used to simultaneously record multiple Methods single-neuronal activities in rodent brains [12]. Extracel- Biotelemetry system experimental setup lular neuronal signals were stored on an SD card using We developed a wireless biologging system that enables a commercially available neurologger (Mouselog-16B, the monitoring of extracellular neuronal signals from Deuteron Technologies, Jerusalem, Israel) placed in a the brains of free-swimming trout. Figure  1a shows the custom-made, 3D-printed, waterproof case (Fig.  1b). experimental setup for the simultaneous monitoring Using dedicated control software for MouseLog-16, we of neuronal activity and swimming trajectory. We used controlled the neurologger via a transceiver that trans- an array of extracellular tetrodes to record extracellular mitted radio signals on the 915-MHz frequency band. neural activities. This device is an electrode consisting T akahashi et al. Anim Biotelemetry (2021) 9:9 Page 3 of 12 The neurologger also recorded external trigger signals skull, fat and tissue fluid were gently extracted with a ® ™ ™ to identify where the fish was located in the water tank Kimwipe (Kimtech Science Kimwipes ) to expose when the action potential occurred from the neuron. the olfactory bulb, telencephalon, and optic tectum. These signals were recorded for each frame captured by For the anchor screw, a maximum of 12 holes equally a USB 3.0 video camera (ace acA3088-57uc, Basler AG, spaced were drilled along the edge of the oval hole. Ahrensburg,  Germany) mounted 1.5  m above the water The number of holes was determined on a subject-by- tank and transmitted from a personal computer via subject basis to avoid scattered cartilaginous tissues a radio link. As was the case in the previous study, the that mainly reside in the rostral area. After the anchor radio link was available within a few tens of centimeters was screwed into the holes, the sites were covered with below the water surface. The details are described in the dental acrylic resin (Unifast III, GC Inc., Japan). The following subsections. ground wire was implanted into the medial optic tec- tum. The tetrode array described below was implanted into the targeted recording locations of the telencepha- Animals lon with micromanipulators (SM-15L or SMM-200, The three fish (rainbow trout [Oncorhynchus mykiss]; Narishige Inc., Japan, respectively) (Fig.  2b). Since the body weights: 1,548–2,230  g) used in the experiment brain depth was approximately 3–4 cm below the skin, were purchased from the Fuji Trout farm in Fujinomiya the precise coordination of electrode locations was per- City, Shizuoka Prefecture, Japan, where they were fed formed on a case-by-case basis. commercial pellets once a day. To facilitate the neu- The space above the brain was filled with Vaseline, rologger attachment, we conducted our experiments at while the space between the head and guide tubes the Fuji Nature Education Center of Nihon University. was filled with small, 3D-printed pieces and then cov - We chose our experimental fish without discriminat - ered with dental acrylic resin. Finally, the neurologger ing for sex. After purchase, they were transported by case was attached to the electrodes, fixed, and cov - car to the Education Center, where they were acclimated ered with dental acrylic resin (Fig.  2c). All operations in spring water at approximately 9  °C until required for were completed within 90  min. After surgery, the fish experimentation. were immediately moved to an outdoor experimental The fish were not fed during the experiment, and all tank (145 cm × 105 cm) and acclimated until they were procedures were approved by the University of Tokyo observed to swim normally. Institutional Animal Care and Use Committee. Surgery Electrode fabrication The experimental fish were immersed in an anesthetic Based on the experience acquired in rodent studies solution, prepared by adjusting FA 100 (eugenol; Tan- [10, 11], we used tetrodes containing four tungsten abe Seiyaku Co. Ltd, Osaka, Japan) to a concentration of microwires (12.5  μm, HML-coated, California Fine 0.5  mL/L, for 5–10  min, until their gill lids ceased mov- Wire, CA, USA), twisted and bundled with a heat gun, ing. When the experimental fish were immobilized, they to record extracellular neuronal activity. Each tetrode were fixed to an acrylic apparatus on a specific mount - was inserted into a polyimide tube (inner diameter: ing table to facilitate the electrode implantation and 0.04 mm, outer diameter: 0.20 mm). The tube array was neurologger attachment (Fig.  2a). The anesthesia, pre - constructed by gluing the tubes in a concentric circle pared with FA 100 at a concentration of 0.25  mL/L, was arrangement, with a distance of approximately 0.2  mm refluxed from the mouth to the gills by tube, using a peri - between them (Fig.  3a). To stabilize the electrode drift, staltic pump for circulation. The fish back was covered a flexible electrode tip (approximately 0.5  cm) was with a towel, kept wet during the experiment by pump- exposed from the tubes to absorb the torque caused by ing water onto it to prevent the body from drying. During the trout’s powerful and fast swimming. Each microw- the experiment, a bag of ice was added to the water tank ire within a tetrode was crimped to the corresponding every 30  min to prevent sudden increases in water tem- hole in the electrode interface board (EIB) using a gold perature, maintaining it at approximately 9 °C. pin (Fig.  3b, c). Immediately before surgery, the tip of With the help of an imaginary line connecting the the tetrode was cut at a right angle. The impedance back of the eyes as a starting point, a rectangular por- was approximately 600 kΩ at a frequency of 1  kHz. tion of the epidermis was removed with a scalpel for a A ground electrode, an insulated stainless-steel wire length of 3–4  cm toward the caudal fin. A micro-drill (diameter: 0.2  mm) implanted into the medial optic was then used to prepare an oval hole in the exposed tectum, also served as a reference point for neuronal skull, followed by a meticulous procedure intended to recordings (Fig. 3c, green wire). avoid damage to the brain. After removing the excised Takahashi et al. Anim Biotelemetry (2021) 9:9 Page 4 of 12 Fig. 2 Surgery for tetrode implantation and case fixation. a To implant the tetrodes into the brain, each anesthetized fish was fixed to an acrylic fixture on a custom-made mounting table (arrow). The anesthetic solution was circulated using a peristaltic pump. The fish back was covered with a wet towel (pink) to prevent the body from drying, and water was poured onto it with a small pump during the experiments. A bag of ice was added to the water tank every 30 min during the experiments to prevent sudden water temperature increases. b Oval hole drilled into the exposed fish skull (white arrow). A scalpel was used to excise a rectangle of epidermis in the direction of the caudal fin, starting from an imaginary guideline between each fish’s eyes. To expose the olfactory bulb, the telencephalon, optic tectum, fat, and tissue fluid were carefully removed. Anchors were screwed into holes equally spaced along the edge of the oval hole and covered with dental acrylic resin. c A ground wire and tetrode array were implanted into the medial optic tectum and target recording locations of the telencephalon using micromanipulators. The space above the brain was then filled with Vaseline, while the space between the head and electrode interface board (EIB) was filled with small, 3D-printed pieces and then covered with a dental acrylic resin. The neurologger case was attached to the EIB and covered with dental acrylic resin (See figure on next page.) Fig. 3 Electrode array and waterproof case for neurologger. a An array of tetrodes; each tetrode was inserted into a polyimide tube, and the tubes were glued together to construct an array with constant internal spacing. b Housing for connecting the tetrode array to the custom-made electrode interface board (EIB). c Each tetrode microwire was crimped into a corresponding contact hole using a gold pin. d Cover to attach the EIB to the tetrode array. e Tetrode array assembly. f–g Tetrode array with an undercover to connect to the logger case (f). h–j Base (h), support (i), and lid (j) for the custom-made, 3D-printed, waterproof case. k Lid with rubber seal. l The base was attached to the housing and covered with dental acrylic resin (scale bar = 1 cm) T akahashi et al. Anim Biotelemetry (2021) 9:9 Page 5 of 12 Takahashi et al. Anim Biotelemetry (2021) 9:9 Page 6 of 12 Fig. 4 Attaching a logger. a To prevent water ingress, the slight gap between the base and the socket of the neurologger (white arrow) was filled with an ultraviolet-curable adhesive immediately after the electrode was implanted. b–c Each day, before the initial recording session, the neurologger, including its SD card (b) and battery (c), was placed in the waterproof case, while the anesthetized fish was fixed to the mounting table. d After the neurologger and the battery were attached to the base, the lid, complete with its rubber seal, was screwed into the base using four screws Waterproof case Neurologger We manufactured a waterproof neurologger case using Before the extracellular neuronal signals were stored in a pioneering study on goldfish as a reference [8 ]. The an SD card, they were unity-gain buffered, digitized, and base, lid, and tetrode array housing were fabricated continuously sampled at 31.25  kHz with an RHA2000 using a 3D printer (Fig.  3h, j) (Form 2 or 3, Formlabs, chip (Intan Technologies, CA, USA) in the MouseLog- MA, USA). The base and tetrode array housing were 16B, operating in either wide- (1–7000  Hz) or narrow- designed to mesh with each other, and the tetrode band (300–7000 Hz) mode. We simultaneously recorded array was connected to the neurologger through a hole up to four tetrodes (16 channels) for over one hour with a in the base. The slight gap between the base and the battery (3.7 V, 170 mAh). The weight of the neurologger, socket was filled with an ultraviolet-curable adhesive including the battery, was approximately 6  g. We used (Bondic , Bondic Co., NY, USA) (Fig.  4a, arrow). After a magnetic switch to turn on the neurologger imme- the base and housing were covered with dental acrylic diately before each recording session to reduce battery resin, the lid with a rubber seal (chloroprene, thickness: consumption. 2  mm) (Fig.  3k) was placed on the base and tightened using four screws (Figs.  3l, 4d). For logger protection Offline preprocessing against high torque, we added a lid, in addition to the After the neuronal signals were downloaded from the SD base and supports, to close the case. The outer dimen - card, action potential (spike) data were digitally filtered sions of the waterproof case were 5  cm × 5  cm × 3  cm. at 800–7500  Hz. The tetrode recording contained mul - The total weight of the case was approximately 17  g. tiple single-neuronal activities. The spikes were isolated Before the initial recording session, the neurologger using spike-sorting software (KlustaKwik, open-source and battery were placed in the waterproof case, and software by Harris Lab, UCL, London, UK, and avail- the anesthetized fish was fixed to the mounting table able from https ://sourc eforg e.net/proje cts/klust akwik ) (Fig. 4b, c). and manually verified [13] to extract individual neuronal T akahashi et al. Anim Biotelemetry (2021) 9:9 Page 7 of 12 Fig. 5 Histological identification of recording locations in the brain. a Illustrations showing the positions of the brains of trout (top) and goldfish (bottom). Unlike goldfish, the trout brain is located far below the head surface. b Sample coronal section stained to show Nissl material from a trout telencephalon ( Trout B). The arrowheads depict the estimated recording locations. For reference, a mirror image outline of the brain section was drawn on the left. c All estimated locations for the three trout (color-coded dots) are depicted on the brain section outlines (Dm = pars medialis; Dl = pars lateralis, in the dorsal pallium). d Color-coded planes indicate the locations of the coronal brain sections where the recording locations were estimated for the three trout shown in c through imagery captured at 30 frames per second. We activity. Cells with ≤ 99 spikes were excluded from the used a USB 3.0 digital video camera with a non-distorting analysis. Cell type classification using the extracellular lens (C-Mount, Manual Iris, Wide Angle Lens, # 89–524, spike waveform feature was not performed, as the neces- Edmund Optics, Japan) mounted 1.5  m above the water sary criteria have not been established for fish neurobiol - tank (Fig.  1a); then, by concatenating the tracked fixed ogy. The unit isolation quality was quantified for each cell base of the case, we were able to reconstruct swimming based on the isolation distance index [14]. An example of trajectories (Fig.  7a). The image definition was set to the spike-sorting performance quality is shown in Fig. 5. 800 × 800 pixels. We computed the head direction from the tracked leading edge and fixed base using the inverse Video tracking of the tangent function. 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 Takahashi et al. Anim Biotelemetry (2021) 9:9 Page 8 of 12 Fig. 6 Neuronal activities extracellularly recorded from free-swimming trout telencephalons with the wireless neurologging system. a Examples of well-isolated neuronal activities. Spikes recorded from a tetrode were plotted as dots in four selected 2D feature spaces (1st to 3rd principal components [PCs], or energies). Spikes were color-coded to indicate two different neurons. b-d Corresponding superimposed spike waveforms with averaged spike waveforms (solid black line) and spike-timing auto-correlograms [bin widths = 1 ms (c) and 0.1 ms (d)]. The superimposed spike waveform exhibits a signal-to-noise ratio. The spike auto-correlograms, especially those shown in d, indicate that the spike-timing intervals had a 1–2 ms refractory period. e Mean firing rate distributions for the entire suite of neurons observed in the test trout dorsal palliums T akahashi et al. Anim Biotelemetry (2021) 9:9 Page 9 of 12 Fig. 7 Trout head direction cells. a Representative trajectory (gray line) with positions showing neuron-generated spikes (red dots) as recorded from the telencephalon of trout swimming in a water tank (scale bar = 10 cm). Remarkable visible objects (pipe for water supply [black filled rectangle], hole for water sink [black filled circle], and radio transceiver [blue filled rectangle]), and compass bearing (bottom, left) are illustrated. b Polar plots of firing rate as a function of head direction, using examples from two representative neurons. The mean vector length (Mv) and peak firing rate (P) are indicated in the images. Visible objects are illustrated as in a. c–d Distribution of mean vector length for randomly shuffled data (c) and for the entire suite of neurons observed in our test trout dorsal palliums (d). The red line and the number indicate the 95th percentile for the shuffled data. e Circular distribution of the number of cells on the head orientation for seven individual head direction cells identified from trout G and #2. Head direction cells exist in the brains of trout G (red) and #2 (blue). In contrast, trout B had no head direction cells. Visible objects are illustrated as in a Table 1 Identified cells from the telencephalon of trout Trout G #2 B # of isolated cells 3 6 14 # of head direction cells 3 4 0 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 begin- ning. A head-direction-tuning function was then con- structed, 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 th set of permutations from all examined cells, and the 95 percentile was determined. Analysis software All analyses were performed using custom-made pro- grams 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- Head direction cell analysis buffered formalin fixative (3.5–3.8% formaldehyde). The The directional tuning function for each cell was obtained extracted brains were post-fixed overnight with David - by plotting its firing rate as a function of the fish head son’s fixative (also known as Hartmann’s fixative) solution direction, divided into bins of 0.5°, and smoothed using a (22.2  mL 10% buffered formalin, 32.0  mL 99% ethanol, 14.5° mean window filter. 11.1  mL acetic acid, and 100  mL distilled water) at 4 ℃. The directional tuning strength was estimated by com - The brains were incubated in gelatin solution (10% gela - puting the mean vector length for the circular distribu- tin in phosphate-buffered saline [PBS]) at 37  °C for 4  h tion of the firing rate. Head direction-modulated cells and solidified at 4  °C. The brain embedded in the gela - were defined as cells in the recorded data with mean vec - tin block was fixed in 10% phosphate-buffered forma - tor lengths > 95th percentile of the shuffled data. For each lin fixative (3.5–3.8% formaldehyde) at 4  °C and sank in permutation trial, the entire sequence of spikes fired by a ▸ Takahashi et al. Anim Biotelemetry (2021) 9:9 Page 10 of 12 30% sucrose in PBS at 4  °C. The brains were cut coro - found and called head direction cells [3]. Recently, a pio- nally with a microtome (Ritratome REM-710, Yamato neering study also reported the presence of head direc- Koki Co., Saitama, Japan) set at 40  µm. The resulting tion cells in the telencephalon of goldfish [9]. Similarly, brain slices were then stained with cresyl violet (Sigma- the neuronal activity of some cells in the trout telenceph- Aldrich, C5042-10G) to facilitate the examination of their alons exhibited head direction preferences, becoming cytoarchitecture. more active when their heads were oriented in a specific direction (Fig. 7b). Seven (30.4%) of the 23 cells recorded Results passed the criterion to be classified as head direction cells As a proof of principle, we recorded the activity of 23 (the mean vector length exceeded the 95th percentile of cells from the telencephalons of three trout using a wire- the mean vector lengths distribution in shuffled data). less biologging system with a lightweight neurologger This number was significantly larger than expected for a enclosed in a 3D-printed, waterproof case. All recordings random selection from the distribution (P < 0.001, bino- were made while the trout were voluntarily swimming mial test with an expected P of 0.05). Whereas cells in in a rectangular water tank, and the fish did not exhibit one trout did not exhibit head direction preferences, the reward-seeking behaviors during the recording periods. head direction cells were found in the telencephalon of Nissl staining showed that the electrode tracks were dis- two of the three trout (Table 1). The heading orientations tributed around the dorsal pallium of the telencepha- covered a wide span (Rayleigh’s test for nonuniform- lon (Fig.  5). Previous fish studies have reported that the ity, P > 0.05, z = − 4.68; Fig.  7c), suggesting that the head dorsal pallium is deeply involved in spatial learning and direction preference was not due to artifacts. Overall, the memory [9, 16–19]. head direction cells were tuned to neither a specific com - pass bearing nor visible objects, including pipes for water Neuronal activity recorded by the wireless logging system sink and source, and radio transceiver (Fig. 7e). However, To confirm whether the signals recorded from the test at least one cell from trout G or #2 mainly fired whenever trout brains originated from neurons, we examined spike the head pointed south or to the water supply. Further- statistics, including spike shape, firing rate, and spike- more, neighboring head direction cells recorded from the timing intervals. The recorded neurons typically exhib - same electrode exhibited a similar heading trigger (trout ited a narrow spike shape [mean peak-to-trough spike G: two of three neighboring head direction cells; trout #2: width: 0.18 ± 0.11 ms (mean ± SD)]. However, cells show- two of two neighboring head direction cells). ing a wider spike shape were also observed (widest spike width: 0.44  ms). In the telencephalons of rodents, cells Discussion exhibiting a narrow spike shape and high firing rates are We demonstrated the capacity of our novel wireless log- categorized as fast-spiking cells [20]. Although the neces- ging system to record neuronal activity in the brains of sary criteria have not been established for fish neurobiol - large salmonids. A previous pioneering study devel- ogy, these results appeared to show that our biologging oped a wireless logging system for goldfish [8]; however, system could record different neuron types. unlike goldfish, salmonids exhibit remarkable mother- The mean firing rate distributions were skewed river homing on a global scale. While swimming tra- (median: 1.45 Hz, Fig. 6e). The typical spike-timing auto- jectories and accompanying external cues have been correlogram for identified single neurons demonstrated researched previously [21], neuronal activity in the that the spike-timing intervals had a clear refractory brains of free-swimming salmonids has not been stud- period (1–2  ms) (Fig.  6d), suggesting that the identified ied before because of the difficult access to the salmonid neurons belonged physiologically to a single type. These brain, located deep in the head, and the trout weight of results suggest that our system was capable of simultane- several kilograms generating high torque. Thus, our sys - ously recording multiple neuronal activities in the brains tem extends the wireless logging in goldfish, providing of free-swimming trout. the possibility to examine the neuronal underpinning of underwater behavior in large salmonids. Trout head direction cells In a previous study on the telencephalon of goldfish Finally, to demonstrate our logging system capacity to [9], we also found head direction cells in large salmo- examine neuronal correlation with fish movement, we nids. However, we could not investigate whether the head examined the neuron firing rates as a function of fish direction preference was tuned to specific cues, including swimming trajectory and heading (Fig. 7a). In some brain geomagnetic compass bearing and landmarks, because of regions of rodents, including the anterior thalamus, sub- the lack of environmental manipulation. Further studies iculum, retrosplenial cortex, and entorhinal cortex, cells will be required to elucidate the neuronal underpinning that maximally fire at a specific heading direction were of spatial information processing in large salmonids. T akahashi et al. Anim Biotelemetry (2021) 9:9 Page 11 of 12 Availability of data and materials Despite its exceptional potential in the field of ani - The 3D CAD designs for the waterproof case and tetrode housing are available mal biotelemetry, we could not test the ability to record at https ://githu b.com/Takah ashiL ab/Trout . The datasets used and/or analyzed neuronal activity in natural rivers where unpredictable during the current study are available from the corresponding author upon reasonable request. events occur, such as higher water pressure, unexpected rocks, and animal recapture. Therefore, the question Ethics approval and consent to participate of whether our method can be used for mother-river All procedures were approved by the University of Tokyo Institutional Animal Care and Use Committee (#A-19–8). homing remains unanswered. Technical improvements are required to address this question. For instance, the Consent for publication waterproof case square box shape must be formed with Not applicable. a streamlined shape against hydraulic resistance in riv- Competing interests ers. Furthermore, since our system cannot synchronize The authors declare no competing interests. external event signals in deeper water and only record Author details neuronal activity for a few hours, neuronal recordings Laboratory of Cognitive and Behavioral Neuroscience, Graduate School need to be operated automatically and locally above the of Brain Science, Doshisha University, Kyotanabe City, Kyoto 610-0394, Japan. fish’s head. A state-of-the-art AI-assisted biologger [22] Graduate School of Environmental Studies, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan. Department of Cell Biology can accomplish such tasks, detect interest behaviors in and Neuroscience, Graduate School of Medicine, Juntendo University, Bun- real time, and activate sensors such as gyroscopes, accel- kyo-ku, Tokyo 113-8421, Japan. International Coastal Research Center, The eration, and water depth. In a natural environment, it is Atmosphere and Ocean Research Institute, The University of Tokyo, 1–19–8, Akahama, Otsuchi, Iwate 028–1102, Japan. College of Bioresource Sciences, desirable to run this system with an AI-assisted biologger Nihon University, Kanagawa 252-0813, Japan. to start recording neuronal activity at the time of interest. Received: 19 October 2020 Accepted: 27 January 2021 Conclusions In the present study, we developed a wireless logging References system capable of recording multiple neuronal activities 1. O’Keefe J, Nadel L. The hippocampus as a cognitive map. New York: from the brains of free-swimming trout, a large salmo- Oxford University Press; 1978. nid. The spike-timing intervals of the recorded neurons 2. Hafting T, Fyhn M, Molden S, Moser M, Moser EI. Microstructure of a spatial map in the entorhinal cortex. Nature. 2005;436(7052):801–6. with a specific refractory period suggested that the sys - 3. Taube JS, Muller RU, Ranck JB. 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Published: Feb 12, 2021

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